Citation
Use of biodegradable pH-sensitive surfactants in liposome mediated oligonucleotide delivery

Material Information

Title:
Use of biodegradable pH-sensitive surfactants in liposome mediated oligonucleotide delivery
Creator:
Liang, Chih-Wei Earvin, 1971-
Publication Date:
Language:
English
Physical Description:
ix, 188 leaves : ill. ; 29 cm.

Subjects

Subjects / Keywords:
Antisense oligonucleotides ( jstor )
Fluorescence ( jstor )
Imidazoles ( jstor )
Lipids ( jstor )
Liposomes ( jstor )
Oligonucleotides ( jstor )
pH ( jstor )
Propionates ( jstor )
Surfactants ( jstor )
Teeth ( jstor )
Department of Pharmaceutics thesis Ph.D ( mesh )
Dissertations, Academic -- College of Pharmacy -- Department of Pharmaceutics -- UF ( mesh )
Drug Delivery Systems ( mesh )
Hydrogen-Ion Concentration ( mesh )
Liposomes ( mesh )
Oligonucleotides -- therapeutic use ( mesh )
Research ( mesh )
Surface-Active Agents ( mesh )
Vehicles ( mesh )
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph.D.)--University of Florida, 1998.
Bibliography:
Bibliography: leaves 168-187.
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Chih-Wei Earvin Liang.

Record Information

Source Institution:
University of Florida
Holding Location:
University of Florida
Rights Management:
The University of Florida George A. Smathers Libraries respect the intellectual property rights of others and do not claim any copyright interest in this item. This item may be protected by copyright but is made available here under a claim of fair use (17 U.S.C. §107) for non-profit research and educational purposes. Users of this work have responsibility for determining copyright status prior to reusing, publishing or reproducing this item for purposes other than what is allowed by fair use or other copyright exemptions. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder. The Smathers Libraries would like to learn more about this item and invite individuals or organizations to contact the RDS coordinator (ufdissertations@uflib.ufl.edu) with any additional information they can provide.
Resource Identifier:
029445277 ( ALEPH )
51586443 ( OCLC )

Downloads

This item has the following downloads:


Full Text









USE OF BIODEGRADABLE pH-SENSITIVE SURFACTANTS IN LIPOSOME
MEDIATED OLIGONUCLEOTIDE DELIVERY














By

CHIH-WEI EARVIN LIANG












A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 1998































Dedicated to my parents Tu-Jow and Chow-Son Liang.














ACKNOWLEDGMENTS


I would like to express my sincere appreciation to Dr. Jeffrey A. Hughes, chairman of my committee. Without his patience, understanding, guidance, and encouragement, I could not receive my Ph.D. degree. I extend my gratitude to my other committee members, Dr. Hartmut Derendorf, Dr. Laszlo Prokai, Dr. Sheldon Schuster, and Dr. Ian R. Tebbett, for their suggestions and criticisms, and especially Dr. Gayle A. Brazeau for her consistent care and concern.

I also give my acknowledgment to all the personnel in the Department of

Pharmaceutics including the secretaries, graduate students, and in particular the Brazeau & Hughes group who helped me throughout my entire graduate life and research at the University of Florida. Finally, I want to thank my parents and siblings for their unselfish love and continuous support during both my good and bad times in the past 26 years.





















111














TABLE OF CONTENTS
page


ACKNOWLEDGMENTS ...................................................................... iii

KEY TO ABBREVIATIONS................................................................... vi

ABSTRACT................................................................................... viii

CHAPTERS

1 INTRODUCTION ................................................................... 1

Objective .............................................................................. 2
Hypothesis............................................................................. 2

2 BACKGROUND AND SIGNIFICANCE ......................................... 3

Oligonucleotide Therapy Overview ................................................ 3
Toxic Effects of Oligonucleotide ................................................... 6
Barriers to Oligonucleotide Transfer and Activity................................ 6
Strategies Available to Deliver Oligonucleotides................................ 10
Origin of Biodegradable pH-Sensitive Surfactants-Lysosomotropic
Detergents....................................................................... 21
Significance of Biodegradable pH-Sensitive Surfactants ...................... 22
Specific Aims........................................................................ 23

3 DESIGN AND SYNTHESIS OF BIODEGRADABLE pHSENSITIVE SURFACTANTS .................................................... 28

Introduction.......................................................................... 28
Rationale............................................................................. 28
Selection of Biodegradable pH-Sensitive Surfactants.......................... 33
Syntheis of Biodegradable pH-Sensitive Surfactants............................ 35
Identification of Biodegradable pH-Sensitive Surfactants..................... 39

4 PHYSICOCHEMICAL CHARACTERIZATION OF
BIODEGRADABLE pH-SENSITIVE SURFACTANTS ..................... 49

Introduction.......................................................................... 49
Materials............................................................................. 49


iv








M ethods ............................................................................................................ 52
Results .............................................................................................................. 57
Discussion ........................................................................................................ 78
Conclusion ....................................................................................................... 83

5 DELIVERY SYSTEM EVALUATION OF BIODEGRADABLE pHSEN SITIV E SU RFA CTAN TS ........................................................................ 84

Introduction ...................................................................................................... 84
M aterials .......................................................................................................... 85
M ethods ............................................................................................................ 86
Results .............................................................................................................. 95
D iscussion ...................................................................................................... 121
Conclusion ..................................................................................................... 125

6 MECHANISM OF ACTION INVESTIGATION OF
BIODEGRADABLE pH-SENSITIVE SURFACTANTS ............................. 126

Introduction .................................................................................................... 126
M aterials ........................................................................................................ 127
M ethods .......................................................................................................... 129
Results ............................................................................................................ 133
D iscussion ...................................................................................................... 151
Conclusion ..................................................................................................... 160

7 CONCLUSION AND FUTURE PROSPECT ............................................... 162

Conclusion ..................................................................................................... 162
Future Aim s ................................................................................................... 165

REFEREN CES .................................................................................................... 168

BIO GR APH ICAL SK ETCH ............................................................................... 188


















v














KEY TO ABBREVIATIONS


ANTS................................................................ 1-aminonaphthalene-3,6,8-trisulfonic acid

BPS ......................................................................biodegradable pH-sensitive surfactant(s)

CMC.......................................................................................critical micelle concentration

DDAB................................................................. dimethyldioctadecylammonium bromide

DC-Chol.................................3 (3-[N-(N',N'-dimethylaminoethane)carbamoyl]cholesterol

D I .......................................................................................................N -dodecyl im idazole

DIP............................................................................dodecyl 2-(1 '-imidazolyl) propionate

DMF..............................................................................................N,N-dimethylformamide

DMRIE............... 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide

DMPC.......................................................... 1,2-dimyristoyl-sn-glycero-3-phosphocholine

D M SO .................................................................................................... dim ethyl sulfoxide

DOPE............................................................................dioleoylphosphatidylethanolamine

DOSPA...............2,3-dioleyloxy-sperminecarboxamido-N,N-dimethyl- 1 -propanaminium

DOTAP......... N-[1-(1-2,3-dioleoloxy)propyl]-N,N,N-trimethylammonium methylsulfate

DOTMA...................N-[ 1-(2,3-dioleoloxy)-propyl]-N,N,N-trimethylammonium chloride

DPX..................................................................N, N'-p-xylylenebis-(pyridinium bromide)

FITC............................................................................................fluorescein isothiocyanate

HVJ...................................................................................hemagglutinating virus of Japan

MIL........................................................................................ methyl 1-imidazolyl laureate



vi








NBD-PE .........................N-(7-nitro-2,1,3-benzoxadiazol-4-yl)-phosphatidylethanolamine

NM R ........................................................................................ nuclear magnetic resonance

PBS ............................................................................................. phosphate buffered saline

PE ............................................................................................... phosphatidylethanolamine

R ................ molar ratio of the biodegradable pH-sensitive surfactants to the other lipids

Re ...................................................................................................... effective release ratio

Rh-PE ...............................N-(lissamine rhodamine B sulfonyl)-phosphatidylethanolamine







































vii













Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

USE OF BIODEGRADABLE pH-SENSITIVE SURFACTANTS IN LIPOSOME
MEDIATED OLIGONUCLEOTIDE DELIVERY By

Chih-Wei Earvin Liang

August, 1998


Chairman: Jeffrey A. Hughes, Ph.D.
Major Department: Pharmaceutics

Oligonucleotide therapy is a promising approach for the treatment of a variety of disorders. A factor limiting the therapeutic effect of oligonucleotides is their inefficient transport to the cytoplasm or nucleus. Most oligonucleotides with or without a delivery system enter cells by endocytosis. Initially they accumulate in endosomes which are intracellular compartments with acidic intraluminal pH. In time, the endosomes mature into lysosomes and oligonucleotides are eliminated. Consequently, the development of a delivery system that increases oligonucleotide transfer from endosome to cytoplasm is essential. Biodegradable pH-sensitive surfactants (BPS) were therefore designed to enhance liposome mediated oligonucleotide delivery. BPS are a unique family of easily metabolized compounds that demonstrate pH-dependent surface activity. Through simple and fast chemical reactions, three BPS, dodecyl 2-(l'-imidazolyl) propionate (DIP), methyl 1-imidazolyl laureate (MIL), and N-dodecyl imidazole (DI), were


viii








synthesized for use in subsequent studies. Critical micelle concentration and effective release ratio of the ionized BPS were determined to identify their surface active properties. Surfactant like behaviors of BPS in a pH-dependent manner were then assessed. Also, several physicochemical parameters of BPS were measured and compared including chemical stability, biodegradability, and cytotoxicity. Utilizing an in vitro model and confocal microscopy, liposomes combined with BPS demonstrated better quantitative and qualitative effects in enhancing oligonucleotide cellular delivery than liposomes without BPS. The enhanced effect due to the presence of BPS, as shown by flow cytometry, was not due to increased cellular uptake of oligonucleotides, but rather their redistribution upon entering the cells. A liposomal model was employed to better understand the membrane defect mechanisms elicited by BPS that enabled the intracellular redistribution of oligonucleotides. This system suggests that depending upon the pH and molar ratio of BPS to membrane lipids, BPS can induce both membrane fusion and rupture which are different from other surfactants and fusogenic compounds. They appear to contribute and participate in the membrane fusion at different stages. In comparison to other similar delivery systems, BPS are more economical, easily produced, and less toxic. Consequently, they provide a potential excellent choice for oligonucleotide delivery.














ix













CHAPTER 1
INTRODUCTION


Oligonucleotides have been used as a gene therapy approach since the late 1970s (Zamecnik & Stephenson, 1978). The theoretical approach of oligonucleotides is very attractive since it allows for the inhibition of a specific protein. Oligonucleotides with or without a carrier are transported into cells mostly by endocytosis (Akhtar & Juliano, 1992; Loke et al., 1988) and accumulate in endosomes, intracellular compartments with an acidic intraluminal pH (Maxfield, 1985; McGraw & Maxfield, 1991). A factor limiting the pharmacological effectiveness of oligonucleotides is their inefficient transport to their sites of action in the cytoplasm or nucleus. The rate and extent of movement from endosomes appear to be important in determining oligonucleotide effects.

Consequently, the development of accessory compounds that enhance endosome to cytoplasm transfer may be vital to oligonucleotide therapy. By taking advantage of the characteristics of surfactants and exploiting a naturally occurring event, the pH gradient, during oligonucleotide transport, we have proposed a delivery system that may increase oligonucleotide cytoplasmic delivery and biological activity.





2

Objective


The main objective of the project was to characterize biodegradable pH-sensitive surfactants (BPS) both physicochemically and biochemically which can serve as adjuvants for improved transfer of oligonucleotides from the endosome to the cytoplasm without associated cellular toxicity.


Hypothesis


The overall hypothesis was that the endosomal membrane presents a major barrier to oligonucleotide delivery. By the addition of biodegradable pH-sensitive surfactants (BPS) to a liposomal delivery system, the low endosomal pH can activate the pHsensitive surfactants and facilitate the transfer of endosomal contents (i.e., oligonucleotide) to the cytoplasm or nucleus. The biodegradable linker can be digested into less toxic metabolites by the endogenous digestive enzymes.













CHAPTER 2
BACKGROUND AND SIGNIFICANCE Oligonucleotide Therapy Overview


The ability of short, synthetic, and single-stranded DNA or RNA oligonucleotides to interfere with individual gene expression in a sequence-specific manner is the foundation for oligonucleotide-based therapy. The first clear exploration of oligonucleotides was reported by Zamecnik and Stephenson (1978). However, due to a number of impediments (e.g., understanding of the sequence and topology of the nucleic acid target, synthesis of research quantities of oligonucleotides, and modification of stabilized oligonucleotides), research in using oligonucleotides for biological studies was limited until the late 1980s. Following advances in oligonucleotide chemistry and initial biological studies (Agrawal et al., 1988; Gao et al., 1989; Smith et al., 1986), interest in oligonucleotide therapy began to increase.

Oligonucleotides, short nucleotide polymer in length (12 to 40 mer) of a synthetic single-stranded nucleic acid, are designed to a specific gene (Cooney et al., 1988; Moser & Dervan, 1987), mRNA (Stein & Cohen, 1988), or protein (Bock et al., 1992; Ellington 1994). After binding to their target in cells, oligonucleotides can prevent the production of a specific protein product (Figure 2-1). Detailed mechanisms are explained in






3





4
















Nucleus





Oligonucleotide



Site of Action
mRNA Protein



Figure 2-1: A depiction of protein synthesis and possible sites of action for designed oligonucleotides. There are three potential sites where oligonucleotides can have actions. First, oligonucleotides (antigenes) can be used to inhibit the transcription process from double stranded DNA to single stranded mRNA through Hoogsteen base pairing interactions. Second, complimentary oligonucleotides (antisense oligonucleotides) can be designed to bind with mRNA to restrain the translation process through Watson-Crick hydrogen bond interactions. Finally, oligonucleotides (aptamers) can interact with a synthesized protein to interfere with its activity via hydrogen bondings.





5


excellent review articles (Crooke, 1993; Helene & Toulme, 1990; Scanlon et al., 1995; Sharma & Narayanan, 1995; Uhlman & Peyman, 1990).

Simply, a triplex forming oligonucleotide (antigene oligonucleotide) is capable of binding to the major groove in double-stranded DNA via Hoogsteen base interactions, thereby causing a triple helical structure and further resulting in sequence-specific inhibition of transcription. On the contrary, an antisense oligonucleotide complementary to a specific sequence of mRNA can hybridize to a given mRNA through Watson-Crick hydrogen bonds (Zamecnik, 1996). It can inhibit translation by several proposed mechanisms including activation of RNase H and blockade of ribosomal reading. RNase H is an endogenous cellular enzyme which can recognize a hybrid duplex between DNA and RNA (Ghosh et al., 1993). RNase H leads to RNA cleavage and release of the DNAoligonucleotide. The freed DNA-oligonucleotide is then able to hybridize to another RNA strand and repeat the RNase H dependent degradation, thus forming the basis for a catalytic effect.

Oligonucleotide therapy is most often directed at inhibiting production of disease causing proteins. The fact that very distinct interactions of oligonucleotides to target sequences occur suggests that oligonucleotide therapy have the potential to be orders of magnitude more specific than conventional drug therapy. Therefore, it may yield a greater therapeutic effect and is an exciting technology for manipulating gene expression in the treatment of human gene diseases.





6


Toxic Effects of Oligonucleotide


High doses of oligonucleotides have been reported harmful in animal studies and the toxicity of oligonucleotides appears to be species dependent. Administration of 100 mg/kg i.p. three times weekly for two weeks in mice and rats resulted in significant toxicity including acute renal failure, liver damage, spleen damage, immune stimulation, severe thrombocytopaenia, and death (Krieg et al., 1995; Sarmiento et al., 1994). Bolus i.v. administration of oligonucleotides in monkeys produced a transient decrease in peripheral total white blood cell, neutrophil counts, prolongation of acitvated partialthromboplastin time, hypotension, and death (Galbraith et al., 1994). As a result of the relatively long retention time in the reticuloendothelial system organ, accumulation of oligonucleotides and their metabolites may be responsible for these toxicities (Zhang et al., 1995).



Barriers to Oligonucleotide Transfer and Activity


The therapeutic promise of specific oligonucleotide interaction is great. However, several technical problems including stability and delivery must be overcome before oligonucleotides can be useful drugs.


In Vitro/In Vivo Stability


Of all the possible obstacles, rapid degradation of unmodified DNA and RNA phosphorodiester oligonucleotides in the biological milieu is the first problem encountered (Akhtar et al., 1991b; Shaw et al., 1991). Enzymes, non-specific endo- and





7


exo-nucleases, limit phosphorodiester oligonucleotides' physiological half-life to a few minutes (Akhtar et al., 1991 b). This short biological half-life makes the therapeutic use of phosphodiester oligonucleotides unlikely. Biologically stable oligonucleotides are achievable by chemically altering the phosphorodiester backbone. To maximize the effect, the modified oligonucleotides should be stable in both serum and inside the cell, able to reach their site of action, and form stable Watson-Crick or Hoogsteen complexes with target sequences.

These modifications summarily occur in three locations (for detailed review see Uhlman et al., 1997):

* Internucleotide phosphodiester bridge

* Base group

* Sugar group

Based upon the above criteria, a number of structural analogues with nuclease resistance have been developed including phosphorothioate (Connolly et al., 1984; Cowsert et al., 1993) and methyl phosphonate (Blake et al., 1985; Murakami et al., 1985). Of these modified oligonucleotides, phosphorothioate oligonucleotides are possibly the most potent because they are highly resistant to nucleases, retain a net charge, are soluble in water, and can act as substrates for RNase H. However, phosphorothioate oligonucleotides may also cause a variety of non-sequence dependent effects (Guvakova et al., 1995; Khaled et al., 1996; Perez et al., 1994).





8


Cellular Transport


Another major encumbrance to the therapeutic use of oligonucleotides is the inefficient delivery of oligonucleotides to the cytoplasm or nucleus. There are two transport aspects that need to be distinguished:

* Cellular uptake

" Entry into the cytoplasm/nucleus

Cellular uptake refers to both oligonucleotide membrane binding and general

internalization within the cell. Entry into the cytoplasm/nucleus concerns the amount of oligonucleotides that reach a pharmacological active compartment. Oligonucleotides internalization by cultured cells is inefficient (Akhtar et al., 1991b; Stein & Cheng, 1993). Only a small fraction of added oligonucleotides can actually gain entry into cells and it is commonly assumed that most oligonucleotides can be brought into cells through (receptor mediated, adsorptive, or fluid phase) endocytosis (Akhtar & Juliano, 1992; Loke et al., 1989).

After entry into cells, oligonucleotides must penetrate the endosomal membrane to exert their effects in the nucleus or cytoplasm. Not all of the internalized oligonucleotides are necessarily available to interact with intended subcellular targets. Indeed, most of them are eliminated by lysosomes, the later endocytotic stage (Figure 22). Unlike gene delivery, however, following cellular entry and escape from endosomal compartments with an effective nuclear pore size of approximately 10 nm in diameter (Aronsohn & Hughes, 1997), oligonucleotides are able to migrate to the nucleus without difficulty (Chin et al., 1990; Leonetti et al., 1991). An issue that needs to be addressed is





9




pH 5 Oligonucleotide

7.4

Cell Membrane

7.0



4- Endosome


6.0





5.0 Lysosome

Oligonucleotide Elimination

Oligonucleotide Liposome+Oligonucleotide

(a) (b)

Figure 2-2: Possible oligonucleotide fates in a cell for two delivery systems. a) Limited amount of oligonucleotides can be taken into cells when not used with any delivery system. For those oligonucleotides that can be brought into cells, the mechanism is mostly through endocytosis along with subcellular compartments (i.e., endosome and lysosome) of a pH-gradient profile. Most of the endocytosed oligonucleotides would then be eliminated. b) When using a delivery system (e.g., liposome), more oligonucleotides can be brought into cells, thereby increasing their probability of escaping from lysosomes. Still, most of the oligonucleotides would be eliminated through the entire endocytosis process.





10


that, like other drugs, oligonucleotides may bind to intracellular proteins which can cause side effects and limit free fraction. Only free unbound oligonucleotides can interact with targets at the sites of action and demonstrate biological effects.

Oligonucleotides traveled to sites of action face several barriers. By optimizing oligonucleotide transfer at each stage of the delivery process, the amount of oligonucleotides with or without their carrier to achieve the same biological effect can be minimized in comparison to unmodified oligonucleotides. Increasing the amount of cellular uptake and/or escape of oligonucleotides from the endosomes may be of considerable value in improving the extent of oligonucleotides at their sites of action and the inhibition of certain protein expression. Hence, the optimization can decrease the cytotoxicity associated with a large amount of oligonucleotides and delivery systems.


Strategies Available to Deliver Oligonucleotides


A number of strategies have been pursued to facilitate the entry of

oligonucleotides into the cytoplasm. The strategies are used either alone or in combination with others to optimize the effect. Each system has its own advantages and drawbacks. According to the two mentioned oligonucleotide transport aspects, these strategies can generally be separated into two parts.

The first group renders delivery systems that increase the amount of

oligonucleotides that associate with target cells. They include conjugation of molecules to oligonucleotides (i.e., conjugating agents), complexation of oligonucleotides with cationic molecules (i.e., complexing agents), encapsulation of oligonucleotides into vesicles (i.e., encapsulating agents), and labeling targets to either oligonucleotides or their





11


delivery carriers (i.e., targeting agents). These systems increase the probability of oligonucleotides escaping endocytotic degradation and reaching the cytoplasm or nucleus.

Irrespective of the above methods, the underlying principle is to increase uptake of oligonucleotides. Thus, the increased oligonucleotide concentration in endosomes enhances the chance of oligonucleotides to reach cytoplasms. These delivery systems therefore exhibit superior effects compared to plain oligonucleotides in tissue culture systems. However, the majority of oligonucleotides that are brought into cells would still be eliminated during endocytosis (Figure 2-2). The biological activity able to be observed results from a diminutive amount of oligonucleotides that escape from the endosomal compartments.

To further optimize oligonucleotide delivery, endosome destabilizing (escaping) systems have been developed. This group applies devices (i.e., oligonucleotide cytoplasmic transfer techniques) or offers delivery systems (i.e., membrane destabilizing agents) that improve oligonucleotide efflux to the cytoplasm. Conjugating Agents


Emphasis on the ability of oligonucleotides to penetrate biological membranes is one of the major elements in making oligonucleotide therapy possible. An strategy is to conjugate hydrophobic anchor groups at either end of the oligonucleotide through chemical reactions to extend their hydrophobicity and/or exo-nuclease resistance, thereby increasing the interaction with target cells.





12


Cholesterol is a typical conjugating agent that has been used as a hydrophobic anchor group at either the 3'- or 5'- terminus of oligonucleotides (Alahari et al., 1996; Boutorin et al., 1989; Godard et al., 1995; Letsinger et al., 1989). Alkyl side chains are another commonly used conjugating agent. Examples include hexadecyl moieties affixed to the 5'-end (Shea et al., 1990), dodecyl moieties to the 3'-end (Saison-Behmoaras et al., 1991), hexanol to the 3'-end (Gamper et al., 1993), aminohexyl to the 3'-end (Gamper et al., 1993), and an undecyl derivative to the 5'-end of oligonucleotides (Kabanov et al., 1990).

Poly(L-lysine) is another type of conjugating agent. By attaching

oligonucleotides to poly(L-lysine) at the 3'-end (Degols et al., 1991; Degols et al., 1989; Lemaitre et al., 1987; Leonetti et al., 1988; Stevenson & Iversen, 1989), cellular uptake is increased most likely due to a better interaction with the negative charge cellular membrane. In addition to the possible permeability mechanism, the biological effect improved by poly(L-lysine) conjugates can also be a consequence of better protective properties against nucleases.

As mentioned above, one major advantage of using conjugating agents is to

increase the initial membrane interaction which leads to greater cellular accumulation of oligonucleotides. However, there are also a number of disadvantages that hinder the use of conjugating agents such as the chemical synthesis of the connector between the oligonucleotides agents. This process is both time consuming and expensive. Furthermore, the manipulation of the conjugating agents (e.g., poly(L-lysine)) can account for increased cytotoxic effects.





13


Complexing Agents


Unlike conjugating agents, the basic principle behind the use of complexing agents is to bind oligonucleotides to their carrier in a strong but non-covalent manner based upon an electrostatic attraction. This system carries more oligonucleotides into cells through endocytosis and hence increases their probability of reaching the cytoplasm.

Cationic polymers such as poly(L-lysine) (Deshpande et al., 1996; Ginobbi et al., 1997; Stewart et al., 1996), polyethylenimine (Boussif et al., 1995), polyamidoamine PAMAM starburst dendrimers (Bielinska et al., 1996; Delong et al., 1997; Hughes et al., 1996; Kukowska-Latallo et al., 1996; Poxon et al., 1996), avidin (Pardridge & Boado, 1991), polyisohexylcyanoacrylate nanoparticles (Chavany et al., 1994), and polyalkylcyanoacrylate nanoparticles (Chavany et al., 1992; Godard et al., 1995; Schwab et al., 1994) are some comlexing agents that have been used in oligonucleotide delivery. Cationic liposomes are other complexing agents that have been investigated. Liposomes are vesicles comprised of lipid bilayer(s) similar in structure to biological membranes. Utilizing their versatility (e.g., size, charge, and composition) and several advantages (e.g., economical, ability to attach chemicals to their surface, and easily produced), different systems involving liposomes can be applied to increase the delivery of oligonucleotides to their sites of action. Cationic liposomes are among one of these strategies. Cationic liposomes that have improved oligonucleotide cellular delivery include N-[ 1 -(2,3-dioleoloxy)-propyl]-N,N,N-trimethylammonium chloride (DOTMA) (Bennett et al., 1992; Hughes et al., 1996; Konopka et al., 1996; Perlaky et al., 1993; Saijo et al., 1993), N-[ 1-(1-2,3-dioleoloxy)propyl]-N,N,N-trimethylammonium





14


methylsulfate (DOTAP) (Capaccioli et al., 1993; Lappalainen et al., 1994; Liang & Hughes, 1998; Quattrone et al., 1994; Takle et al., 1997; Zelphati & Szoka, 1996a), 33[N-(N',N'-dimethylaminoethane)carbamoyl]cholesterol (DC-Chol) (Litzinger et al., 1996), spermine-cholesterol (Guy Caffey et al., 1995), spermidine-cholesterol (Guy Caffey et al., 1995), 2,3-dioleoyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl- 1propanaminum trifluoroacetate (DOSPA) (Lappalainen et al., 1997; Lappalainen et al., 1996), 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide (DMRIE) (Konopka et al., 1996), and dimethyldioctadecylammonium bromide (DDAB) (Jaaskelainen et al., 1994; Ollikainen et al., 1996; Rose et al., 1991).

In contrast to conjugating agents, the ease of production of the complexing agents is the biggest advantage. No chemical linkage between oligonucleotides and complexing agents is required. In addition, they provide high capacity to retain oligonucleotides. Complexing agents may also prevent oligonucleotides from enzymatic degradation by forming poor substrates (Gershon et al., 1993). However, a major concern in using complexing agents is their possible toxic effects. Cationic polymers and cationic liposomes eventually are more toxic to cells than neutral counterparts as their concentrations are increased (Barry et al., 1993; Clarenc et al., 1993; Wagner et al., 1993; Yeoman et al., 1992). In addition, the intrinsic properties of the carriers such as liposomes or nanoparticles lead to increased immunological problems with the oligonucleotide complex (Chavany et al., 1994; Phillips & Emili, 1991).





15

Encapsulating Agents


Encapsulating and complexing agents are possibly the most popular systems used in the delivery of oligonucleotides. Not only do both methods protect oligonucleotides from degradation (Leonetti et al., 1990; Schwab et al., 1994; Thierry & Dritschilo, 1992), but they also increase cellular uptake (Juliano & Akhtar, 1992). However, the preparation behind the similar outcomes is quite different. Complexing agents bind to oligonucleotides through an electrostatic attraction while encapsulating agents entrap oligonucleotides within vesicles.

The most popular encapsulating approach currently being investigated is through the use of liposomes. In addition to the above mentioned advantages, liposomes offer the potential to reach specific targets via attached ligands. They may control or sustain oligonucleotide release (Akhtar et al., 1991b). Abundant examples using liposomes illustrate the improved effect of oligonucleotides (Akhtar et al., 1991b; Anazodo et al., 1995; Hatta et al., 1997; Hatta et al., 1996; Marzo et al., 1997; Ogo et al., 1994; Wielbo et al., 1996). Furthermore, cyclodextrin analogs including 2-hydroxypropyl betacyclodextrin (Habus et al., 1995; Zhao et al., 1995), hydroxyethyl beta-cyclodextrin (Zhao et al., 1995), and encapsin (Zhao et al., 1995) have also been demonstrated as possible carrier candidates for oligonucleotide delivery.

Similar to complexing agents, the biggest advantage of encapsulating agents in oligonucleotide delivery is their ease of production. Unlike complexing agents, encapsulating agents are believed to be less cytotoxic. However, compared to





16


complexing agents, they have a lower capacity to bring oligonucleotides into cells which may reduce the efficiency of the encapsulating agent system. Targeting Agents


Targeting agents may be categorized into two groups. The first group targeting at the nucleic acid level is known as intercalating agents. These agents are mostly often attached at the 3'- or 5'-end of oligonucleotides. The moieties linked to oligonucleotides interact strongly and nonspecifically with nucleic acids. After entering into cells and interacting with target nucleic acids, the hybrids are stabilized by the intercalation of the agents in the RNA-DNA duplex. Hence, they increase the affinity of oligonucleotides to their targets.

Of all the intercalating agents, acridine is most widely used and investigated as a possible means to increase the effect of oligonucleotides (Fukui & Tanaka, 1996; Grigoriev et al., 1992; Klysik et al., 1997; Lacoste et al., 1997; McConnaughie & Jenkins, 1995; Stein et al., 1988; Toulme et al., 1986; Verspieren et al., 1987; Walter, 1995). Other examined intercalators are chlorambucil (Belousov et al., 1997), benzopyridoquinoxaline (Marchand et al., 1996; Silver et al., 1997), benzopyridoindole (Giovannangeli et al., 1996; Silver et al., 1997), benzophenanthridine (Chen et al., 1995), and phenazinium (Levina et al., 1993).

The second group of targeting agents is accessed by utilizing moieties that can selectively and specifically transport oligonucleotides to a target cell population. Therefore, their accumulation in intracellular compartments is increased. The moieties





17


can be either conjugated to oligonucleotides or attached to a carrier system (e.g., poly(Llysine) or liposomes) linked to the oligonucleotides.

For cells that express the characteristics of receptor mediated endocytosis, ligands represent good candidates as targeting agents to initiate cellular uptake of oligonucleotides. Glycoproteins and neoglycoproteins bearing an appropriate sugar residue specifically attach to sugar binding receptors (Sharon & Lis, 1989). By labeling oligonucleotides at the 3'-end to the neoglycoprotein (6-phosphomannosylated glycoprotein), an improved effect was observed (Bonfils et al., 1992). Similarly, asialoorosomucoid (Bunnell et al., 1992; Wu & Wu, 1992) or mannosylated glycoprotein (Liang et al., 1996) conjugated to poly(L-lysine) has been employed to target and enhance cellular uptake of oligonucleotides.

Since malignant cells are correlated with an increased need for essential nutrients (e.g., folic acid and transferrin) relative to benign cells, these nutrients can be used as potential candidates to target oligonucleotides in the inhibition of cancerous cell growth. Further improved oligonucleotide cellular uptake is seen when folic acid (Citro et al., 1994; Ginobbi et al., 1997), epidermal growth factor (Deshpande et al., 1996), and transferrin (Citro et al., 1992) is linked to poly(L-lysine). Liposomes coated with maleylated bovine serum albumin (Chaudhuri, 1997), folic acid (Wang et al., 1995), or ferric protoporphyrin IX (Talke et al., 1997) have been shown to increase the cellular uptake of oligonucleotides.

In order to increase the specificity of oligonucleotides, liposomes can also be

attached to antibodies to reach the desired targets. Several monoclonal antibody-targeted liposomes, immunoliposomes, have been developed and used for mediating





18


oligonucleotides to specific receptors on targeted cells (Lefebvre-d'Hellencourt et al., 1995; Leonetti et al., 1990; Loke et al., 1989; Ma & Wei, 1996; Renneisen et al., 1990; Selvam et al., 1996; Zelphati et al., 1994; Zelphati et al., 1993).

The major advantage of targeting agents is to enhance oligonucleotide cellular uptake specifically. The targeting strategy can be incorporated with other systems to further increase the cellular biological activity of oligonucleotides. Similar to conjugating agents, a disadvantages that may hamper the development of targeting agents is the synthetic linking process. Furthermore, targeting particular routes of endocytosis is strongly dependent upon receptor subtype thereby limiting the use of targeting agents. Oligonucleotide Cytoplasmic Transfer Techniques


Even if cellular uptake of oligonucleotides through a delivery system was

increased, escape from endosome must still be accomplished. One way to avoid this barrier is to transfer them directly into cytoplasm or nucleus. This has been accomplished through electroporation (Bergan et al., 1996; Flanagan & Wagner, 1997; Griffey et al., 1996; Schaal et al., 1995) and microinjection (Blondel et al., 1990; Fenster et al., 1994; Lamprecht et al., 1997; O'Keefe et al., 1994; Sagata et al., 1988).

Electroporation involves delivering a high-voltage pulse of a defined magnitude and length to the oligonucleotide-cell system. The membrane structures of the cells are loosened and oligonucleotides can be introduced directly into the cell's cytoplasm. On the other hand, microinjection was performed by injecting oligonucleotides directly into the nucleus.





19


The above methods prevent lysosomal elimination without falling into the trap of the endocytosis pathway. However, these techniques have limited use from the standpoint of clinical therapy. Therefore, it is necessary to develop more practical delivery systems to improve oligonucleotide therapy. Membrane Destabilizing Agents


Membrane destabilizing agents provide a means to disrupt endosomal

membranes. Some agents are conjugated directly to oligonucleotides through chemical reactions while other agents may be a part of liposome composition to which oligonucleotides are either complexed or encapsulated. Fusogenic and pH-sensitive lipids

Fusogenic and pH-sensitive lipids have been used together as liposomes (i.e., encapsulating agents) to promote efflux of oligonucleotides from the endosomal compartment (Bentz et al., 1985; Connor et al., 1984; Duzgunes et al., 1985). Fusogenic lipids include phosphatidylethanolamine (PE) derivatives while pH-sensitive lipids that have titratable carboxylic acids contain oleic acid (Cristina De Oliveira et al., 1997; Ma & Wei, 1996; Ropert et al., 1996; Ropert et al., 1993; Ropert et al., 1992) and cholesteryl hemisuccinate (Chu et al., 1990; Slepushkin et al., 1997).

A fusogenic lipid is able to form hexagonal II phase that influences membrane fusion and oligonucleotide release. Before the disruption of the endosomal membrane occurs inside the cells, however, liposomes must maintain their integrity to encapsulate oligonucleotides. A pH-sensitive lipid is therefore introduced into the liposomal matrix. With a chemical structure complementary to the hexagonal II phase (e.g.,





20


dioleoylphosphatidylethanolamine (DOPE)), the pH-sensitive lipid will assist in retaining the bilayer vesicle structure of the liposomes at an alkaline pH. When the pH decreases as a result of the acidification of the endosome, the titratable head group of the pHsensitive lipid is protonated. Hence, it destabilizes the bilayer structure and PE promotes membrane fusion (Duzgunes et al., 1985). Eventually, oligonucleotides are released out of the endosomes. Also, cationic liposomes (i.e., complexing agents) usually comprise a fusogenic lipid (e.g., DOPE) and a cationic lipid (e.g., 2,3 dioleyloxy-N[2(sperminecarboxamido)ethyl]-N,N-dimethyl propanaminium (DOSPA), N-[1-(1-2,3dioleoloxy)propyl]-N,N,N-trimethylammonium methylsulfate (DOTAP), N-[ 1-(2,3dioleoloxy)-propyl]-N,N,N-trimethylammonium chloride (DOTMA), dimethyldioctadecylammonium bromide (DDAB), and 3p-[N-(N',N'dimethylaminoethane)carbamoyl]cholesterol (DC-Chol)) to improve oligonucleotide delivery (Guy Caffey et al., 1995; Lappalainen et al., 1997; Lappalainen et al., 1996; Litzinger et al., 1996; Ollikainen et al., 1996; Takle et al., 1997; Zalphati & Szoka, 1996b).

In addition to increasing cellular uptake of oligonucleotides, pH-sensitive

liposomes further increase the entry of oligonucleotides to the cytoplasm. However, since both pH-sensitive anionic lipids and nucleic acids have a negative charge, they may have limited capacity to entrap nucleic acids. Viral peptides

Oligonucleotides incubated with or coupled to viral peptides derived from the hemagglutinin envelop protein of the Influenza virus (Bongartz et al., 1994; Hughes et





21


al., 1996; Yu et al., 1994) provide a route to improve their cytoplasmic delivery. These peptides are able to form a transmembrane channel through a conformational change induced by the acidification following endocytosis (Carrasco, 1994; Fattal et al., 1994; Marsh, 1984; Parente et al., 1990). Viral peptides can therefore help to transfer oligonucleotides into the cytoplasmic compartment.

When liposomes are decorated with viral peptides from the Sendai virus

(hemagglutinating virus of Japan (HVJ)), oligonucleotide delivery can also be improved (Aoki et al., 1997; Dzau et al., 1996; Kumar et al., 1997; Mann et al., 1997; Morishita et al., 1993; Yonemitsu et al., 1997). Unlike the system from the Influenza virus, most HVJ-liposomes appear to employ a cell-membrane fusion mechanism (Okada et al., 1975), thus averting oligonucleotides from the endocytotic pathway and releasing them directly into the cytoplasm.

The advantage of fusogenic peptides is the ability to follow the natural virus entry pathway. However, fusogenic peptides are expensive to produce and could also pose the problem of immunogenicity on repeat administration.


Origin of Biodegradable pH-Sensitive Surfactants-Lysosomotropic Detergents


The use of detergents to disrupt phospholipid bilayers (e.g., endosomal

membranes) is efficient (Pinnaduwage et al., 1989) and provide a rationale approach to enhance oligonucleotide release from endosomes, but most detergents are indiscriminate of membrane type and attack the first cellular membrane they contact. In order to provide selectivity, a trigger is required to activate detergents in specific subcellular locations. A lysosomotropic amine whose pKa value is between approximately 5 and 7 (De Duve,





22


1983; De Duve et al., 1974) bearing a hydrophobic tail group is classified as a lysosomotropic detergent (Firestone et al., 1979). At an alkaline environment, the molecule is predominated by its hydrophobic tail making it simply an oily substance with limited surface active properties. The non-charged lysosomotropic detergent can be passively diffused across cellular membranes. Due to the low intralysosomal pH usually between 4 and 6 (McGraw & Maxfield, 1991), the detergent is protonated and trapped once inside lysosomes, allowing a continuous gradient for drug entry (Dean et al., 1984; Forster et al., 1987; Wilson, 1989). When accumulation of the protonated form of the compound progresses to a certain concentration, the material disrupts the lysosomal membrane, releasing a variety of lysosomal enzymes into the cytoplasm. Once released within the cells, these digestive enzymes are able to degrade cellular structures resulting in cell death (Miller et al., 1983; Wilson et al., 1987).

Consequently, a series of lysosomotropic detergents were synthesized and tested (Firestone et al., 1982a; Firestone et al., 1982b; Firestone et al., 1979). The development of lysosomotropic detergents originated as a potential way to destroy tumor cells under the assumption that the malignant cells carry more lysosomes than benign cells (Trouet et al., 1972). Although later abandoned due to problems with nonspecific lysosomal cellular destruction, the theory behind this approach has provided the basis for the development of biodegradable pH-sensitive surfactants (BPS).


Significance of Biodegradable pH-Sensitive Surfactants


Due to the possible pitfalls in nucleic acid delivery, the lysosomotropic detergents were modified and biodegradable pH-sensitive surfactants (BPS) were developed to





23


induce endosomal membrane defects. The novelty of the delivery system stems from the two following reasons:

* Exploitation of a naturally occurring oligonucleotide-liposome transport mechanism

(endocytosis) with the incorporation of BPS into the delivery system (e.g., liposomes) Biodegradable drug approach to decrease toxicity

Developing BPS that can be activated at the endosome (early lysosome) stage will enable the destabilization of the endosomal membranes and liberate oligonucleotides to their sites of action in the cytoplasm or nucleus (Figure 2-3). However, unlike lysosomotropic detergents, BPS may be cleaved into less toxic metabolites by the endogenous digestive enzymes after being released into the cytoplasm due to their biodegradability. Furthermore, BPS can be quickly synthesized by a one to two-step standard chemical reaction (e.g., esterification and substitution) with commercially available inexpensive starting materials (e.g., dodecanol and imidazole). Thus, compared to other delivery agents, BPS may be more economical, easily produced, and less toxic.


Specific Aims


The inefficiency of nucleic acid delivery systems is likely due, in part, to the

failure of endosomes to release oligonucleotides before reaching degradative lysosomes. A solution is to incorporate compounds in a delivery vector that will selectively increase the release of encapsulated nucleic acids from the endosome. To meet the above criteria, we developed a group of biodegradable pH-sensitive surfactants (BPS).




24




pH
S Oligonucleotide 74
o Un-ionized BPS S
* Ionized BPS Cell Membrane


7.0


Endosome


6.0



Oligonucleotide Release
BPS-Liposome
Figure 2-3: Proposed mechanism of the biodegradable pH-sensitive surfactants (BPS)liposome system. Like the regular liposome system, similar amount of oligonucleotides will be brought into cells. After being activated during endocytosis, protonated BPS can destabilize the endosomal membrane which would release the oligonucleotide into the cytoplasm.





25


The proposed studies focused on four parts. First, BPS were designed and synthesized to provide an alternative to oligonucleotide delivery. Second, the physicochemical properties of three synthesized BPS were evaluated and compared. Third, the oligonucleotide biological effect derived from the BPS-liposome delivery system was assessed. Finally, the mechanisms behind how BPS destabilize the endosomal membrane were investigated. Specific Aim 1: Design and Synthesis of Biodegradable pH-Sensitive Surfactants Objective

We designed biodegradable pH-sensitive surfactants (BPS) by varying the head groups, linkage bridges, and tail groups to investigate the impact on the physicochemical characteristics. By changing the combinations of different linkage bridges and hydrocarbon chains with a lysosomotropic amine, imidazole, on the surfactant molecules, three BPS were synthesized.


Specific Aim 2: Physicochemical Characterization of Biodegradable pH-Sensitive
Surfactants


Objective

The objective was to acquire the surface active properties of the ionized

biodegradable pH-sensitive surfactants (BPS), investigate the membrane destabilization ability of BPS at varied pHs, determine their stability, and screen their cytotoxicity.





26


Hypothesis

The first hypothesis was that as the pH of the environment decreased, the surface activity of BPS would increase, thereby destabilizing the liposomal membrane. The second hypothesis was that the linkage connector of BPS was mostly responsible for their stability which would further determine their cytotoxicity. Specific Aim 3: Delivery System Evaluation of Biodegradable pH-Sensitive
Surfactants


Objective

The objective was to quantitatively and qualitatively evaluate the cellular delivery of oligonucleotides using a biodegradable pH-sensitive surfactants (BPS)-liposomal delivery system.

Hypothesis

The hypothesis was that when using liposomes to deliver oligonucleotides in

vitro, the effect would be further enhanced in the presence of BPS as a component of the liposome composition.


Specific Aim 4: Mechanism of Action Investigation of Biodegradable p1--Sensitive
Surfactants


Objective

With the different chemical structures from other fusogenic compounds and surfactants, biodegradable pH-sensitive surfactants (BPS) were expected to have disparate membrane activities. The possible mechanisms of how BPS caused membrane defects were therefore investigated.





27


Hypothesis

The hypothesis was that BPS could induce both membrane fusion and rupture and eventually release liposomal contents in a pH-dependent manner.













CHAPTER 3
DESIGN AND SYNTHESIS OF BIODEGRADABLE pH-SENSITIVE SURFACTANTS


Introduction


The idea of using lysosomotropic detergents to gain access to cells exploits the fact that surfactants lyse lysosomal membranes. Biodegradable pH-sensitive surfactants (BPS) expand this concept by providing a mechanism to control the lytic properties of the surfactants. By gathering information of the potential effect on which each individual component of BPS has impact, a rational approach may be under taken. It is hypothesized that using the correct design and synthesis of a series of original BPS, the potency of BPS can be predicted. To begin this project, three BPS, dodecyl 2-(1 'imidazolyl) propionate (DIP), methyl 1-imidazolyl laureate (MIL), N-dodecyl imidazole

(DI), were synthesized using standard well understood reactions (esterification and substitution).


Rationale


A surfactant (surface active agent) is a substance that adsorbs onto the surfaces or interfaces of a system and alters the free energies of those surfaces or interfaces to a marked degree (Rosen, 1989). Surface active agents have a characteristic molecular structure consisting of a head group that is hydrophilic and a tail group that is



28





29

hydrophobic thus making the compounds amphoteric. Depending on the number and nature of the polar and nonpolar groups present, the agents can be designed to be more hydrophilic or lipophilic.

In order to design biodegradable pH-sensitive surfactants (BPS), there are three critical requirements or structural components (Figure 3-1). 1.) The first one is a lysosomotropic amine as the head group of BPS. 2.) To make the lysosomotropic agent amphoteric, it is necessary to attach a hydrocarbon chain to the amine as the tail group. At an alkaline environment, the pH-sensitive surfactant will be un-ionized and lipophilic. When incorporating the surfactant into liposomes as a delivery system, the lipophilicity of the surfactant will enhance its chance to remain within the lipid bilayers. After the amine is protonated due to a pH gradient (e.g., endocytosis), the pH-sensitive surfactant will increase its surface activity significantly. This change is strong enough to induce membrane destabilization. 3.) The pH-sensitive surfactant would then be degraded by the endogenous enzymes into less toxic metabolites with the introduction of a biodegradable connector. By understanding the relationship among the specific characteristic of each individual BPS component (head group, tail group, and linkage bridge), it should be possible to optimize the design of BPS.


Head Group


The head group of biodegradable pH-sensitive surfactants (BPS) is the major factor determining its ionization constant (pKa) controlling the amount of activated surfactant at endosomal pH. Two important criteria influence the pKa:




30










A Lipophilic
Hydrocarbon Chain
A pH Sensitive T Low pH
Lysosomotropic Amine
An Enzymatically
Cleavable Connector Enzyme

+

Figure 3-1: Three individual requirements to create biodegradable pH-sensitive surfactants (BPS).





31

* Type of lysosomotropic amines

* Presence of substituents on the amine head groups

Since imidazole and morpholine were used as the head groups in the first

generation lysosomotropic detergents, information exists about their chemical properties and biological effects (De Duve et al., 1974). Other heterocyclic ring compounds, such as indole, may also have lysosomotropic properties. When compared to morpholine, imidazole has a higher pKa since the aromatic ring formed by the imidazolyl group decreases the nucleophilicity and the basicity of the amine. However, compared to indole, imidazole has higher basicity due to its extra electron lone pair (Table 3-1).




Table 3-1: Predicted effect on pKa from the head group of biodegradable pH-sensitive surfactants (BPS).

Head Group Effect on pKa Value
Imidazole
Morpholine ""
Indole ,




All substituents near the titratable amine can affect pKa. Electron donating

groups generally increase pKa while electron withdrawing groups generally decrease the pKa of an amine. Figure 3-2 shows the order of the nucleophilicity, basicity, and pKa for the substituents at 2' position of the imidazolyl group.





32




















,CH 3
-N CH3 > -NH2 > -OCH3 > -OH > -alkyl
>CH 3
O

> H > X (I, Br, C) > CN > C-R' > NO2

Figure 3-2: Order of nucleophilicity, basicity, and pKa for different substitutes on the head group of biodegradable pH-sensitive surfactants (BPS).





33

Tail Group


The hydrocarbon chain of biodegradable pH-sensitive surfactants (BPS) partially determines their hydrophilicities. The stronger the interaction between the tail groups, the more lipophilic BPS. As the lipophilicity increases, a lower critical micelle concentration (CMC) will be observed. Figure 3-3 gives the order of the expected CMC values when BPS have the same head group by changing the tail group. Linkage Bridge


The effectiveness of biodegradable pH-sensitive surfactants (BPS) is related to its rate of hydrolysis which is controlled by the linkage bridge of the molecule. An ester bond is subject to hydrolysis with the rate dependent upon the extent of steric hindrance caused by the substituents. An amide bond, however, is stable and unlikely to undergo hydrolysis quickly in aqueous solutions. For a drug to be effective, an optimum hydrolysis rate is regulated which must allow the molecule to stay intact long enough to have an effect while also allowing it to break down after releasing oligonucleotides to their sites of action.


Selection of Biodegradable pH-Sensitive Surfactants


With the different combinations of head groups, tail groups, and linkers possible for biodegradable pH-sensitive surfactants (BPS), the number of potential BPS is numerous. In the subsequent studies, however, three BPS were selected, synthesized, and compared.





34


















R R




R> R




> R > .Am R


Figure 3-3: Biodegradable pH-sensitive surfactants (BPS) with the same head group but different tail groups in order of decreasing hydrophilicity and critical micelle concentration (CMC).





35

* Dodecyl 2-(l'-imidazolyl) propionate (DIP)

* Methyl 1-imidazolyl laureate (MIL)

* N-dodecyl imidazole (DI)

DIP, the first member of the BPS family, was originally devised by Hughes and co-workers (1996). MIL was designed to compare with DIP when the ester linker between head and tail groups is positioned into the other direction. DI, lacking a biodegradable connector, was loosely grouped as a BPS member. DI was originally synthesized by Firestone and co-workers (1979) and compared to other BPS to address the importance of the linker with respect to cytotoxicity.


Synthesis of Biodegradable pH-Sensitive Surfactants Chemicals


N,N-dimethylformamide (DMF) was purchased from Aldrich (Milwaukee, WI). Dodecanol, 2-bromopropionyl bromide, imidazole, 12-bromo-1-dodecanol, triethylamine, lauric acid, and N,N'-dicyclohexylcarbodimide were purchased from Fluka (Ronkonkoma, NY). 1-imidazolyl methanol was a gift from Dr. Kenneth Sloan (Department of Medicinal Chemistry, University of Florida). All chemicals were used directly without additional purification.


Dodecyl 2-(1l'-Imidazolyl) Propionate


Dodecyl 2-(1 '-imidazolyl) propionate (DIP) was synthesized as modified from a previous report (Hughes et al., 1996). Briefly, dodecanol (0.05 mole), 2-bromopropionyl





36

bromide (0.025 mole), and triethylamine (0.025 mole) were mixed and stirred in 50 ml of chloroform for 24 h to yield crude dodecyl 2-bromopropionate (Figure 3-4). The crude product was washed three times with adequate water to remove impurities. The organic phase was dried by adding anhydrous sodium sulfate and distilled under vacuum. After this simple extraction, the crude product, dodecyl 2-bromopropionate (0.015 mole), was mixed with imidazole (0.03 mole) in chloroform and refluxed for another 24 h (Figure 34).

The final crude DIP product was washed with an adequate amount of water three times and dried with sodium sulfate. Then, the oily compound was purified through flash chromatography (Still et al., 1978) using silica gel (235-400 mesh size) as the adsorbent and methanol-methylene chloride mixture as the mobile phase at a ratio (v/v) of 3.5 to 96.5, respectively.


Methyl 1-Imidazolyl Laureate


A standard esterificaiton method (Hassner & Alwxanian, 1978) was used to synthesize methyl 1 -imidazolyl laureate (MIL). A mixture of 1 -imidazolyl methanol (0.05 mole), lauric acid (0.025 mole), and N,N'-dicyclohexylcarbodiimide (0.025 mole) in 50 ml of DMF was stirred overnight at 750C to produce MIL (Figure 3-5). N,Ndicyclohexyl urea was filtered and washed three times with water, three times with 5% acetic acid solution, again three times with water, and then dried with anhydrous sodium sulfate. Pure MIL was then obtained through quick flash chromatography with the ratio (v/v) of methanol to methylene chloride at 2.5 to 97.5, respectively.





37















0
H3C -,A Br HO-- CH3

Br
2-bromopropionyl bromide Dodecanol


0

TEA H3C -,A 0" CH3
Br
Dodecyl 2-bromopropionate



N //' N H CH3
\-j N //' N 0,, CH3
Imidazole 0
-Alp
A Dodecyl 2-(l'-imidazolyl) propionate

Figure 3-4: Synthetic pathway of dodecyl 2-(I'-imidazolyl) propionate (DIP).





38
















O
N N 'OH + HO O iCH3

1-imidazolyl methanol Lauric acid

O
N, N o''' CH3
DCC N N
A Methyl 1-imidazolyl laureate

Figure 3-5: Synthetic pathway of methyl 1-imidazolyl laureate (MIL).





39

N-Dodecyl Imidazole


N-dodecyl imidazole (DI) was synthesized by reacting imidazole (0.05 mole) and 12-bromo-l-dodecanol (0.025 mole) in 50 ml of DMF at 75C for 24 h (Figure 3-6). Crude DI was washed with water three times, 5% acetic acid solution three times, water three times, and then dried with anhydrous sodium sulfate. Pure DI was obtained after passing through quick flash chromatography with the ratio (v/v) of methanol to methylene chloride at 3.5 to 96.5, respectively.


Identification of Biodegradable pH-Sensitive Surfactants


After purifying these three agents, their structures were identified through a 300MHz 'H-nuclear magnetic resonance (NMR) in the Center of Structural Biology and mass spectroscopy (FAB) in the Department of Chemistry at the University of Florida. The purities of the three biodegradable pH-sensitive surfactants (BPS) were confirmed by elemental analysis in the Department of Chemistry at the University of Florida.


Dodecyl 2-(l'-Imidazolyl) Propionate


The 'H-NMR (CDC1,) spectrum showed resonances of 7.60 (s, 1H), 7.05 (s, 1H), 7.00 (s, 1H), 4.85 (q, 1H), 4.15 (t, 2H), 1.75 (d, 3H), 1.20-1.40 (m br, 20H), and 0.85 (t, 3H) which was consistent with the proposed structure (Figure 3-7). The mass spectrum (C18H32N202, F.W.: 308.2464) had a molecular ion (M+1) at 309.2537 (Figure 3-8). The elementary analysis indicated similar experimental percentages to the theoretical values (Table 3-2). All these assays were within acceptable margins of error (mass





40
















N //' N H + Br CH3
\,-i

Imidazole 12-bromo- I -dodecanol



N // '- N C H
A \,-i
N-dodecyl imidazole Figure 3-6: Synthetic pathway of N-dodecyl imidazole (DI).





41










































7 6 5 4 11 .
la .20.98 LOO 2.07 3.19 19.62


Figure 3-7: 1H-NMR spectrum of dodecyl 2-(1 '-imidazolyl) propionate (DIP).






42




























100 309.2537 E 06
1.56



80"




6040




20- 141.0698
96.0757
154.9694 209.1249 237.2050 .. _.o100 150 200 250 300 350



Figure 3-8: Mass spectrum of dodecyl 2-(1 '-imidazolyl) propionate (DIP).





43

spectroscopy: 15 mmu; elemental analysis: 0.4% each element) which confirmed the chemical structure and purity of dodecyl 2-(1 '-imidazolyl) propionate (DIP).




Table 3-2: Elemental analysis of dodecyl 2-(1 '-imidazolyl) propionate (DIP). Comparison of experimental and theoretical percentages of each element.

DIP Theoretical % Experimental %
Carbon 70.1 69.8
Hydrogen 10.9 10.9
Nitrogen 9.1 8.7




Methyl 1-Imidazolyl Laureate


The 'H-NMR spectrum showed resonances of 7.50 (s, 1H), 7.00 (d, 2H), 5.90 (s, 2H), 2.35 (t, 2H), 1.20-1.40 (m br, 18H), and 0.90 (t, 3H) (Figure 3-9). The mass spectrum (C16H28N202, F.W.: 280.2151) had a molecular ion (M+I) at 281.2231 (Figure 3-10). The combustion analysis of experimental percentages of elements was in agreement with the theoretical values (Table 3-3). All assays had acceptable margins of error and confirmed the chemical structure and purity of methyl 1-imidazolyl laureate (MIL).





44




































0.88 1.98 2.00 2.04 18.37 3.04

Figure 3-9: 'H-NMR spectrum of methyl 1-imidazolyl laureate (MIL).






45



























281.2223 B+ 06
100 4.70



8060"



4020"
81.0507 147.0704 623.3514
53.0742 361.2618 493.3998

100 200 300 400 500 600


Figure 3-10: Mass spectrum of methyl 1-imidazolyl laureate (MIL).





46

Table 3-3: Elemental analysis of methyl 1-imidazolyl laureate (MIL). Comparison of experimental and theoretical percentages of each element.

MIL Theoretical % Experimental %
Carbon 68.6 68.7
Hydrogen 11.0 11.0
Nitrogen 10.0 9.6




N-Dodecyl Imidazole


1H-NMR spectrum showed resonances of 7.45 (s, 1H), 7.05 (s, 1H), 6.90 (s, 1H),

3.95 (t, 2H), 1.20-1.40 (m br, 20H), and 0.85 (t, 3H) (Figure 3-11). For N-dodecyl imidazole (DI) (C15H28N2, F.W.: 236.2252), the mass spectrum had a molecular ion (M+I) at 237.2324 (Figure 3-12). The above two assays confirmed the chemical structure of DI. The combustion analysis of experimental percentages of elements was in agreement with the theoretical values indicating the purity of this compound (Table 3-4).




Table 3-4: Elemental analysis of N-dodecyl imidazole (DI). Comparison of experimental and theoretical percentages of each element.

DI Theoretical % Experimental %
Carbon 76.3 76.5
Hydrogen 11.9 11.5
Nitrogen 11.9 12.2





47





































1.00 2.04 2.12 20.20 3.18

Figure 3-11: 1H-NMR spectrum of N-dodecyl imidazole (DI).






48




























237.2324 E+ 05
100 8.04




80




60




4020

235.2149 535.3390
82.0868 137.1053 249.2260 372.2544 473.4408 1

100 200 300 400 500 600



Figure 3-12: Mass spectrum of N-dodecyl imidazole (DI).













CHAPTER 4
PHYSICOCHEMICAL CHARACTERIZATION OF BIODEGRADABLE pHSENSITIVE SURFACTANTS


Introduction


In this chapter, we have characterized and compared three members of the BPS family, dodecyl 2'-(l-imidazolyl) propionate (DIP), methyl 1-imidazolyl laureate (MIL) and N-dodecyl imidazole (DI). First, surface active properties including critical micelle concentration (CMC) and effective release ratio (Re) of the ionized BPS were measured and verified. The pH sensitivity of BPS to lyse liposomes from the external environment and the behavior of BPS to destabilize liposomes when incorporated in them were also evaluated. Then, systems were established to decide the chemical and biological stabilities of BPS and the results compared in relation to their chemical structures. Finally, the cellular toxicity of these agents was determined and correlated with their biodegradability (biological stability).


Materials


Chemical


Calcein, ferric chloride, and ammonium thiocyanate were purchased from Aldrich (Milwaukee, WI). Dodecanol, imidazole, and lauric acid were purchased from Fluka



49





50

(Ronkonkoma, NY). 1-imidazole methanol was a gift from Dr. Kenneth Sloan (Department of Medicinal Chemistry, University of Florida). Calcein-AM was purchased from Molecular Probes (Eugene, OR). L-ac-lecithin, dioleoylphosphatidylethanolamine (DOPE), and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) were purchased from Avanti Polar Lipids (Alabaster, AL). Porcine esterase (300 U/mg protein) and cholesterol were purchased from Sigma (St. Louis, MO). All purchased or obtained chemicals were used directly without additional purification.

Ammonium ferrothiocyanate (0.1 M) was prepared by dissolving 8.1 g of ferric chloride and 15.2 g of ammonium thiocyanate in 500 ml of distilled water. Dodecyl 2(1'-imidazolyl) propionate (DIP), Methyl 1-imidazolyl laureate (MIL), and N-dodecyl imidazole (DI) were synthesized as previously reported (Chapter 3).


Cell


The SKnSH (HTB-11) cell line was purchased from American Type Culture

Collection (Rockville, MD). The CV-1 cell line was a generous gift from Dr. M. C. Cho (Department of Pharmaceutics, University of North Carolina). Buffer Preparation


All pH buffers were adjusted with NaC1 to an equal ionic strength (Bodor et al., 1980). The pHs of the buffers (and their chemical compositions) used were as follows: pH 1.4 (420 mM potassium chloride and 80 mM hydrochloric acid), pH 2.8 (123 mM citric acid and 60 mM sodium hydroxide), pH 4.2 (150 mM sodium acetate and 350 mM





51

glacial acetic acid), pH 5.0 (300 mM KH2PO4 and 50 mM Na2HPO4), pH 6.0 (150 mM KH2PO4 and 100 mM Na2HPO4), pH 6.5 (120 mM KH2PO4 and 100 mM Na2HPO4), pH

7.0 (80 mM KH2PO4 and 120 mM Na2HPO4), and pH 8.0 (200 mM KH2PO4 and 188 mM sodium hydroxide).


Liposome Preparation


Instead of serving as a nucleic acid delivery system, liposomes (L-c-lecithin: 1,2dimyristoyl-sn-glycero-3-phosphocholine (DMPC): cholesterol; molar ratio 6:1:8) were used as a model membrane system. To maintain simplicity, only neutral lipids were employed in the liposomal membrane system. While the liposomes may not fully represent events occurring in biological situations they still served as excellent models in addressing potential mechanisms of lipid membrane disruption. Calcein (100 mM) was entrapped within the liposomes as a fluorescent marker to monitor membrane lysis events and unentrapped calcein was removed through centrifugation (14,000 rpm, 5 min) five times and washed with a pH 7.4 phosphate buffered saline (PBS) each time.

Reverse-phase evaporation vesicle method (Szoka & Papahadjopoulos, 1978) was used to produce unilamellar vesicles (600 nm) using polycarbonate membranes (Poretics; Livermore, CA) through a high pressure extruder three times (Lipex Biomembrane Inc.; Vancouver, Canada). The size of the liposomes (volume-weight Gaussian distribution) was measured to be 609158 nm (standard deviation) by a dynamic light scattering method using a NICOMP Model 380 ZLS Zeta Potential/Particle Sizer (Santa Barbara, CA). The concentration of phospholipid in each experiment was measured by a





52


modification of a spectrophotometric technique (Stewart, 1980). Briefly, varying amounts of L-cx-lecithin (0-50 mg/ml) were added to test tubes containing 2 ml of 0.1 M ammonium ferrothiocyanate and 2 ml of chloroform. The contents were mixed vigorously for 1 min and centrifuged at 6,000 rpm (Safeguard Centrifuge, Clay-Adams Inc.) for 5 min to fully separate the two phases. The aqueous phase was removed and the absorbance of the remaining organic phase was measured at 488 nm with a spectrophotometer (UV/Vis Perkin-Elmer spectrophotometer Lambda 3) to establish a calibration curve. The concentrations of unknown samples were then determined from the calibration curve.


Methods


Critical Micelle Concentration Determination


To determine the critical micelle concentration (CMC) of the ionized

biodegradable pH-sensitive surfactants (BPS), surface tension measurements were performed using a CRC-DuNotiy interfacial tensiometer (Martin, 1993). The pH was adjusted to pH 3.0 in different counter ion solutions (HF, HC1, HBr, and HI) at a constant room temperature (220C). Increasing amounts of BPS were added into different solutions and surface tension measured. The concentration region in which surface tension stopped changing was recorded as the CMC.





53

Effective Release Ratio Determination of Biodegradable pH-Sensitive Surfactants


Unilamellar liposomes (600 nm; 10 nmol/ml) containing 100 mM calcein (>selfquenching concentration) were suspended in a pH 4.2 acetate buffer solution with increasing amounts of biodegradable pH-sensitive surfactants (BPS). Equilibrium was allowed to occur for 30 min at room temperature. A substantial portion to cause membrane lysis from most detergents has taken place after 30 min of incubation with liposomes (Ruiz et al., 1988). Complete equilibrium between surfactants and lipids can take several hours (Lichtenberg et al., 1979). After this time, however, surfactant induced release of liposomal contents can be masked by the concomitant spontaneous diffusion of solutes out of the vesicles. Therefore, long time incubation with complete equilibrium may not be appropriate in this study.

Released calcein was excited at 496 un and observed at 517 nm in a Perkin-Elmer luminescence spectrophotometer LS-50B at room temperature. The percentage of released calcein was calculated by the equation 1(%) (ia _b) 100% (Liu & Regen,



1993). I, is the 100% fluorescence intensity value when adding excess Triton X-100 (10 mM); Ia and Ib are the fluorescence intensities after incubation with and without BPS, respectively. Effective release ratio (Re) was defined at the molar ratio (surfactant/liposome) when 50% calcein was released. During this process, the surfactant must come in contact with the lipid bilayer and partition into the hydrophobic environment.





54

pH Sensitivity of Biodegradable pH-Sensitive Surfactants on Liposomal Calcein
Release


To determine the ability of biodegradable pH-sensitive surfactants (BPS) to cause membrane lysis/leakage at different pHs, studies were conducted that varied the BPS concentration in a solution of calcein containing liposomes (10 nmol/ml) as described above. Increasing BPS concentrations (0.5 nmol/ml-500 nmol/ml) were added to four buffer systems (pH 4.2, 6.0, 6.5, and 8.0) containing liposomes with calcein. The suspensions were incubated for 30 min at room temperature and the percentage of calcein release was calculated by the equation I(%) = (I Ib) *100% as previously described (x b)

(Liu & Regen, 1993).


Membrane Lysis Profile of Biodegradable pH-Sensitive Surfactants when
Incorporated into Liposomes


Different molar ratios (R=0, R=0.2, and R=0.4) of biodegradable pH-sensitive surfactants (BPS) were incorporated into liposomes (10 nmol/ml) containing 100 mM calcein to observe the induced calcein release at different pHs. The various BPSliposomes were incubated in phosphate buffer solutions (pH 5-8) for 30 min to determine the release characteristics. The percentage of release was then recorded and corrected by the equation I(%) = (Ia Ib) *100% as mentioned previously.
(, lb)





55

Chemical and Biological Stabilities of Biodegradable pH-Sensitive Surfactants


The aqueous stability of biodegradable pH-sensitive surfactants (BPS) was

determined by incubating different concentrations of BPS in pH buffers (pH 1.4-8.0) with 5% dimethyl sulfoxide (DMSO) as a co-solvent at 37C. Samples were removed periodically and BPS concentration was quantified using an HPLC method. The HPLC system consisted of a Milton Roy CM 4000 pump, an LDC Analytical 3200 absorbance detector, a Hewlett Packard 3395 integrator, and a Spectra Physics SP 8780 autosampler. A 3.9*75 mm C8 column (Nova-Pak) along with a mobile phase consisting of 60% acetonitrile and 40% 10 mM pH 2.8 NaH2PO4 solution was used to separate and determine intact BPS from their degradation products at 210 nm (Buyuktimkin et al., 1993). The flow rate was set at 1.0 ml/min. The chemical degradation rate constant was obtained after plotting the peak height of the intact BPS over time and used to create BPS pH hydrolysis rate profile.

To determine the hydrolysis rate of BPS in biological systems, a porcine esterase was used. Varying units of esterase were incubated at 37C with constant amount of BPS in a 5% DMSO pH 7.0 buffer solution. Aliquot was removed at a periodic interval and peak height of the intact BPS was measured with the HPLC method described above. The biological rate constants of three BPS were calculated to compare their biodegradability.





56

Cellular Toxicity Test of Biodegradable pH-Sensitive Surfactants


The cellular toxicity of biodegradable pH-sensitive surfactants (BPS) was

monitored in an SKnSH human neuroblastoma cell line and in a CV-1 monkey kidney fibroblast cell line with a calcein-AM assay (Lichtenfels et al., 1994). To confirm the toxic effect of DIP and MIL as a result of their metabolites, imidazole, 1-imidazole methanol, dodecanol, and lauric acid were used to test their individual cytotoxicity in a CV-1 cell line.

Briefly, for the SKnSH cell line, subconfluent monolayered cultures were

incubated in a 96-well plate (10' cells/well) with 200 tl of RPMI 1640 growth medium (100 U/ml penicillin, 100 ig/ml streptomycin, and 10% fetal bovine serum) at 37C, 5% C02, and 100% humidity environment for 24 h. For the CV-1 cell line, MEM growth medium (100 U/ml penicillin, 100 jtg/ml streptomycin, 1 mM MEM sodium pyruvate solution, 1X MEM amino acids solution, and 10% heated fetal bovine serum) was used instead of RPMI 1640. The growth medium was then removed and BPS added from 1 tM to 1 M in 200 [d of fresh growth medium. The cells were maintained for an additional 48 h. After the incubation, cells were washed three times with phosphate buffered saline and incubated with 100 l of calcein-AM (1 jtM) for 30 min at room temperature.

Calcein fluorescence intensity was then measured at an excitation wavelength of 496 nm and observed at 517 nm on a Perkin Elmer LS 50B Spectrophotometer. The percentage of live cells was calibrated as in the following equation:





57


Live(%) =(Sample -Min) 100% where Max is the fluorescence signal from cells
(Max Min)

without any treatment, Min is the fluorescence signal without cells, and Sample is fluorescence signal from each sample. To compare the difference among different treatments, a parameter ID,,, was used. ID,0 was defined as the drug concentration required to reduce the absorbance of calcein by 50%, thus indicating 50% cell death.


Statistical Analysis


Statistical differences between the treatments were determined using analysis of variance where appropriate (StatView 4.53, Abacus Concepts, Inc., Berkeley, CA) with p<0.05 considered statistically significant and Fisher's (PLSD) post hoc t-test was applied.


Results


Critical Micelle Concentration Determination


An important parameter in characterizing surfactants is the concentration at which micelles form. All experiments were conducted at pH 3.0 to ensure that all biodegradable pH-sensitive surfactants (BPS) were in an ionized state (>99.9%). The critical micelle concentrations (CMCs) of the ionized BPS were determined by using surface tension measurements in different counter ion solution. As BPS concentration increased, the surface tension of the solution sharply decreased until the formation of micelles occurred (Figure 4- 1). In general, N-dodecyl imidazole (DI) had the highest CMCs while methyl





58















80

70

E 60

S50
V
40

30 20

10

0 I
0 1 2 3 4
Conc (m M)


Figure 4-1: Critical micelle concentration (CMC) measurement of three ionized biodegradable pH-sensitive surfactants (BPS). The CMC (mean+standard deviation
(SD)) was calculated in a pH 3.0 hydrofluoric acid solution at room temperature with an interfacial tensiometer (n=3). The concentration region at which surface tension stabilized was recorded as the CMC. The CMC was determined to be 1.0-1.2 mM for ionized dodecyl 2-(1l'-imidazolyl) propionate (DIP) (0), 0.6-0.8 mM for ionized methyl 1-imidazolyl laureate (MIL) (*), and 1.0-1.2 mM for N-dodecyl imidazole (DI) (A).





59

1-imidazolyl laureate (MIL) had the lowest CMCs (Table 4-1) in all four counter ion solutions. For all three BPS, HI, HBr, HC1, and HF caused the CMC to decrease in descending order.




Table 4-1: The critical micelle concentration of dodecyl 2-(1 '-imidazolyl) propionate (DIP), methyl 1-imidazolyl laureate (MIL), and N-dodecyl imidazole (DI) in four different counter ion solutions (n=3).

BPS (mM)
Solution DIP MIL DI
HF 1.0-1.2 0.6-0.8 1.0-1.2
HC1 0.9-1.1 0.6-0.7 0.9-1.1
HBr 1.7-1.9 1.0-1.2 1.8-2.2
HI 1.6-1.7 0.8-1.0 2.5-2.5




Effective Release Ratio Determination of Biodegradable pH-Sensitive Surfactants


Effective release ratio (Re) describes the molar ratio of a surfactant to the total

amount of lipid required to release 50% liposomal contents. Re was determined by fitting a sigmoidal curve of calcein release from liposomes at increasing molar ratios of biodegradable pH-sensitive surfactants (BPS) to lipid using the Scientist computer program (Micromath; Salt Lake City, Utah). Re was determined to be 3.0, 6.0, and 13.2 for dodecyl 2-(1 '-imidazolyl) propionate (DIP), methyl 1-imidazolyl laureate (MIL), and N-dodecyl imidazole (DI), respectively (Figure 4-2).




60








I I i I I i t 1 I I i 1 11 I i ii i

100





II I.I.I...I..l.... Iii ......l..ll...
80 /

_60
/
~40
2 0 ..................... .. .........
0
0 I 11111'''
10-2 10-1 100 101 102
Molar Ratio (BPS/Lipid)
Figure 4-2: Ability of various biodegradable pH-sensitive surfactants (BPS) to induce calcein release (mean+SD) at increasing molar ratios of ionized BPS from an external environment to liposomes when incubated in a pH 4.2 buffer solution for 30 min (n=3). The effective release ratio was then determined to be 3.0, 6.0 and 13.2 for dodecyl 2-(1 'imidazolyl) propionate (DIP) (0), methyl 1-imidazolyl laureate (MIL) (0), and Ndodecyl imidazole (DI) (A), respectively.





61

pH Sensitivity of Biodegradable pH-Sensitive Surfactants on Liposomal Calcein
Release


To determine whether biodegradable pH-sensitive surfactants (BPS) become effective in acidic environments but have limited effect at extracellular biological pH, calcein containing liposomes were incubated with increasing amounts of BPS at four pHs (4.2, 6.0, 6.5, and 8.0). An increase in fluorescence intensity indicated calcein release that correlated to membrane lysis.

Calcein was released sigmoidally at pH 4.2 as the concentration of dodecyl 2-(1'imidazolyl) propionate (DIP) increased (Figure 4-3a). When the pH of the incubation environment was increased, the percentage of calcein release was decreased at the same molar ratio of DIP to lipid. With the decreased surface active properties of DIP at pH 8.0, calcein release was only slightly increased at higher DIP concentration. This release was most likely due to saturation of the space between the lipid bilayers with increasing amount of DIP. Since distribution between the aqueous environment and lipid bilayer must occur for DIP to elicit membrane lysis, no significant differences between calcein release and pH were observed until the molar ratio reached 2 (p<0.01).

Similar profiles were seen with methyl 1-imidazolyl laureate (MIL) induced

calcein release (Figure 4-3b). Significant differences (p<0.01) on calcein release were observed at different pHs when the molar ratio of MIL to lipid equaled to 2 or above. However, compared to the calcein release caused by DIP at the same molar ratio, MIL showed less pH sensitivity and less calcein release at pH 6.0 and 6.5.





62













120 110 100

90 80
S 70
I/)
2 60
50

40 30 20 10
0
0.01 0.1 1 10 100
Molar Ratio (DIP/Lipid)

(a)

Figure 4-3: Effect of different biodegradable pH-sensitive surfactants (BPS) on calcein release from liposomes when added from an external environment (n=3). The percentages (mean+SD) of release were calculated after 30 min of incubation in four buffer solutions (pH 4.2 (*), 6.0 (X), 6.5 (A), and 8.0 (0)). a) Dodecyl 2-(1 'imidazolyl) propionate (DIP); b) Methyl 1-imidazolyl laureate (MIL); c) N-dodecyl imidazole (DI).





63
















100 90 80

70

S60 .2 50 40
40

30

20 10

0
0.01 0.1 1 10 100
Molar Ratio (MIL/Lipid)

(b)
Figure 4-3--continued





64
















100 90 80 70

60 .2 50

S40

30

20 10

0
0.01 0.1 1 10 100
Molar Ratio (DI/Lipid)

(c)
Figure 4-3--continued





65

When DIP was replaced by N-dodecyl imidazole (DI), similar profiles were seen on calcein release at low pH (Figure 4-3c). However, no significant difference was seen on the calcein release among the four tested pHs at all observed molar ratios. Membrane Lysis Profile of Biodegradable pH-Sensitive Surfactants when
Incorporated into Liposomes


Since the ultimate goal was to incorporate biodegradable pH-sensitive surfactants (BPS) into liposomes, we determined the ability of unionized BPS incorporated into liposomes to be protonated at lower pHs, thereby facilitating the release of entrapped materials. Liposomes containing calcein were prepared with increasing molar ratios (R=O, R=0.2, and R=0.4) of BPS and incubated at decreasing pHs.

Minimal calcein release was observed when no dodecyl 2-(l '-imidazolyl) propionate (DIP) was incorporated into the liposomes (Figure 4-4a). As the pH decreased, calcein release increased gradually in all groups. At each ratio group, calcein release increased significantly (p<0.05) as pH dropped from 6.0 to 5.0. Significant differences (p<0.05) of the calcein release were also observed among all molar ratio groups (R=O, R=0.2, and R=0.4) at all observed pHs. Like DIP, both methyl 1imidazolyl laureate (MIL) (Figure 4-4b) and N-dodecyl imidazole (DI) (Figure 4-4c) caused calcein release in a similar pH-dependent manner. Compared to other groups, the system at the R=0.4 group was relatively unstable at physiological pH probably due to the alternations in lipid packing of the liposomes.

When comparing the calcein release caused by three BPS, significant differences (p<0.05) were observed at the R=0.4 group as pH was 6.0 or 5.0. However, no





66










45 40 35 30

S25 S 20

15 10

5 0
4 5 6 7 8
pH
(a)

Figure 4-4: Membrane lysis profile of various biodegradable pH-sensitive surfactants (BPS) when incorporated into liposomes (n=3). The effect of pH and BPS/liposome molar ratio (R=0 (*), R=0.2 (N), and R=0.4 (A)) on BPS-induced calcein release from liposomes were plotted after 30 min (meanSD). a) Dodecyl 2-(1l'-imidazolyl) propionate (DIP); b) Methyl 1-imidazolyl laureate (MIL); c) N-dodecyl imidazole (DI).





67













45 40 35 30

S25 S20

15 10 5

0
4 5 6 7 8

pH

(b) Figure 4-4--continued





68













45

40 35 30

S25 S20

15 10

5 0
4 5 6 7 8
pH
(c) Figure 4-4--continued





69

significant difference of the calcein release was seen at the other molar ratio groups (R=O. I and R=0.2) among these three BPS. Chemical and Biological Stabilities of Biodegradable p11-Sensitive Surfactants


After releasing oligonucleotides to cytoplasm, an ideal pH-sensitive surfactants must be degraded in the intercellular milieu, thus limiting its potential toxicity. Biodegradable pH-sensitive surfactants (BPS) should be able to be degraded by ester hydrolysis either chemically or enzymatically. The hydrolytic stability of BPS was assessed by incubating the compound in pH buffers and monitoring the amount of the starting material remaining intact (Figure 4-5). Using the Scientist program to fit the degradation curve, the pH-dependent pseudo-first order degradation rate constants (k) was calculated.

Dodecyl 2-(1 -imidazolyl) propionate (DIP) was at its most stable state (k=0.055 day') at pH 2.8. The degradation rate constant reached a plateau after pH 6.0 (k=0.70 day') (Figure 4-6). Similar to DIP, methyl I1-imidazolyl laureate (MIL) and N-dodecyl imidazole (DI) showed the greatest stability at pH 2.8 (k=0.050 day-' and k=0.0055 day', respectively). DI exhibited the lowest rate constant among the three BPS from pH 1.3 to pH 7.0 solutions. No difference in the degradation rate constant of BPS was seen when the pH was adjusted to 8.0. However, since each pH buffer solution was composed of different compounds, it might have its individual catalyst effect on the chemical rate constant.

To assess the enzymatic stability of BPS, we used various amounts of porcine esterase as a model enzyme. Similar to the determination of chemical degradation rate





70













50.


40

Q
30

0)
20



10


0
0 1 2 3 4 5 6
Time (day)

Figure 4-5: Chemical degradation profile of dodecyl 2-(l '-imidazolyl) propionate (DIP) at pH 1.4 and 370C over time (n=3). The peak heights of intact DIP were obtained at various time points (meanSD). Using the Scientist program to fit the degradation curve, the pseudo-first order degradation rate constant (k) was determined to be 0.36 (/day).





71










10




1




0.1

-o


0.01




0.001 i l7
1 2 3 4 5 6 7 8
pH

Figure 4-6: Chemical degradation pH-rate profiles of three biodegradable pH-sensitive surfactants (BPS). The rate constant (mean+SD) measured at 370C of dodecyl 2-(1 'imidazolyl) propionate (DIP) (i), methyl 1-imidazolyl laureate (MIL) (*), and Ndodecyl imidazole (DI) (A) was plotted against pH (n=3).





72

stability of BPS was assessed by monitoring the amount of the starting material remaining intact in a pH 7.0 buffer solution (Figure 4-7). Figure 4-8 showed the biological degradation rate profile of BPS at various ratios of esterase/BPS. MIL was more biodegradable than DIP through out the tested ratios. Furthermore, DI was almost insensitive to the addition of the esterase.


Cellular Toxicity Test of Biodegradable pH-Sensitive Surfactants


A more important parameter than biodegradability of biodegradable pH-sensitive surfactants (BPS) is the cytotoxicity. Biodegradability of BPS relates to levels of cellular toxicity. A commonly used calcein-AM assay was used to measure the cellular toxicity of BPS. Calcein-AM is a lipophilic ester compound without fluorescent activity. After diffusion into cells, calcein-AM is degraded into calcein, a fluorescence chemical, by endogenous enzymes. The percentage of live cells is then calculated indirectly from the fluorescence signal of calcein. The higher the signal, the more cells surviving.

In SKnSH cells, the ID,0 (Figure 4-9a) was determined to be 1 mM, 8 mM, and 0.07 mM for dodecyl 2-(1 '-imidazolyl) propionate (DIP), methyl 1-imidazolyl laureate (MIL) and N-dodecyl imidazole (DI), respectively. In CV-1 cells, the ID50 (Figure 4-9b) was measured at 20 mM, 300 mM, and 0.3 mM for DIP, MIL, and DI, respectively. For the possible metabolites from DIP and MIL, the ID50 was 70 mM for imidazole, 7 mM for dodecanol, 800 mM for 1-imidazole methanol, and 9 mM for lauric acid in CV-1 cells (Figure 4-10).

DI showed the highest cytotoxic effect and was one and two orders of the

magnitude more toxic than DIP in SKnSH and CV- 1 cells, respectively. On the contrary,





73












25




2 0 1 - - -- -- -

0 5 - - - -- . .
0M



ZD
0- - . - -
5






0 20 40 60 80
Time (minute)

Figure 4-7: Biological degradation profile of 0.65 gtmole of dodecyl 2 (1 '-imidazolyl) propionate (DIP) when incubated with 0.9 U of porcine esterase at pH1 7.0 and 370C over time. The peak heights of the intact DIP were obtained at various time points. Using the Scientist program to fit the degradation curve, the pseudo-first order degradation rate constant (k) was determined to be 0.99 (/h).





74









5
y = 1.7453x + 0.1221 R2 = 0.9826
4



3
L..


2



1
y 0.7476x 0.0004 R2 = 0.9973

0 Ar i_
0 0.5 1 1.5 2 2.5
U/m icrom ole


Figure 4-8: Biological degradation rate profile of biodegradable pH-sensitive surfactants (BPS). Various ratios (U/imole) of porcine esterase to three BPS (dodecyl 2-(1 'imidazolyl) propionate (DIP) (0), methyl 1-imidazolyl laureate (MIL) (4*), and Ndodecyl imidazole (DI) (A)) were plotted against degradation rate constants (mean+SD) in a pH 7.0 buffer solution at 370C (n=4). The linear relationship between the degradation rate constant of each BPS and the ratio of esterase to BPS was also shown.





75











100



80



(n




40




20



0
0.001 0.01 0.1 1 10 100 1000
Conc. (mM)

(a)

Figure 4-9: Cytotoxic effect of biodegradable pH-sensitive surfactants (BPS) on two cell lines measured by a calcein-AM assay after 48 h of incubation. Data were expressed as mean+SD. a) In SKnSH cells, the ID50 (50% live cells) of methyl 1-imidazolyl laureate (MIL) (A), dodecyl 2-(1 '-imidazolyl) propionate (DIP) (0), and N-dodecyl imidazole
(DI) (*) was visually determined to be 8 mM, 1 mM, and 0.07 mM, respectively (n=4). b) In CV-1 cells, the ID50 of MIL (A), DIP (M), and DI (4) was visually determined to be 300 mM, 20 mM, and 0.3 mM, respectively (n=4).





76
















100 80



60
0



40 20



0 -
0.001 0.01 0.1 1 10 100 1000
Conc. (mM)

(b) Figure 4-9--continued





77











100




80




5 60



40




20




0
0.01 0.1 1 10 100 1000
Conc. (mM)


Figure 4-10: Cytotoxic effect of possible metabolites from biodegradable pH-sensitive surfactants (BPS) on CV-1 cells measured by a calcein-AM assay after 48 h of incubation. The ID50 (50% live cells) was determined to be 70 mM for imidazole (*), 7 mM for dodecanol (0), 800 mM for 1-imidazole methanol (0), and 9 mM for lauric acid
(X), respectively (n=4). Data are expressed as mean-SD.





78

MIL exhibited the least cytotoxicity compared to the other two BPS and was one order of the magnitude less toxic than DIP in both cell lines. The possible metabolites, imidazole and dodecanol, from DIP were more toxic than those metabolites, 1-imidazole methanol and lauric acid, from MIL in CV-1 cells.


Discussion


Imidazolyl based lipids have been used successfully for in vitro delivery of

nucleic acids (Solodin et al., 1995) establishing a rational for the use of imidazole. Most non-viral delivery systems enter cells through endocytosis. The most significant characteristic of endosomes is the pH gradient from inside the endosome to the intracellular space.

Dodecyl 2-(1 '-imidazolyl) propionate (DIP), methyl 1-imidazolyl laureate (MIL), and N-dodecyl imidazole (DI) are imidazolyl based surfactants in the biodegradable pHsensitive surfactants (BPS) family which have been proposed to facilitate the transport of nucleic acid through the endosomal pathway. BPS take advantage of the acidic environment within endosomes to protonate a lysosomotropic amine thus increasing their surface active properties. After BPS become ionized, they can assist the destabilization of the endosomal membrane. To lessen adverse effects of the ionized BPS, an ester bond was introduced into DIP and MIL's structure making it biodegradable. In a preliminary study (Hughes et al., 1996), DIP has been shown to reduce the concentration of oligonucleotides required to produce a biological effect using a tissue culture system. In this report, the physicochemical properties of three BPS were systematically characterized.





79

The number of possible analogs of the BPS family can be immense. In order to determine what physicochemical parameters influence the biological activity of BPS, a series of evaluative tests associated with surfactants were established. Presently, it is not clear which measured parameter would be useful in the characterization of BPS for nucleic acid delivery. The current established methods have been optimized for studying small molecule transport instead of macromolecules such as oligonucleotides and plasmid DNA.

The first parameter determined for BPS was the critical micelle concentration

(CMC). The more hydrophilic a surfactant, the higher the CMC (Rosen, 1989). From the chemical structures, DI, DIP, and MIL had decreasing orders of hydrophilicity. As a result of this difference, DI, DIP, and MIL had decreasing orders of CMC in general. As the size of the counter ion increased, the surfactant became more hydrophilic thus obtaining the higher CMC.

CMC is an essential parameter in describing surfactants (i.e., hydrophilicity, surface excess); however, it may not be the best parameter to measure the ability of a surfactant to cause membrane lysis (Lichtenberg, 1985; Ruiz et al., 1988). Therefore, the effective release ratio (Re) was utilized to describe the ability of BPS to lyse membranes. The lower the Re, the less surfactant required to lyse membranes.

For basic amines such as BPS, the intrinsic ionization constant (pKa 5-7) and

local pH environment determine the percent ionized. The relationship can be expressed [BH ]
in Henderson-Hasselbach equation: pKa = pH + log [ where [BH ] and [B] represents the ionized and un-ionized bases, respectively. From the perspective of their





80

chemical structures, DI, DIP, and MIL were expected to have a decreasing order of pKa. This difference between the pKa reflects the different amount of ionized BPS that can be converted at the same pH environment. While pKa is an important factor that determines the amount of ionized BPS and further influence the liposomal content release, the ability of different BPS partition into the liposomal membranes at different pHs should be also considered.

Comparing these three agents, with the highest pKa and no chemical hindrance (i.e., straight hydrocarbon chain) that facilitated partition into liposomes more easily, DI was almost indiscriminate to various pH environments resulting in a similar calcein release profile. For the MIL treated liposome group, on the other hand, a limited amount of calcein was detected at pH 6.0, 6.5, and 8.0 because of its lowest pKa and chemical hindrance effect (i.e., ester linker) that might decrease the partition of MIL into liposomes.

However, with the median pKa value and chemical hindrance (i.e., ester linker and branched methyl group) of DIP, more calcein was released than MIL as pH was decreased from 8.0 to 6.5 or 6.0. With these two factors (i.e., pKa and partition coefficient), the calcein release profiles were therefore established in various pH environments.

The ability of BPS to release liposome-entrapped molecules was tested as a proof of the compound's pH sensitivity based on the principle experiment. As the BPS/liposome molar ratio was increased and/or pH decreased, more calcein was released from the liposome model system. Calcein release induced by BPS when incorporated into the liposome system was slightly higher than that caused by BPS when added to a





81


solution containing calcein liposomes. The greater release could be due to the dilution of BPS or may be related to partitioning barriers.

An interesting finding was observed between the release of liposome entrapped calcein when BPS were added externally as compared to direct incorporation into the liposome matrix. All three BPS demonstrated similar release profiles when the uninoized BPS agent was incorporated into the liposome illustrating they most likely have similar mobility within the bilayer. When the three agents were added from the external phase to preformed liposomnes there was a drastic difference between the release profiles of BPS with ester linkages (e.g., DIP and MIL) as compared to the alkyl chain analog (DI) which demonstrated little pH dependency.

This effect is also possibly due to the change of pKa at the interface between the monolayer of the liposome and the solution. However, it is unlikely that this lack of pH dependency is solely due to changes in the pKa of BPS. The different profiles may reflect the ease of BPS to partition into the bilayer. BPS with a more polar head group (e.g., ester containing) would be expected to have added resistance in membrane partitioning.

After releasing membrane entrapped molecules in a pH-sensitive manner, the

biodegradability of BPS was confirmed. Due to its possible increased stable metabolites and lack of chemical hindrance, MIL was more biodegradable than DIP (Figure 4-8). On the other hand, without any biodegradable bond (e.g., ester), DI showed no difference of the degradation rate constants when incubated in biological media. The degradation rate of BPS in vivo would be expected to be greater due to the higher number of esterase (e.g.,





82

lipase) molecules. After the release of BPS from the liposomal membrane, these molecules would be available for BPS metabolism.

A major concern with the use of agents to enhance oligonucleotide delivery is that any compound added to a delivery vector might contribute to the toxicity of the system. The toxicity of a given compound is often related to its stability; therefore, the biodegradability of BPS may decrease its cellular toxicity. In the calcein-AM assay, the addition of an ester group to the surfactant resulted in a less toxic effect as compared to N-dodecyl imidazole, a first generation lysosomotropic detergent (De Duve et al., 1974), by one to two orders of magnitude in two different cell lines. It was also shown that the less toxic effect of MIL compared to DIP was the possible result of the less toxic metabolites from MIL than those from DIP. While this study implies that ester containing imidazole based surfactants are less toxic than their straight chain analogs, it is unclear how the toxicity of BPS alone will compare to cationic liposome mediated delivery.

The rate and efficiency of oligonucleotide release from lipid compartments

depend on the characteristics of the surfactant. Biodegradable pH-sensitive surfactants (BPS) must be stable enough in the biological milieu to induce a therapeutic effect. However, after facilitating oligonucleotides to their sites of action, BPS must be metabolized into less toxic lysosomotropic amines and hydrocarbon chain components to prevent cellular toxicity. BPS are similar to the first generation lysosomotropic detergents with their long lasting surface active property in a pH-dependent manner. However, they have much higher biodegradability because of the bridge connector which can be hydrolyzed into less toxic compounds.





83



Conclusion


The ultimate goal of this project was to adjust chemical characteristics of BPS components, head and tail groups, in order to optimize BPS-induced release of oligonucleotides from membrane compartments. Furthermore, BPS would have minimum cellular toxicity with the introduction of a biodegradable linker. At present, the critical micelle concentration (CMC), effective release ratio (Re), biodegradability, and cytotoxicity of these three BPS have little biological relevance. As more diverse BPS are established and characterized with these mentioned parameters, it is hoped that particular physicochemical properties will be predictive of promoting oligonucleotide biological activity.













CHAPTER 5
DELIVERY SYSTEM EVALUATION OF BIODEGRADABLE pH-SENSITIVE SURFACTANTS


Introduction


Oligonucleotides must reach their sites of action (cytoplasm or nucleus) to

hybridize with their targets to exert their effect (Agrawal & Iyer, 1997). However, the cellular delivery of free oligonucleotides is very poor (Akhtar et al., 1991b; Stein & Cheng, 1993). One strategy to improve the delivery of the oligonucleotide is to use liposomes which can carry oligonucleotides with the vehicles and increase the intracellular accumulation via endocytosis. Liposomes can deliver oligonucleotides to the cells but the obstacle of escaping from endosomes still remains. A series of biodegradable pH-sensitive surfactants (BPS) were developed to conquer the potential pitfall that the liposome delivery system might have and to further enhance cellular delivery of the oligonucleotide.

In previous studies, the physicochemical properties of BPS were characterized. They were shown to be able to induce membrane lysis in a pH- and molar ratiodependent manner (Chapter 4). Nevertheless, the BPS-liposome system has not yet been tested using a biological system. Therefore, we further evaluated the BPS-liposome delivery system in vitro.





84





85

To evaluate oligonucleotide cellular uptake with the BPS-liposome system, flow cytometry was employed. Another screening method that addressed oligonucleotide cellular delivery was the use of laser scanning confocal microscopy (Fisher et al., 1993). We used this technique to investigate oligonucleotide cellular uptake and distribution that the BPS-liposome delivery system affected. Additionally, we quantitatively monitored the inhibition effect of oligonucleotides in BPS-liposomes on luciferase enzyme activity (Brasier et al., 1989).


Materials


Chemical


Dihydrogen potassium phosphate, EDTA, formaldehyde solution, and Triton X100 were bought from Fisher Scientific (Pittsburgh, PA). Adenosine triphosphate, dithiothreitol (DTT), magnesium sulfate, and tricine were purchased from Sigma (St. Louis, MO). BCA Protein Assay Kit was obtained from Pierce (Rockford, IL) and Label IT fluorescein Nucleic Acid Labeling Kit from Mirus (Madison, WI). The pGL3 plasmid DNA was obtained from Mr. Fuxing Tang in the Department of Pharmaceutics, University of Florida. D-luciferin was purchased from Molecular Probes (Eugene, OR). L-cx-lecithin and N-[1-(1-2,3-dioleoloxy)propyl]-N,N,N-trimethylammonium methylsulfate (DOTAP) was purchased from Avanti Polar Lipids (Alabaster, AL). Three biodegradable pH-sensitive surfactants (BPS), dodecyl 2-(1'-imidazolyl) propionate (DIP), methyl 1-imidazolyl laureate (MIL), and dodecyl imidazole (DI) were synthesized





86

as previously reported (Chapter 3). All purchased or obtained chemicals were used directly without additional purification.


Cell


The CV-1 luciferase expressing cell line was a generous gift from Dr. M. C. Cho of the University of North Carolina. The RAW 264.7 (TIB-71) cell line was purchased from the American Type Culture Collection (Rockville, MD).



Methods


Oligonucleotide and Plasmid DNA Cellular Uptake Evaluation Using Flow
Cytometry


Oligonucleotide synthesis and liposome preparation

Phosphorothioate oligonucleotides (15 bases; 5'-TGG CGT CTT CCA TTT-3')

labeled with fluorescein isothiocyanate (FITC) at the 5'-end were synthesized in the DNA Core Synthesis Lab at the University of Florida. Oligonucleotides were used directly without additional purification. pGL3 plasmid DNA with a size of 5256 bp and purity of

1.9 (A260/A280) was labeled with fluorescein using a Mirus Label IT Nucleic Acid labeling Kit. Simply, 100 ptg of the plasmid DNA (2.25 mg/ml) reacted with 100 Jl of fluorescein reagent at 1:1 (w/v) ratio was dilute to a final concentration of 0.1 mg/ml in 1X labeling buffer and incubated at 370C for 1 h. The unreacted labeling reagent was removed from labeled nucleic acid by sephadex G50 spin columns at a force of 720*g for

2 min and then the purified sample collected.





87

Cationic N-[ 1-(1-2,3-dioleoloxy)propyl]-N,N,N-trimethylammonium

methylsulfate (DOTAP) liposomes were made as a control vector for nucleic acid delivery. Biodegradable pH-sensitive surfactants (BPS) at various molar ratios were combined with DOTAP to create liposomes. After liposomes were rehydrated, a Sonic Dismembrator 60 probe was used to form small unilamellar vesicles by applying 5 Watts of power for 10 s to the liposome suspension and keeping on ice for 30 s. The cycle was repeated until a clear solution was seen. The size of the liposomes (volume-weight Gaussian distribution) was determined to be 55.226.1 nm (standard deviation) by a dynamic light scattering method using a NICOMP 380 ZLS Zeta Potential/Particle Sizer (Santa Barbara, CA).

Cell preparation

Monolayered CV-1 (monkey kidney fibroblast) cells were incubated in 24-well plates (5*10' cells/well) with 1 ml of MEM growth medium in each well at 370C, 5% CO2, and a 100% humidity environment for 24 h. The medium included 100 U/ml penicillin, 100 jtg/ml streptomycin, 1 mM MEM sodium pyruvate solution, 1X MEM amino acids solution, and 10% heated fetal bovine serum. Experimental procedures

Either 37.5 nmole of the liposome (DOTAP or DOTAP-dodecyl 2-(1 '-imidazolyl) propionate (DIP) at molar ratio 0.3 (R=0.3)) was complexed with 0.25 nmole of the FITC-oligonucleotide or 20 tg of the liposome with 1 tg of the plasmid DNA in each well for 30 min. The charge ratio (+/-) of DOTAP to oligonucleotide was set to be 10 which was proposed as the optimal ratio in other similar study (Zelphati & Szoka,





88

1996a). The charge ratio (+/-) of DOTAP to plasmid DNA was also set to be 10 to exclude the possible charge interference when these two types of nucleic acid were compared. The liposome-nucleic acid complex was added into 500 p1 of serum free MEM growth medium after the MEM growth medium in each well was removed. After 4 h of incubation, the serum free MEM growth medium including the liposome-nucleic acid complex was discarded and replaced with 1 ml of fresh growth medium. The cells were harvested at various time points and analyzed by flow cytometry.

In the second part of the experiments, all three BPS (DIP, methyl 1-imidazolyl laureate (MIL), and N-dodecyl imidazole (DI)) were incorporated into cationic DOTAP liposomes at four molar ratios (R=0, R=0.1, R=0.2, and R=0.3). After a 30-min period of complexation, either 37.5 nmole of the liposome with 0.25 nmole of the fluoresceinlabeled oligonucleotide or 20 g of the liposome with 1 tg of the fluorescein-labeled plasmid DNA with at a charge ratio of 10 (+/-) was incubated with CV-l cells in 500 ptl of serum free growth medium. After 4 h, the cells were harvested and then analyzed by flow cytometry.

Flow cytometry

In order to minimize the nucleic acids adsorbed onto cells, the cells were washed twice with phosphate buffered saline (PBS) and lifted from the wells before analyzing the samples. The cells were then transferred to tubes and centrifuged at 1,200 rpm for 5 min. The supernatant (800 j,1) was decanted and resuspended with 800 [1 of PBS. The above procedure was repeated twice and the samples kept on ice until analysis. To address





89

nucleic acid cellular uptake in some studies, rather than using PBS in the final step, 2% formaldehyde was added into the tubes for 30 min to fix the cells.

The signals emitted from the FITC-oligonucleotide (or fluorescein-plasmid DNA) were performed by a Becton Dickinson FACSort flow cytometer (San Jose, CA). Green fluorescence was monitored with a 530/30 nm bandpass filter, and photomultiplier tube pulses were amplified logarithmically. Ten thousand cells were counted at a flow rate between 100 and 200 cells per second. Cells were gated with their morphological properties, forward scatter and side scatter, set on logarithmic mode. The mean fluorescence intensity of the related populations of cells was calculated using histograms and expressed in arbitrary units corresponding to an intensity channel number ranging from 0 to 1,023 using a LYSYS II program (Becton Dickinson; San Jose, CA).


Oligonucleotide Cellular Uptake and Distribution Evaluation with Confocal
Microscopy


Oligonucleotide synthesis and liposome preparation

Fifteen bases of poly-A phosphorothioate oligonucleotides labeled with

fluorescein isothiocyanate (FITC) at the 5'-end were synthesized in the DNA Core Synthesis Lab at the University of Florida. They were used directly without additional purification.

Two different lipid formulations were used: L-a-lecithin and L-a-lecithin with dodecyl 2-(1 '-imidazolyl) propionate (DIP) at molar ratio 0.3 (R=0.3). The lipid rehydration method was used to form neutral liposomes as FITC-labeled oligonucleotides dissolved in the aqueous solvent. To increase the encapsulation efficiency of





90


oligonucleotides into the liposomes, five freeze-and-thaw cycles were employed after a 30-min period of hand shaking.

The liposomes were then passed three times through 600-nm polycarbonate membranes (Poretics; Livermore, CA) using a high pressure extruder (Lipex Biomembrane Inc.; Vancouver, Canada). The concentration of the phospholipid was calculated as previously reported (Chapter 4). The volume-weight Gaussian distribution of the liposome size was determined to be 630+265 nm (standard deviation) by a dynamic light scattering method using a NICOMP 380 ZLS Zeta Potential/Particle Sizer (Santa Barbara, CA).

Cell preparation

Before plating RAW 264.7 (mouse monocyte-macrophage) cells, a cover slip was placed in each well of 12-well plates for later observation on a confocal microscope. Each well containing a cover slip was treated with 300 [tl of collagen in a 0.02 M acetic acid solution (50 pg/ml) and incubated at room temperature. After 1 h, the solution was removed by rinsing with phosphate buffered saline (PBS) three times. The plates were then ready for use.

Subconfluent monolayered RAW 264.7 cells were cultured in the 12-well plates (2* 10' cells/well) with 1 ml of DMEM growth medium in each well at 370C, 5% CO2, and a 100% humidity environment for 24 h. The medium included 100 U/ml penicillin, 100 sg/ml streptomycin, and 10% fetal bovine serum.





91


Experimental procedures

After the incubation, the growth medium in each well was replaced with 500 pl of serum free medium containing same amount of starting FITC-oligonucleotides as the following preparations.

* FITC-oligonucleotide

* FITC-oligonucleotide+liposome (100 nmole)

* FITC-oligonucleotide+DIP-liposome (R=0.3) (120 nmole) Confocal microscopy

After 4 h of incubation in serum free medium, 10% fetal bovine serum was added. At the end of each sampling time (4 h, 8 h, and 24 h), the wells were washed with PBS and cells fixed in 2% formaldehyde for 30 min. The cover slips were transferred to the slides with Gel/Mount. Cellular uptakes and distributions of the oligonucleotides were then viewed and compared.

Cells incubated with FITC-labeled oligonucleotides were imaged using a Biorad MRC-600 laser scanning confocal microscope equipped with a krypton/argon laser at the Center of Structural Biology, University of Florida. Images were collected on an Olympus IMT-2 inverted microscope using the 488/568 nm line at which the excitation light was attenuated with a 1% neutral density filter to minimize photobleaching and photodamage.




Full Text

PAGE 1

86( 2) %,2'(*5$'$%/( S+6(16,7,9( 685)$&7$176 ,1 /,32620( 0(',$7(' 2/,*218&/(27,'( '(/,9(5< %\ &+,+:(, ($59,1 /,$1* $ ',66(57$7,21 35(6(17(' 72 7+( *5$'8$7( 6&+22/ 2) 7+( 81,9(56,7< 2) )/25,'$ ,1 3$57,$/ )8/),//0(17 2) 7+( 5(48,5(0(176 )25 7+( '(*5(( 2) '2&725 2) 3+,/2623+< 81,9(56,7< 2) )/25,'$

PAGE 2

'HGLFDWHG WR P\ SDUHQWV 7X-RZ DQG &KRZ6RQ /LDQJ

PAGE 3

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t +XJKHV JURXS ZKR KHOSHG PH WKURXJKRXW P\ HQWLUH JUDGXDWH OLIH DQG UHVHDUFK DW WKH 8QLYHUVLW\ RI )ORULGD )LQDOO\ ZDQW WR WKDQN P\ SDUHQWV DQG VLEOLQJV IRU WKHLU XQVHOILVK ORYH DQG FRQWLQXRXV VXSSRUW GXULQJ ERWK P\ JRRG DQG EDG WLPHV LQ WKH SDVW \HDUV P

PAGE 4

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

PAGE 5

0HWKRGV 5HVXOWV 'LVFXVVLRQ &RQFOXVLRQ '(/,9(5< 6<67(0 (9$/8$7,21 2) %,2'(*5$'$%/( S+ 6(16,7,9( 685)$&7$176 ,QWURGXFWLRQ 0DWHULDOV 0HWKRGV 5HVXOWV 'LVFXVVLRQ &RQFOXVLRQ 0(&+$1,60 2) $&7,21 ,19(67,*$7,21 2) %,2'(*5$'$%/( S+6(16,7,9( 685)$&7$176 ,QWURGXFWLRQ 0DWHULDOV 0HWKRGV 5HVXOWV 'LVFXVVLRQ &RQFOXVLRQ &21&/86,21 $1' )8785( 35263(&7 &RQFOXVLRQ )XWXUH $LPV 5()(5(1&(6 %,2*5$3+,&$/ 6.(7&+ Y

PAGE 6

.(< 72 $%%5(9,$7,216 $176 ODPLQRQDSKWKDOHQHWULVXOIRQLF DFLG %36 ELRGHJUDGDEOH S+VHQVLWLYH VXUIDFWDQWVf &0& FULWLFDO PLFHOOH FRQFHQWUDWLRQ ''$% GLPHWK\OGLRFWDGHF\ODPPRQLXP EURPLGH '&&KRO S>11?1fGLPHWK\ODPLQRHWKDQHfFDUEDPR\O@FKROHVWHURO ', 1GRGHF\O LPLGD]ROH ',3 GRGHF\O O fLPLGD]RO\Of SURSLRQDWH '0) 11GLPHWK\OIRUPDPLGH '05,( OGLP\ULVW\OR[\SURS\OGLPHWK\OK\GUR[\HWK\O DPPRQLXP EURPLGH '03& GLP\ULVWR\OVQJO\FHURSKRVSKRFKROLQH '062 GLPHWK\O VXOIR[LGH '23( GLROHR\OSKRVSKDWLG\OHWKDQRODPLQH '263$ GLROH\OR[\VSHUPLQHFDUER[DPLGR11GLPHWK\OOSURSDQDPLQLXP '27$3 1>OOGLROHROR[\fSURS\O@111WULPHWK\ODPPRQLXP PHWK\OVXOIDWH '270$ 1>OGLROHROR[\fSURS\O@111WULPHWK\ODPPRQLXP FKORULGH '3; 1 1fS[\O\OHQHELVS\ULGLQLXP EURPLGHf ),7& IOXRUHVFHLQ LVRWKLRF\DQDWH +9KHPDJJOXWLQDWLQJ YLUXV RI -DSDQ 0,/ PHWK\O LPLGD]RO\O ODXUHDWH YL

PAGE 7

1%'3( 1QLWUROEHQ]R[DGLD]RO\OfSKRVSKDWLG\OHWKDQRODPLQH 105 QXFOHDU PDJQHWLF UHVRQDQFH 3%6 SKRVSKDWH EXIIHUHG VDOLQH 3( SKRVSKDWLG\OHWKDQRODPLQH 5 PRODU UDWLR RI WKH ELRGHJUDGDEOH S(OVHQVLWLYH VXUIDFWDQWV WR WKH RWKHU OLSLGV 5H HIIHFWLYH UHOHDVH UDWLR 5K3( 1OLVVDPLQH UKRGDPLQH % VXOIRQ\OfSKRVSKDWLG\OHWKDQRODPLQH YLL

PAGE 8

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f ZHUH WKHUHIRUH GHVLJQHG WR HQKDQFH OLSRVRPH PHGLDWHG ROLJRQXFOHRWLGH GHOLYHU\ %36 DUH D XQLTXH IDPLO\ RI HDVLO\ PHWDEROL]HG FRPSRXQGV WKDW GHPRQVWUDWH S+GHSHQGHQW VXUIDFH DFWLYLW\ 7KURXJK VLPSOH DQG IDVW FKHPLFDO UHDFWLRQV WKUHH %36 GRGHF\O OfLPLGD]RO\Of SURSLRQDWH ',3f PHWK\O LPLGD]RO\O ODXUHDWH 0,/f DQG 1GRGHF\O LPLGD]ROH ',f ZHUH

PAGE 9

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

PAGE 10

&+$37(5 ,1752'8&7,21 2OLJRQXFOHRWLGHV KDYH EHHQ XVHG DV D JHQH WKHUDS\ DSSURDFK VLQFH WKH ODWH V =DPHFQLN t 6WHSKHQVRQ f 7KH WKHRUHWLFDO DSSURDFK RI ROLJRQXFOHRWLGHV LV YHU\ DWWUDFWLYH VLQFH LW DOORZV IRU WKH LQKLELWLRQ RI D VSHFLILF SURWHLQ 2OLJRQXFOHRWLGHV ZLWK RU ZLWKRXW D FDUULHU DUH WUDQVSRUWHG LQWR FHOOV PRVWO\ E\ HQGRF\WRVLV $NKWDU t -XOLDQR /RNH HW DK f DQG DFFXPXODWH LQ HQGRVRPHV LQWUDFHOOXODU FRPSDUWPHQWV ZLWK DQ DFLGLF LQWUDOXPLQDO S+ 0D[ILHOG 0F*UDZ t 0D[ILHOG f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

PAGE 11

2EMHFWLYH 7KH PDLQ REMHFWLYH RI WKH SURMHFW ZDV WR FKDUDFWHUL]H ELRGHJUDGDEOH S+VHQVLWLYH VXUIDFWDQWV %36f ERWK SK\VLFRFKHPLFDOO\ DQG ELRFKHPLFDOO\ ZKLFK FDQ VHUYH DV DGMXYDQWV IRU LPSURYHG WUDQVIHU RI ROLJRQXFOHRWLGHV IURP WKH HQGRVPH WR WKH F\WRSODVP ZLWKRXW DVVRFLDWHG FHOOXODU WR[LFLW\ +\SRWKHVLV 7KH RYHUDOO K\SRWKHVLV ZDV WKDW WKH HQGRVRPDO PHPEUDQH SUHVHQWV D PDMRU EDUULHU WR ROLJRQXFOHRWLGH GHOLYHU\ %\ WKH DGGLWLRQ RI ELRGHJUDGDEOH S+VHQVLWLYH VXUIDFWDQWV %36f WR D OLSRVRPDO GHOLYHU\ V\VWHP WKH ORZ HQGRVRPDO S+ FDQ DFWLYDWH WKH S+ VHQVLWLYH VXUIDFWDQWV DQG IDFLOLWDWH WKH WUDQVIHU RI HQGRVRPDO FRQWHQWV LH ROLJRQXFOHRWLGHf WR WKH F\WRSODVP RU QXFOHXV 7KH ELRGHJUDGDEOH OLQNHU FDQ EH GLJHVWHG LQWR OHVV WR[LF PHWDEROLWHV E\ WKH HQGRJHQRXV GLJHVWLYH HQ]\PHV

PAGE 12

&+$37(5 %$&.*5281' $1' 6,*1,),&$1&( 2OLJRQXFOHRWLGH 7KHUDS\ 2YHUYLHZ 7KH DELOLW\ RI VKRUW V\QWKHWLF DQG VLQJOHVWUDQGHG '1$ RU 51$ ROLJRQXFOHRWLGHV WR LQWHUIHUH ZLWK LQGLYLGXDO JHQH H[SUHVVLRQ LQ D VHTXHQFHVSHFLILF PDQQHU LV WKH IRXQGDWLRQ IRU ROLJRQXFOHRWLGHEDVHG WKHUDS\ 7KH ILUVW FOHDU H[SORUDWLRQ RI ROLJRQXFOHRWLGHV ZDV UHSRUWHG E\ =DPHFQLN DQG 6WHSKHQVRQ f +RZHYHU GXH WR D QXPEHU RI LPSHGLPHQWV HJ XQGHUVWDQGLQJ RI WKH VHTXHQFH DQG WRSRORJ\ RI WKH QXFOHLF DFLG WDUJHW V\QWKHVLV RI UHVHDUFK TXDQWLWLHV RI ROLJRQXFOHRWLGHV DQG PRGLILFDWLRQ RI VWDELOL]HG ROLJRQXFOHRWLGHVf UHVHDUFK LQ XVLQJ ROLJRQXFOHRWLGHV IRU ELRORJLFDO VWXGLHV ZDV OLPLWHG XQWLO WKH ODWH V )ROORZLQJ DGYDQFHV LQ ROLJRQXFOHRWLGH FKHPLVWU\ DQG LQLWLDO ELRORJLFDO VWXGLHV $JUDZDO HW DO *DR HW DO 6PLWK HW DO f LQWHUHVW LQ ROLJRQXFOHRWLGH WKHUDS\ EHJDQ WR LQFUHDVH 2OLJRQXFOHRWLGHV VKRUW QXFOHRWLGH SRO\PHU LQ OHQJWK WR PHUf RI D V\QWKHWLF VLQJOHVWUDQGHG QXFOHLF DFLG DUH GHVLJQHG WR D VSHFLILF JHQH &RRQH\ HW DO 0RVHU t 'HUYDQ f P51$ 6WHLQ t &RKHQ f RU SURWHLQ %RFN HW DO (OOLQJWRQ f $IWHU ELQGLQJ WR WKHLU WDUJHW LQ FHOOV ROLJRQXFOHRWLGHV FDQ SUHYHQW WKH SURGXFWLRQ RI D VSHFLILF SURWHLQ SURGXFW )LJXUH f 'HWDLOHG PHFKDQLVPV DUH H[SODLQHG LQ

PAGE 13

2OLJRQXFOHRWLGH 6LWH RI $FWLRQ )LJXUH $ GHSLFWLRQ RI SURWHLQ V\QWKHVLV DQG SRVVLEOH VLWHV RI DFWLRQ IRU GHVLJQHG ROLJRQXFOHRWLGHV 7KHUH DUH WKUHH SRWHQWLDO VLWHV ZKHUH ROLJRQXFOHRWLGHV FDQ KDYH DFWLRQV )LUVW ROLJRQXFOHRWLGHV DQWLJHQHVf FDQ EH XVHG WR LQKLELW WKH WUDQVFULSWLRQ SURFHVV IURP GRXEOH VWUDQGHG '1$ WR VLQJOH VWUDQGHG P51$ WKURXJK +RRJVWHHQ EDVH SDLULQJ LQWHUDFWLRQV 6HFRQG FRPSOLPHQWDU\ ROLJRQXFOHRWLGHV DQWLVHQVH ROLJRQXFOHRWLGHVf FDQ EH GHVLJQHG WR ELQG ZLWK P51$ WR UHVWUDLQ WKH WUDQVODWLRQ SURFHVV WKURXJK :DWVRQ&ULFN K\GURJHQ ERQG LQWHUDFWLRQV )LQDOO\ ROLJRQXFOHRWLGHV DSWDPHUVf FDQ LQWHUDFW ZLWK D V\QWKHVL]HG SURWHLQ WR LQWHUIHUH ZLWK LWV DFWLYLW\ YLD K\GURJHQ ERQGLQJV

PAGE 14

H[FHOOHQW UHYLHZ DUWLFOHV &URRNH +HOHQH t 7RXOPH 6FDQORQ HW DO 6KDUPD t 1DUD\DQDQ 8KOPDQ t 3H\PDQ f 6LPSO\ D WULSOH[ IRUPLQJ ROLJRQXFOHRWLGH DQWLJHQH ROLJRQXFOHRWLGHf LV FDSDEOH RI ELQGLQJ WR WKH PDMRU JURRYH LQ GRXEOHVWUDQGHG '1$ YLD +RRJVWHHQ EDVH LQWHUDFWLRQV WKHUHE\ FDXVLQJ D WULSOH KHOLFDO VWUXFWXUH DQG IXUWKHU UHVXOWLQJ LQ VHTXHQFHVSHFLILF LQKLELWLRQ RI WUDQVFULSWLRQ 2Q WKH FRQWUDU\ DQ DQWLVHQVH ROLJRQXFOHRWLGH FRPSOHPHQWDU\ WR D VSHFLILF VHTXHQFH RI P51$ FDQ K\EULGL]H WR D JLYHQ P51$ WKURXJK :DWVRQ&ULFN K\GURJHQ ERQGV =DPHFQLN f ,W FDQ LQKLELW WUDQVODWLRQ E\ VHYHUDO SURSRVHG PHFKDQLVPV LQFOXGLQJ DFWLYDWLRQ RI 51DVH + DQG EORFNDGH RI ULERVRPDO UHDGLQJ 51DVH + LV DQ HQGRJHQRXV FHOOXODU HQ]\PH ZKLFK FDQ UHFRJQL]H D K\EULG GXSOH[ EHWZHHQ '1$ DQG 51$ *KRVK HW DO f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

PAGE 15

7R[LF (IIHFWV RI 2OLJRQXFOHRWLGH +LJK GRVHV RI ROLJRQXFOHRWLGHV KDYH EHHQ UHSRUWHG KDUPIXO LQ DQLPDO VWXGLHV DQG WKH WR[LFLW\ RI ROLJRQXFOHRWLGHV DSSHDUV WR EH VSHFLHV GHSHQGHQW $GPLQLVWUDWLRQ RI PJNJ LS WKUHH WLPHV ZHHNO\ IRU WZR ZHHNV LQ PLFH DQG UDWV UHVXOWHG LQ VLJQLILFDQW WR[LFLW\ LQFOXGLQJ DFXWH UHQDO IDLOXUH OLYHU GDPDJH VSOHHQ GDPDJH LPPXQH VWLPXODWLRQ VHYHUH WKURPERF\WRSDHQLD DQG GHDWK .ULHJ HW DO 6DUPLHQWR HW DO f %ROXV LY DGPLQLVWUDWLRQ RI ROLJRQXFOHRWLGHV LQ PRQNH\V SURGXFHG D WUDQVLHQW GHFUHDVH LQ SHULSKHUDO WRWDO ZKLWH EORRG FHOO QHXWURSKLO FRXQWV SURORQJDWLRQ RI DFLWYDWHG SDUWLDO WKURPERSODVWLQ WLPH K\SRWHQVLRQ DQG GHDWK *DOEUDLWK HW DO f $V D UHVXOW RI WKH UHODWLYHO\ ORQJ UHWHQWLRQ WLPH LQ WKH UHWLFXORHQGRWKHOLDO V\VWHP RUJDQ DFFXPXODWLRQ RI ROLJRQXFOHRWLGHV DQG WKHLU PHWDEROLWHV PD\ EH UHVSRQVLEOH IRU WKHVH WR[LFLWLHV =KDQJ HW DO f %DUULHUV WR 2OLJRQXFOHRWLGH 7UDQVIHU DQG $FWLYLW\ 7KH WKHUDSHXWLF SURPLVH RI VSHFLILF ROLJRQXFOHRWLGH LQWHUDFWLRQ LV JUHDW +RZHYHU VHYHUDO WHFKQLFDO SUREOHPV LQFOXGLQJ VWDELOLW\ DQG GHOLYHU\ PXVW EH RYHUFRPH EHIRUH ROLJRQXFOHRWLGHV FDQ EH XVHIXO GUXJV ,Q 9LWUR,Q 9LYR 6WDELOLW\ 2I DOO WKH SRVVLEOH REVWDFOHV UDSLG GHJUDGDWLRQ RI XQPRGLILHG '1$ DQG 51$ SKRVSKRURGLHVWHU ROLJRQXFOHRWLGHV LQ WKH ELRORJLFDO PLOLHX LV WKH ILUVW SUREOHP HQFRXQWHUHG $NKWDU HW DO 6KDZ HW DO f (Q]\PHV QRQVSHFLILF HQGR DQG

PAGE 16

H[RQXFOHDVHV OLPLW SKRVSKRURGLHVWHU ROLJRQXFOHRWLGHVf SK\VLRORJLFDO KDOIOLIH WR D IHZ PLQXWHV $NKWDU HW DO f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f f ,QWHPXFOHRWLGH SKRVSKRGLHVWHU EULGJH f %DVH JURXS f 6XJDU JURXS %DVHG XSRQ WKH DERYH FULWHULD D QXPEHU RI VWUXFWXUDO DQDORJXHV ZLWK QXFOHDVH UHVLVWDQFH KDYH EHHQ GHYHORSHG LQFOXGLQJ SKRVSKRURWKLRDWH &RQQROO\ HW DK &RZVHUW HW DK f DQG PHWK\O SKRVSKRQDWH %ODNH HW DK 0XUDNDPL HW DK f 2I WKHVH PRGLILHG ROLJRQXFOHRWLGHV SKRVSKRURWKLRDWH ROLJRQXFOHRWLGHV DUH SRVVLEO\ WKH PRVW SRWHQW EHFDXVH WKH\ DUH KLJKO\ UHVLVWDQW WR QXFOHDVHV UHWDLQ D QHW FKDUJH DUH VROXEOH LQ ZDWHU DQG FDQ DFW DV VXEVWUDWHV IRU 51DVH + +RZHYHU SKRVSKRURWKLRDWH ROLJRQXFOHRWLGHV PD\ DOVR FDXVH D YDULHW\ RI QRQVHTXHQFH GHSHQGHQW HIIHFWV *XYDNRYD HW DK .KDOHG HW DK 3HUH] HW DK f

PAGE 17

&HOOXODU 7UDQVSRUW $QRWKHU PDMRU HQFXPEUDQFH WR WKH WKHUDSHXWLF XVH RI ROLJRQXFOHRWLGHV LV WKH LQHIILFLHQW GHOLYHU\ RI ROLJRQXFOHRWLGHV WR WKH F\WRSODVP RU QXFOHXV 7KHUH DUH WZR WUDQVSRUW DVSHFWV WKDW QHHG WR EH GLVWLQJXLVKHG f &HOOXODU XSWDNH f (QWU\ LQWR WKH F\WRSODVPQXFOHXV &HOOXODU XSWDNH UHIHUV WR ERWK ROLJRQXFOHRWLGH PHPEUDQH ELQGLQJ DQG JHQHUDO LQWHUQDOL]DWLRQ ZLWKLQ WKH FHOO (QWU\ LQWR WKH F\WRSODVPQXFOHXV FRQFHUQV WKH DPRXQW RI ROLJRQXFOHRWLGHV WKDW UHDFK D SKDUPDFRORJLFDO DFWLYH FRPSDUWPHQW 2OLJRQXFOHRWLGHV LQWHUQDOL]DWLRQ E\ FXOWXUHG FHOOV LV LQHIILFLHQW $NKWDU HW DO 6WHLQ t &KHQJ f 2QO\ D VPDOO IUDFWLRQ RI DGGHG ROLJRQXFOHRWLGHV FDQ DFWXDOO\ JDLQ HQWU\ LQWR FHOOV DQG LW LV FRPPRQO\ DVVXPHG WKDW PRVW ROLJRQXFOHRWLGHV FDQ EH EURXJKW LQWR FHOOV WKURXJK UHFHSWRU PHGLDWHG DGVRUSWLYH RU IOXLG SKDVHf HQGRF\WRVLV $NKWDU t -XOLDQR /RNH HW DK f $IWHU HQWU\ LQWR FHOOV ROLJRQXFOHRWLGHV PXVW SHQHWUDWH WKH HQGRVRPDO PHPEUDQH WR H[HUW WKHLU HIIHFWV LQ WKH QXFOHXV RU F\WRSODVP 1RW DOO RI WKH LQWHUQDOL]HG ROLJRQXFOHRWLGHV DUH QHFHVVDULO\ DYDLODEOH WR LQWHUDFW ZLWK LQWHQGHG VXEFHOOXODU WDUJHWV ,QGHHG PRVW RI WKHP DUH HOLPLQDWHG E\ O\VRVRPHV WKH ODWHU HQGRF\WRWLF VWDJH )LJXUH f 8QOLNH JHQH GHOLYHU\ KRZHYHU IROORZLQJ FHOOXODU HQWU\ DQG HVFDSH IURP HQGRVRPDO FRPSDUWPHQWV ZLWK DQ HIIHFWLYH QXFOHDU SRUH VL]H RI DSSUR[LPDWHO\ QP LQ GLDPHWHU $URQVRKQ t +XJKHV f ROLJRQXFOHRWLGHV DUH DEOH WR PLJUDWH WR WKH QXFOHXV ZLWKRXW GLIILFXOW\ &KLQ HW DK /HRQHWWL HW DK f $Q LVVXH WKDW QHHGV WR EH DGGUHVVHG LV

PAGE 18

/\VRVRPH 2OLJRQXFOHRWLGH (OLPLQDWLRQ 2OLJRQXFOHRWLGH /LSRVRPH2OLJRQXFOHRWLGH )LJXUH 3RVVLEOH ROLJRQXFOHRWLGH IDWHV LQ D FHOO IRU WZR GHOLYHU\ V\VWHPV Df /LPLWHG DPRXQW RI ROLJRQXFOHRWLGHV FDQ EH WDNHQ LQWR FHOOV ZKHQ QRW XVHG ZLWK DQ\ GHOLYHU\ V\VWHP )RU WKRVH ROLJRQXFOHRWLGHV WKDW FDQ EH EURXJKW LQWR FHOOV WKH PHFKDQLVP LV PRVWO\ WKURXJK HQGRF\WRVLV DORQJ ZLWK VXEFHOOXODU FRPSDUWPHQWV LH HQGRVPH DQG O\VRVRPHf RI D S+JUDGLHQW SURILOH 0RVW RI WKH HQGRF\WRVHG ROLJRQXFOHRWLGHV ZRXOG WKHQ EH HOLPLQDWHG Ef :KHQ XVLQJ D GHOLYHU\ V\VWHP HJ OLSRVRPHf PRUH ROLJRQXFOHRWLGHV FDQ EH EURXJKW LQWR FHOOV WKHUHE\ LQFUHDVLQJ WKHLU SUREDELOLW\ RI HVFDSLQJ IURP O\VRVRPHV 6WLOO PRVW RI WKH ROLJRQXFOHRWLGHV ZRXOG EH HOLPLQDWHG WKURXJK WKH HQWLUH HQGRF\WRVLV SURFHVV

PAGE 19

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f FRPSOH[DWLRQ RI ROLJRQXFOHRWLGHV ZLWK FDWLRQLF PROHFXOHV LH FRPSOH[LQJ DJHQWVf HQFDSVXODWLRQ RI ROLJRQXFOHRWLGHV LQWR YHVLFOHV LH HQFDSVXODWLQJ DJHQWVf DQG ODEHOLQJ WDUJHWV WR HLWKHU ROLJRQXFOHRWLGHV RU WKHLU

PAGE 20

GHOLYHU\ FDUULHUV LH WDUJHWLQJ DJHQWVf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f 7KH ELRORJLFDO DFWLYLW\ DEOH WR EH REVHUYHG UHVXOWV IURP D GLPLQXWLYH DPRXQW RI ROLJRQXFOHRWLGHV WKDW HVFDSH IURP WKH HQGRVRPDO FRPSDUWPHQWV 7R IXUWKHU RSWLPL]H ROLJRQXFOHRWLGH GHOLYHU\ HQGRVPH GHVWDELOL]LQJ HVFDSLQJf V\VWHPV KDYH EHHQ GHYHORSHG 7KLV JURXS DSSOLHV GHYLFHV LH ROLJRQXFOHRWLGH F\WRSODVPLF WUDQVIHU WHFKQLTXHVf RU RIIHUV GHOLYHU\ V\VWHPV LH PHPEUDQH GHVWDELOL]LQJ DJHQWVf WKDW LPSURYH ROLJRQXFOHRWLGH HIIOX[ WR WKH F\WRSODVP &RQMXJDWLQJ $JHQWV (PSKDVLV RQ WKH DELOLW\ RI ROLJRQXFOHRWLGHV WR SHQHWUDWH ELRORJLFDO PHPEUDQHV LV RQH RI WKH PDMRU HOHPHQWV LQ PDNLQJ ROLJRQXFOHRWLGH WKHUDS\ SRVVLEOH $Q VWUDWHJ\ LV WR FRQMXJDWH K\GURSKRELF DQFKRU JURXSV DW HLWKHU HQG RI WKH ROLJRQXFOHRWLGH WKURXJK FKHPLFDO UHDFWLRQV WR H[WHQG WKHLU K\GURSKRELFLW\ DQGRU H[RQXFOHDVH UHVLVWDQFH WKHUHE\ LQFUHDVLQJ WKH LQWHUDFWLRQ ZLWK WDUJHW FHOOV

PAGE 21

&KROHVWHURO LV D W\SLFDO FRQMXJDWLQJ DJHQW WKDW KDV EHHQ XVHG DV D K\GURSKRELF DQFKRU JURXS DW HLWKHU WKH f RU f WHUPLQXV RI ROLJRQXFOHRWLGHV $ODKDUL HW DO %RXWRULQ HW DK *RGDUG HW DK /HWVLQJHU HW DK f $ON\O VLGH FKDLQV DUH DQRWKHU FRPPRQO\ XVHG FRQMXJDWLQJ DJHQW ([DPSOHV LQFOXGH KH[DGHF\O PRLHWLHV DIIL[HG WR WKH fHQG 6KHD HW DK f GRGHF\O PRLHWLHV WR WKH fHQG 6DLVRQ%HKPRDUDV HW DK f KH[DQRO WR WKH fHQG *DPSHU HW DK f DPLQRKH[\O WR WKH fHQG *DPSHU HW DK f DQG DQ XQGHF\O GHULYDWLYH WR WKH fHQG RI ROLJRQXFOHRWLGHV .DEDQRY HW DK f 3RO\/O\VLQHf LV DQRWKHU W\SH RI FRQMXJDWLQJ DJHQW %\ DWWDFKLQJ ROLJRQXFOHRWLGHV WR SRO\/O\VLQHf DW WKH fHQG 'HJROV HW DK 'HJROV HW DK /HPDLWUH HW DK /HRQHWWL HW DO 6WHYHQVRQ t ,YHUVHQ f FHOOXODU XSWDNH LV LQFUHDVHG PRVW OLNHO\ GXH WR D EHWWHU LQWHUDFWLRQ ZLWK WKH QHJDWLYH FKDUJH FHOOXODU PHPEUDQH ,Q DGGLWLRQ WR WKH SRVVLEOH SHUPHDELOLW\ PHFKDQLVP WKH ELRORJLFDO HIIHFW LPSURYHG E\ SRO\/O\VLQHf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ff FDQ DFFRXQW IRU LQFUHDVHG F\WRWR[LF HIIHFWV

PAGE 22

&RPSOH[LQJ $JHQWV 8QOLNH FRQMXJDWLQJ DJHQWV WKH EDVLF SULQFLSOH EHKLQG WKH XVH RI FRPSOH[LQJ DJHQWV LV WR ELQG ROLJRQXFOHRWLGHV WR WKHLU FDUULHU LQ D VWURQJ EXW QRQFRYDOHQW PDQQHU EDVHG XSRQ DQ HOHFWURVWDWLF DWWUDFWLRQ 7KLV V\VWHP FDUULHV PRUH ROLJRQXFOHRWLGHV LQWR FHOOV WKURXJK HQGRF\WRVLV DQG KHQFH LQFUHDVHV WKHLU SUREDELOLW\ RI UHDFKLQJ WKH F\WRSODVP &DWLRQLF SRO\PHUV VXFK DV SRO\/O\VLQHf 'HVKSDQGH HW DK *LQREEL HW DK 6WHZDUW HW DK f SRO\HWK\OHQLPLQH %RXVVLI HW DK f SRO\DPLGRDPLQH 3$0$0 VWDUEXUVW GHQGULPHUV %LHOLQVND HW DK 'HORQJ HW DO +XJKHV HW DK .XNRZVND/DWDOOR HW DK 3R[RQ HW DK f DYLGLQ 3DUGULGJH t %RDGR f SRO\LVRKH[\OF\DQRDFU\ODWH QDQRSDUWLFOHV &KDYDQ\ HW DK f DQG SRO\DON\OF\DQRDFU\ODWH QDQRSDUWLFOHV &KDYDQ\ HW DK *RGDUG HW DK 6FKZDE HW DK f DUH VRPH FRPOH[LQJ DJHQWV WKDW KDYH EHHQ XVHG LQ ROLJRQXFOHRWLGH GHOLYHU\ &DWLRQLF OLSRVRPHV DUH RWKHU FRPSOH[LQJ DJHQWV WKDW KDYH EHHQ LQYHVWLJDWHG /LSRVRPHV DUH YHVLFOHV FRPSULVHG RI OLSLG ELOD\HUVf VLPLODU LQ VWUXFWXUH WR ELRORJLFDO PHPEUDQHV 8WLOL]LQJ WKHLU YHUVDWLOLW\ HJ VL]H FKDUJH DQG FRPSRVLWLRQf DQG VHYHUDO DGYDQWDJHV HJ HFRQRPLFDO DELOLW\ WR DWWDFK FKHPLFDOV WR WKHLU VXUIDFH DQG HDVLO\ SURGXFHGf GLIIHUHQW V\VWHPV LQYROYLQJ OLSRVRPHV FDQ EH DSSOLHG WR LQFUHDVH WKH GHOLYHU\ RI ROLJRQXFOHRWLGHV WR WKHLU VLWHV RI DFWLRQ &DWLRQLF OLSRVRPHV DUH DPRQJ RQH RI WKHVH VWUDWHJLHV &DWLRQLF OLSRVRPHV WKDW KDYH LPSURYHG ROLJRQXFOHRWLGH FHOOXODU GHOLYHU\ LQFOXGH 1>OGLROHROR[\fSURS\O@111WULPHWK\ODPPRQLXP FKORULGH '270$f %HQQHWW HW DK +XJKHV HW DK .RQRSND HW DK 3HUODN\ HW DK 6DLMR HW DK f 1>OOGLROHROR[\fSURS\O@111WULPHWK\ODPPRQLXP

PAGE 23

PHWK\OVXOIDWH '27$3f &DSDFFLROL HW DO /DSSDODLQHQ HW DO /LDQJ t +XJKHV 4XDWWURQH HW DO 7DNOH HW DO =HOSKDWL t 6]RND Df >11f1GLPHWK\ODPLQRHWKDQHfFDUEDPR\O@FKROHVWHURO '&&KROf /LW]LQJHU HW DO f VSHUPLQHFKROHVWHURO *X\ &DIIH\ HW DO f VSHUPLGLQHFKROHVWHURO *X\ &DIIH\ HW DO f GLROHR\OR[\1>VSHUPLQHFDUER[DPLGRfHWK\O@11GLPHWK\OO SURSDQDPLQXP WULIOXRURDFHWDWH '263$f /DSSDODLQHQ HW DO /DSSDODLQHQ HW DO f OGLP\ULVW\OR[\SURS\OGLPHWK\OK\GUR[\HWK\O DPPRQLXP EURPLGH '05,(f .RQRSND HW DO f DQG GLPHWK\OGLRFWDGHF\ODPPRQLXP EURPLGH ''$%f -DDVNHODLQHQ HW DO 2OOLNDLQHQ HW DO 5RVH HW DO f ,Q FRQWUDVW WR FRQMXJDWLQJ DJHQWV WKH HDVH RI SURGXFWLRQ RI WKH FRPSOH[LQJ DJHQWV LV WKH ELJJHVW DGYDQWDJH 1R FKHPLFDO OLQNDJH EHWZHHQ ROLJRQXFOHRWLGHV DQG FRPSOH[LQJ DJHQWV LV UHTXLUHG ,Q DGGLWLRQ WKH\ SURYLGH KLJK FDSDFLW\ WR UHWDLQ ROLJRQXFOHRWLGHV &RPSOH[LQJ DJHQWV PD\ DOVR SUHYHQW ROLJRQXFOHRWLGHV IURP HQ]\PDWLF GHJUDGDWLRQ E\ IRUPLQJ SRRU VXEVWUDWHV *HUVKRQ HW DO f +RZHYHU D PDMRU FRQFHUQ LQ XVLQJ FRPSOH[LQJ DJHQWV LV WKHLU SRVVLEOH WR[LF HIIHFWV &DWLRQLF SRO\PHUV DQG FDWLRQLF OLSRVRPHV HYHQWXDOO\ DUH PRUH WR[LF WR FHOOV WKDQ QHXWUDO FRXQWHUSDUWV DV WKHLU FRQFHQWUDWLRQV DUH LQFUHDVHG %DUU\ HW DO &ODUHQF HW DO :DJQHU HW DO
PAGE 24

(QFDSVXODWLQJ $JHQWV (QFDSVXODWLQJ DQG FRPSOH[LQJ DJHQWV DUH SRVVLEO\ WKH PRVW SRSXODU V\VWHPV XVHG LQ WKH GHOLYHU\ RI ROLJRQXFOHRWLGHV 1RW RQO\ GR ERWK PHWKRGV SURWHFW ROLJRQXFOHRWLGHV IURP GHJUDGDWLRQ /HRQHWWL HW DO 6FKZDE HW DO 7KLHUU\ t 'ULWVFKLOR f EXW WKH\ DOVR LQFUHDVH FHOOXODU XSWDNH -XOLDQR t $NKWDU f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f $EXQGDQW H[DPSOHV XVLQJ OLSRVRPHV LOOXVWUDWH WKH LPSURYHG HIIHFW RI ROLJRQXFOHRWLGHV $NKWDU HW DO $QD]RGR HW DO +DWWD HW DO +DWWD HW DO 0DU]R HW DO 2JR HW DO :LHOER HW DO f )XUWKHUPRUH F\FORGH[WULQ DQDORJV LQFOXGLQJ K\GUR[\SURS\O EHWD F\FORGH[WULQ +DEXV HW DO =KDR HW DO f K\GUR[\HWK\O EHWDF\FORGH[WULQ =KDR HW DO f DQG HQFDSVLQ =KDR HW DO f KDYH DOVR EHHQ GHPRQVWUDWHG DV SRVVLEOH FDUULHU FDQGLGDWHV IRU ROLJRQXFOHRWLGH GHOLYHU\ 6LPLODU WR FRPSOH[LQJ DJHQWV WKH ELJJHVW DGYDQWDJH RI HQFDSVXODWLQJ DJHQWV LQ ROLJRQXFOHRWLGH GHOLYHU\ LV WKHLU HDVH RI SURGXFWLRQ 8QOLNH FRPSOH[LQJ DJHQWV HQFDSVXODWLQJ DJHQWV DUH EHOLHYHG WR EH OHVV F\WRWR[LF +RZHYHU FRPSDUHG WR

PAGE 25

FRPSOH[LQJ DJHQWV WKH\ KDYH D ORZHU FDSDFLW\ WR EULQJ ROLJRQXFOHRWLGHV LQWR FHOOV ZKLFK PD\ UHGXFH WKH HIILFLHQF\ RI WKH HQFDSVXODWLQJ DJHQW V\VWHP 7DUJHWLQJ $JHQWV 7DUJHWLQJ DJHQWV PD\ EH FDWHJRUL]HG LQWR WZR JURXSV 7KH ILUVW JURXS WDUJHWLQJ DW WKH QXFOHLF DFLG OHYHO LV NQRZQ DV LQWHUFDODWLQJ DJHQWV 7KHVH DJHQWV DUH PRVWO\ RIWHQ DWWDFKHG DW WKH f RU fHQG RI ROLJRQXFOHRWLGHV 7KH PRLHWLHV OLQNHG WR ROLJRQXFOHRWLGHV LQWHUDFW VWURQJO\ DQG QRQVSHFLILFDOO\ ZLWK QXFOHLF DFLGV $IWHU HQWHULQJ LQWR FHOOV DQG LQWHUDFWLQJ ZLWK WDUJHW QXFOHLF DFLGV WKH K\EULGV DUH VWDELOL]HG E\ WKH LQWHUFDODWLRQ RI WKH DJHQWV LQ WKH 51$'1$ GXSOH[ +HQFH WKH\ LQFUHDVH WKH DIILQLW\ RI ROLJRQXFOHRWLGHV WR WKHLU WDUJHWV 2I DOO WKH LQWHUFDODWLQJ DJHQWV DFULGLQH LV PRVW ZLGHO\ XVHG DQG LQYHVWLJDWHG DV D SRVVLEOH PHDQV WR LQFUHDVH WKH HIIHFW RI ROLJRQXFOHRWLGHV )XNXL t 7DQDND *ULJRULHY HW DO .O\VLN HW DO /DFRVWH HW DK 0F&RQQDXJKLH t -HQNLQV 6WHLQ HW DK 7RXOPH HW DK 9HUVSLHUHQ HW DK :DOWHU f 2WKHU H[DPLQHG LQWHUFDODWRUV DUH FKORUDPEXFLO %HORXVRY HW DK f EHQ]RS\ULGRTXLQR[DOLQH 0DUFKDQG HW DK 6LOYHU HW DK f EHQ]RS\ULGRLQGROH *LRYDQQDQJHOL HW DK 6LOYHU HW DK f EHQ]RSKHQDQWKULGLQH &KHQ HW DK f DQG SKHQD]LQLXP /HYLQD HW DK f 7KH VHFRQG JURXS RI WDUJHWLQJ DJHQWV LV DFFHVVHG E\ XWLOL]LQJ PRLHWLHV WKDW FDQ VHOHFWLYHO\ DQG VSHFLILFDOO\ WUDQVSRUW ROLJRQXFOHRWLGHV WR D WDUJHW FHOO SRSXODWLRQ 7KHUHIRUH WKHLU DFFXPXODWLRQ LQ LQWUDFHOOXODU FRPSDUWPHQWV LV LQFUHDVHG 7KH PRLHWLHV

PAGE 26

FDQ EH HLWKHU FRQMXJDWHG WR ROLJRQXFOHRWLGHV RU DWWDFKHG WR D FDUULHU V\VWHP HJ SRO\/ O\VLQHf RU OLSRVRPHVf OLQNHG WR WKH ROLJRQXFOHRWLGHV )RU FHOOV WKDW H[SUHVV WKH FKDUDFWHULVWLFV RI UHFHSWRU PHGLDWHG HQGRF\WRVLV OLJDQGV UHSUHVHQW JRRG FDQGLGDWHV DV WDUJHWLQJ DJHQWV WR LQLWLDWH FHOOXODU XSWDNH RI ROLJRQXFOHRWLGHV *O\FRSURWHLQV DQG QHRJO\FRSURWHLQV EHDULQJ DQ DSSURSULDWH VXJDU UHVLGXH VSHFLILFDOO\ DWWDFK WR VXJDU ELQGLQJ UHFHSWRUV 6KDURQ t /LV f %\ ODEHOLQJ ROLJRQXFOHRWLGHV DW WKH fHQG WR WKH QHRJO\FRSURWHLQ SKRVSKRPDQQRV\ODWHG JO\FRSURWHLQf DQ LPSURYHG HIIHFW ZDV REVHUYHG %RQILOV HW DO f 6LPLODUO\ DVLDORRURVRPXFRLG %XQQHOO HW DO :X t :X f RU PDQQRV\ODWHG JO\FRSURWHLQ /LDQJ HW DO f FRQMXJDWHG WR SRO\/O\VLQHf KDV EHHQ HPSOR\HG WR WDUJHW DQG HQKDQFH FHOOXODU XSWDNH RI ROLJRQXFOHRWLGHV 6LQFH PDOLJQDQW FHOOV DUH FRUUHODWHG ZLWK DQ LQFUHDVHG QHHG IRU HVVHQWLDO QXWULHQWV HJ IROLF DFLG DQG WUDQVIHUULQf UHODWLYH WR EHQLJQ FHOOV WKHVH QXWULHQWV FDQ EH XVHG DV SRWHQWLDO FDQGLGDWHV WR WDUJHW ROLJRQXFOHRWLGHV LQ WKH LQKLELWLRQ RI FDQFHURXV FHOO JURZWK )XUWKHU LPSURYHG ROLJRQXFOHRWLGH FHOOXODU XSWDNH LV VHHQ ZKHQ IROLF DFLG &LWUR HW DO *LQREEL HW DO f HSLGHUPDO JURZWK IDFWRU 'HVKSDQGH HW DO f DQG WUDQVIHUULQ &LWUR HW DO f LV OLQNHG WR SRO\/O\VLQHf /LSRVRPHV FRDWHG ZLWK PDOH\ODWHG ERYLQH VHUXP DOEXPLQ &KDXGKXUL f IROLF DFLG :DQJ HW DO f RU IHUULF SURWRSRUSK\ULQ ,; 7DONH HW DO f KDYH EHHQ VKRZQ WR LQFUHDVH WKH FHOOXODU XSWDNH RI ROLJRQXFOHRWLGHV ,Q RUGHU WR LQFUHDVH WKH VSHFLILFLW\ RI ROLJRQXFOHRWLGHV OLSRVRPHV FDQ DOVR EH DWWDFKHG WR DQWLERGLHV WR UHDFK WKH GHVLUHG WDUJHWV 6HYHUDO PRQRFORQDO DQWLERG\WDUJHWHG OLSRVRPHV LPPXQROLSRVRPHV KDYH EHHQ GHYHORSHG DQG XVHG IRU PHGLDWLQJ

PAGE 27

ROLJRQXFOHRWLGHV WR VSHFLILF UHFHSWRUV RQ WDUJHWHG FHOOV /HIHEYUHGf+HOOHQFRXUW HW DO /HRQHWWL HW DO /RNH HW DO 0D t :HL 5HQQHLVHQ HW DO 6HOYDP HW DO =HOSKDWL HW DO =HOSKDWL HW DO f 7KH PDMRU DGYDQWDJH RI WDUJHWLQJ DJHQWV LV WR HQKDQFH ROLJRQXFOHRWLGH FHOOXODU XSWDNH VSHFLILFDOO\ 7KH WDUJHWLQJ VWUDWHJ\ FDQ EH LQFRUSRUDWHG ZLWK RWKHU V\VWHPV WR IXUWKHU LQFUHDVH WKH FHOOXODU ELRORJLFDO DFWLYLW\ RI ROLJRQXFOHRWLGHV 6LPLODU WR FRQMXJDWLQJ DJHQWV D GLVDGYDQWDJHV WKDW PD\ KDPSHU WKH GHYHORSPHQW RI WDUJHWLQJ DJHQWV LV WKH V\QWKHWLF OLQNLQJ SURFHVV )XUWKHUPRUH WDUJHWLQJ SDUWLFXODU URXWHV RI HQGRF\WRVLV LV VWURQJO\ GHSHQGHQW XSRQ UHFHSWRU VXEW\SH WKHUHE\ OLPLWLQJ WKH XVH RI WDUJHWLQJ DJHQWV 2OLJRQXFOHRWLGH &\WRSODVPLF 7UDQVIHU 7HFKQLTXHV (YHQ LI FHOOXODU XSWDNH RI ROLJRQXFOHRWLGHV WKURXJK D GHOLYHU\ V\VWHP ZDV LQFUHDVHG HVFDSH IURP HQGRVPH PXVW VWLOO EH DFFRPSOLVKHG 2QH ZD\ WR DYRLG WKLV EDUULHU LV WR WUDQVIHU WKHP GLUHFWO\ LQWR F\WRSODVP RU QXFOHXV 7KLV KDV EHHQ DFFRPSOLVKHG WKURXJK HOHFWURSRUDWLRQ %HUJDQ HW DO )ODQDJDQ t :DJQHU *ULIIH\ HW DO 6FKDDO HW DO f DQG PLFURLQMHFWLRQ %ORQGHO HW DO )HQVWHU HW DO /DPSUHFKW HW DO 2f.HHIH HW DO 6DJDWD HW DO f (OHFWURSRUDWLRQ LQYROYHV GHOLYHULQJ D KLJKYROWDJH SXOVH RI D GHILQHG PDJQLWXGH DQG OHQJWK WR WKH ROLJRQXFOHRWLGHFHOO V\VWHP 7KH PHPEUDQH VWUXFWXUHV RI WKH FHOOV DUH ORRVHQHG DQG ROLJRQXFOHRWLGHV FDQ EH LQWURGXFHG GLUHFWO\ LQWR WKH FHOOfV F\WRSODVP 2Q WKH RWKHU KDQG PLFURLQMHFWLRQ ZDV SHUIRUPHG E\ LQMHFWLQJ ROLJRQXFOHRWLGHV GLUHFWO\ LQWR WKH QXFOHXV

PAGE 28

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f WR SURPRWH HIIOX[ RI ROLJRQXFOHRWLGHV IURP WKH HQGRVRPDO FRPSDUWPHQW %HQW] HW DO &RQQRU HW DK 'X]JXQHV HW DK f )XVRJHQLF OLSLGV LQFOXGH SKRVSKDWLG\OHWKDQRODPLQH 3(f GHULYDWLYHV ZKLOH S+VHQVLWLYH OLSLGV WKDW KDYH WLWUDWDEOH FDUER[\OLF DFLGV FRQWDLQ ROHLF DFLG &ULVWLQD 'H 2OLYHLUD HW DK 0D t :HL 5RSHUW HW DK 5RSHUW HW DK 5RSHUW HW DK f DQG FKROHVWHU\O KHPLVXFFLQDWH &KX HW DK 6OHSXVKNLQ HW DK f $ IXVRJHQLF OLSLG LV DEOH WR IRUP KH[DJRQDO ,, SKDVH WKDW LQIOXHQFHV PHPEUDQH IXVLRQ DQG ROLJRQXFOHRWLGH UHOHDVH %HIRUH WKH GLVUXSWLRQ RI WKH HQGRVRPDO PHPEUDQH RFFXUV LQVLGH WKH FHOOV KRZHYHU OLSRVRPHV PXVW PDLQWDLQ WKHLU LQWHJULW\ WR HQFDSVXODWH ROLJRQXFOHRWLGHV $ S+VHQVLWLYH OLSLG LV WKHUHIRUH LQWURGXFHG LQWR WKH OLSRVRPDO PDWUL[ :LWK D FKHPLFDO VWUXFWXUH FRPSOHPHQWDU\ WR WKH KH[DJRQDO ,, SKDVH HJ

PAGE 29

GLROHR\OSKRVSKDWLG\OHWKDQRODPLQH '23(ff WKH S+VHQVLWLYH OLSLG ZLOO DVVLVW LQ UHWDLQLQJ WKH ELOD\HU YHVLFOH VWUXFWXUH RI WKH OLSRVRPHV DW DQ DONDOLQH S+ :KHQ WKH S+ GHFUHDVHV DV D UHVXOW RI WKH DFLGLILFDWLRQ RI WKH HQGRVPH WKH WLWUDWDEOH KHDG JURXS RI WKH S+ VHQVLWLYH OLSLG LV SURWRQDWHG +HQFH LW GHVWDELOL]HV WKH ELOD\HU VWUXFWXUH DQG 3( SURPRWHV PHPEUDQH IXVLRQ 'X]JXQHV HW DO f (YHQWXDOO\ ROLJRQXFOHRWLGHV DUH UHOHDVHG RXW RI WKH HQGRVRPHV $OVR FDWLRQLF OLSRVRPHV LH FRPSOH[LQJ DJHQWVf XVXDOO\ FRPSULVH D IXVRJHQLF OLSLG HJ '23(f DQG D FDWLRQLF OLSLG HJ GLROH\OR[\1 >VSHUPLQHFDUER[DPLGRfHWK\O@11GLPHWK\O SURSDQDPLQLXP '263$f 1>OO GLROHROR[\fSURS\O@111WULPHWK\ODPPRQLXP PHWK\OVXOIDWH '27$3f 1>O GLROHROR[\fSURS\O@111WULPHWK\ODPPRQLXP FKORULGH '270$f GLPHWK\OGLRFWDGHF\ODPPRQLXP EURPLGH ''$%f DQG S>11f1f GLPHWK\ODPLQRHWKDQHfFDUEDPR\O@FKROHVWHURO '&&KROff WR LPSURYH ROLJRQXFOHRWLGH GHOLYHU\ *X\ &DIIH\ HW DO /DSSDODLQHQ HW DO /DSSDODLQHQ HW DO /LW]LQJHU HW DO 2OOLNDLQHQ HW DO 7DNOH HW DO =DOSKDWL t 6]RND Ef ,Q DGGLWLRQ WR LQFUHDVLQJ FHOOXODU XSWDNH RI ROLJRQXFOHRWLGHV S+VHQVLWLYH OLSRVRPHV IXUWKHU LQFUHDVH WKH HQWU\ RI ROLJRQXFOHRWLGHV WR WKH F\WRSODVP +RZHYHU VLQFH ERWK S+VHQVLWLYH DQLRQLF OLSLGV DQG QXFOHLF DFLGV KDYH D QHJDWLYH FKDUJH WKH\ PD\ KDYH OLPLWHG FDSDFLW\ WR HQWUDS QXFOHLF DFLGV 9LUDO SHSWLGHV 2OLJRQXFOHRWLGHV LQFXEDWHG ZLWK RU FRXSOHG WR YLUDO SHSWLGHV GHULYHG IURP WKH KHPDJJOXWLQLQ HQYHORS SURWHLQ RI WKH ,QIOXHQ]D YLUXV %RQJDUW] HW DO +XJKHV HW

PAGE 30

DO
PAGE 31

'H 'XYH HW DO f EHDULQJ D K\GURSKRELF WDLO JURXS LV FODVVLILHG DV D O\VRVRPRWURSLF GHWHUJHQW )LUHVWRQH HW DO f $W DQ DONDOLQH HQYLURQPHQW WKH PROHFXOH LV SUHGRPLQDWHG E\ LWV K\GURSKRELF WDLO PDNLQJ LW VLPSO\ DQ RLO\ VXEVWDQFH ZLWK OLPLWHG VXUIDFH DFWLYH SURSHUWLHV 7KH QRQFKDUJHG O\VRVRPRWURSLF GHWHUJHQW FDQ EH SDVVLYHO\ GLIIXVHG DFURVV FHOOXODU PHPEUDQHV 'XH WR WKH ORZ LQWUDO\VRVRPDO S+ XVXDOO\ EHWZHHQ DQG 0F*UDZ t 0D[ILHOG f WKH GHWHUJHQW LV SURWRQDWHG DQG WUDSSHG RQFH LQVLGH O\VRVRPHV DOORZLQJ D FRQWLQXRXV JUDGLHQW IRU GUXJ HQWU\ 'HDQ HW DO )RUVWHU HW DO :LOVRQ f :KHQ DFFXPXODWLRQ RI WKH SURWRQDWHG IRUP RI WKH FRPSRXQG SURJUHVVHV WR D FHUWDLQ FRQFHQWUDWLRQ WKH PDWHULDO GLVUXSWV WKH O\VRVRPDO PHPEUDQH UHOHDVLQJ D YDULHW\ RI O\VRVRPDO HQ]\PHV LQWR WKH F\WRSODVP 2QFH UHOHDVHG ZLWKLQ WKH FHOOV WKHVH GLJHVWLYH HQ]\PHV DUH DEOH WR GHJUDGH FHOOXODU VWUXFWXUHV UHVXOWLQJ LQ FHOO GHDWK 0LOOHU HW DO :LOVRQ HW DO f &RQVHTXHQWO\ D VHULHV RI O\VRVRPRWURSLF GHWHUJHQWV ZHUH V\QWKHVL]HG DQG WHVWHG )LUHVWRQH HW DO D )LUHVWRQH HW DO )LUHVWRQH HW DO f 7KH GHYHORSPHQW RI O\VRVRPRWURSLF GHWHUJHQWV RULJLQDWHG DV D SRWHQWLDO ZD\ WR GHVWUR\ WXPRU FHOOV XQGHU WKH DVVXPSWLRQ WKDW WKH PDOLJQDQW FHOOV FDUU\ PRUH O\VRVRPHV WKDQ EHQLJQ FHOOV 7URXHW HW DO f $OWKRXJK ODWHU DEDQGRQHG GXH WR SUREOHPV ZLWK QRQVSHFLILF O\VRVRPDO FHOOXODU GHVWUXFWLRQ WKH WKHRU\ EHKLQG WKLV DSSURDFK KDV SURYLGHG WKH EDVLV IRU WKH GHYHORSPHQW RI ELRGHJUDGDEOH S+VHQVLWLYH VXUIDFWDQWV %36f 6LJQLILFDQFH RI %LRGHJUDGDEOH S+6HQVLWLYH 6XUIDFWDQWV 'XH WR WKH SRVVLEOH SLWIDOOV LQ QXFOHLF DFLG GHOLYHU\ WKH O\VRVRPRWURSLF GHWHUJHQWV ZHUH PRGLILHG DQG ELRGHJUDGDEOH S+VHQVLWLYH VXUIDFWDQWV %36f ZHUH GHYHORSHG WR

PAGE 32

LQGXFH HQGRVRPDO PHPEUDQH GHIHFWV 7KH QRYHOW\ RI WKH GHOLYHU\ V\VWHP VWHPV IURP WKH WZR IROORZLQJ UHDVRQV f ([SORLWDWLRQ RI D QDWXUDOO\ RFFXUULQJ ROLJRQXFOHRWLGHOLSRVRPH WUDQVSRUW PHFKDQLVP HQGRF\WRVLVf ZLWK WKH LQFRUSRUDWLRQ RI %36 LQWR WKH GHOLYHU\ V\VWHP HJ OLSRVRPHVf f %LRGHJUDGDEOH GUXJ DSSURDFK WR GHFUHDVH WR[LFLW\ 'HYHORSLQJ %36 WKDW FDQ EH DFWLYDWHG DW WKH HQGRVPH HDUO\ O\VRVRPHf VWDJH ZLOO HQDEOH WKH GHVWDELOL]DWLRQ RI WKH HQGRVRPDO PHPEUDQHV DQG OLEHUDWH ROLJRQXFOHRWLGHV WR WKHLU VLWHV RI DFWLRQ LQ WKH F\WRSODVP RU QXFOHXV )LJXUH f +RZHYHU XQOLNH O\VRVRPRWURSLF GHWHUJHQWV %36 PD\ EH FOHDYHG LQWR OHVV WR[LF PHWDEROLWHV E\ WKH HQGRJHQRXV GLJHVWLYH HQ]\PHV DIWHU EHLQJ UHOHDVHG LQWR WKH F\WRSODVP GXH WR WKHLU ELRGHJUDGDELOLW\ )XUWKHUPRUH %36 FDQ EH TXLFNO\ V\QWKHVL]HG E\ D RQH WR WZRVWHS VWDQGDUG FKHPLFDO UHDFWLRQ HJ HVWHULILFDWLRQ DQG VXEVWLWXWLRQf ZLWK FRPPHUFLDOO\ DYDLODEOH LQH[SHQVLYH VWDUWLQJ PDWHULDOV HJ GRGHFDQRO DQG LPLGD]ROHf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f

PAGE 33

2OLJRQXFOHRWLGH 5HOHDVH %36/LSRVRPH )LJXUH 3URSRVHG PHFKDQLVP RI WKH ELRGHJUDGDEOH S+VHQVLWLYH VXUIDFWDQWV %36f OLSRVRPH V\VWHP /LNH WKH UHJXODU OLSRVRPH V\VWHP VLPLODU DPRXQW RI ROLJRQXFOHRWLGHV ZLOO EH EURXJKW LQWR FHOOV $IWHU EHLQJ DFWLYDWHG GXULQJ HQGRF\WRVLV SURWRQDWHG %36 FDQ GHVWDELOL]H WKH HQGRVRPDO PHPEUDQH ZKLFK ZRXOG UHOHDVH WKH ROLJRQXFOHRWLGH LQWR WKH F\WRSODVP

PAGE 34

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f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f LQYHVWLJDWH WKH PHPEUDQH GHVWDELOL]DWLRQ DELOLW\ RI %36 DW YDULHG S+V GHWHUPLQH WKHLU VWDELOLW\ DQG VFUHHQ WKHLU F\WRWR[LFLW\

PAGE 35

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fOLSRVRPDO GHOLYHU\ V\VWHP +\SRWKHVLV 7KH K\SRWKHVLV ZDV WKDW ZKHQ XVLQJ OLSRVRPHV WR GHOLYHU ROLJRQXFOHRWLGHV LQ YLWUR WKH HIIHFW ZRXOG EH IXUWKHU HQKDQFHG LQ WKH SUHVHQFH RI %36 DV D FRPSRQHQW RI WKH OLSRVRPH FRPSRVLWLRQ 6SHFLILF $LP 0HFKDQLVP RI $FWLRQ ,QYHVWLJDWLRQ RI %LRGHJUDGDEOH S+6HQVLWLYH 6XUIDFWDQWV 2EMHFWLYH :LWK WKH GLIIHUHQW FKHPLFDO VWUXFWXUHV IURP RWKHU IXVRJHQLF FRPSRXQGV DQG VXUIDFWDQWV ELRGHJUDGDEOH S+VHQVLWLYH VXUIDFWDQWV %36f ZHUH H[SHFWHG WR KDYH GLVSDUDWH PHPEUDQH DFWLYLWLHV 7KH SRVVLEOH PHFKDQLVPV RI KRZ %36 FDXVHG PHPEUDQH GHIHFWV ZHUH WKHUHIRUH LQYHVWLJDWHG

PAGE 36

+\SRWKHVLV 7KH K\SRWKHVLV ZDV WKDW %36 FRXOG LQGXFH ERWK PHPEUDQH IXVLRQ DQG UXSWXUH DQG HYHQWXDOO\ UHOHDVH OLSRVRPDO FRQWHQWV LQ D S+GHSHQGHQW PDQQHU

PAGE 37

&+$37(5 '(6,*1 $1' 6<17+(6,6 2) %,2'(*5$'$%/( S+6(16,7,9( 685)$&7$176 ,QWURGXFWLRQ 7KH LGHD RI XVLQJ O\VRVRPRWURSLF GHWHUJHQWV WR JDLQ DFFHVV WR FHOOV H[SORLWV WKH IDFW WKDW VXUIDFWDQWV O\VH O\VRVRPDO PHPEUDQHV %LRGHJUDGDEOH S+VHQVLWLYH VXUIDFWDQWV %36f H[SDQG WKLV FRQFHSW E\ SURYLGLQJ D PHFKDQLVP WR FRQWURO WKH O\WLF SURSHUWLHV RI WKH VXUIDFWDQWV %\ JDWKHULQJ LQIRUPDWLRQ RI WKH SRWHQWLDO HIIHFW RQ ZKLFK HDFK LQGLYLGXDO FRPSRQHQW RI %36 KDV LPSDFW D UDWLRQDO DSSURDFK PD\ EH XQGHU WDNHQ ,W LV K\SRWKHVL]HG WKDW XVLQJ WKH FRUUHFW GHVLJQ DQG V\QWKHVLV RI D VHULHV RI RULJLQDO %36 WKH SRWHQF\ RI %36 FDQ EH SUHGLFWHG 7R EHJLQ WKLV SURMHFW WKUHH %36 GRGHF\O O LPLGD]RO\Of SURSLRQDWH ',3f PHWK\O LPLGD]RO\O ODXUHDWH 0,/f 1GRGHF\O LPLGD]ROH ',f ZHUH V\QWKHVL]HG XVLQJ VWDQGDUG ZHOO XQGHUVWRRG UHDFWLRQV HVWHULILFDWLRQ DQG VXEVWLWXWLRQf 5DWLRQDOH $ VXUIDFWDQW VXUIDFH DFWLYH DJHQWf LV D VXEVWDQFH WKDW DGVRUEV RQWR WKH VXUIDFHV RU LQWHUIDFHV RI D V\VWHP DQG DOWHUV WKH IUHH HQHUJLHV RI WKRVH VXUIDFHV RU LQWHUIDFHV WR D PDUNHG GHJUHH 5RVHQ f 6XUIDFH DFWLYH DJHQWV KDYH D FKDUDFWHULVWLF PROHFXODU VWUXFWXUH FRQVLVWLQJ RI D KHDG JURXS WKDW LV K\GURSKLOLF DQG D WDLO JURXS WKDW LV

PAGE 38

K\GURSKRELF WKXV PDNLQJ WKH FRPSRXQGV DPSKRWHULF 'HSHQGLQJ RQ WKH QXPEHU DQG QDWXUH RI WKH SRODU DQG QRQSRODU JURXSV SUHVHQW WKH DJHQWV FDQ EH GHVLJQHG WR EH PRUH K\GURSKLOLF RU OLSRSKLOLF ,Q RUGHU WR GHVLJQ ELRGHJUDGDEOH S+VHQVLWLYH VXUIDFWDQWV %36f WKHUH DUH WKUHH FULWLFDO UHTXLUHPHQWV RU VWUXFWXUDO FRPSRQHQWV )LJXUH f f 7KH ILUVW RQH LV D O\VRVRPRWURSLF DPLQH DV WKH KHDG JURXS RI %36 f 7R PDNH WKH O\VRVRPRWURSLF DJHQW DPSKRWHULF LW LV QHFHVVDU\ WR DWWDFK D K\GURFDUERQ FKDLQ WR WKH DPLQH DV WKH WDLO JURXS $W DQ DONDOLQH HQYLURQPHQW WKH S+VHQVLWLYH VXUIDFWDQW ZLOO EH XQLRQL]HG DQG OLSRSKLOLF :KHQ LQFRUSRUDWLQJ WKH VXUIDFWDQW LQWR OLSRVRPHV DV D GHOLYHU\ V\VWHP WKH OLSRSKLOLFLW\ RI WKH VXUIDFWDQW ZLOO HQKDQFH LWV FKDQFH WR UHPDLQ ZLWKLQ WKH OLSLG ELOD\HUV $IWHU WKH DPLQH LV SURWRQDWHG GXH WR D S+ JUDGLHQW HJ HQGRF\WRVLVf WKH S+VHQVLWLYH VXUIDFWDQW ZLOO LQFUHDVH LWV VXUIDFH DFWLYLW\ VLJQLILFDQWO\ 7KLV FKDQJH LV VWURQJ HQRXJK WR LQGXFH PHPEUDQH GHVWDELOL]DWLRQ f 7KH S+VHQVLWLYH VXUIDFWDQW ZRXOG WKHQ EH GHJUDGHG E\ WKH HQGRJHQRXV HQ]\PHV LQWR OHVV WR[LF PHWDEROLWHV ZLWK WKH LQWURGXFWLRQ RI D ELRGHJUDGDEOH FRQQHFWRU %\ XQGHUVWDQGLQJ WKH UHODWLRQVKLS DPRQJ WKH VSHFLILF FKDUDFWHULVWLF RI HDFK LQGLYLGXDO %36 FRPSRQHQW KHDG JURXS WDLO JURXS DQG OLQNDJH EULGJHf LW VKRXOG EH SRVVLEOH WR RSWLPL]H WKH GHVLJQ RI %36 +HDG *URXS 7KH KHDG JURXS RI ELRGHJUDGDEOH S+VHQVLWLYH VXUIDFWDQWV %36f LV WKH PDMRU IDFWRU GHWHUPLQLQJ LWV LRQL]DWLRQ FRQVWDQW S.Df FRQWUROOLQJ WKH DPRXQW RI DFWLYDWHG VXUIDFWDQW DW HQGRVRPDO S+ 7ZR LPSRUWDQW FULWHULD LQIOXHQFH WKH S.D

PAGE 39

$ /LSRSKLOLF +\GURFDUERQ &KDLQ $ S+ 6HQVLWLYH /\VRVRPRWURSLF $PLQH $Q (Q]\PDWLFDOO\ &OHDYDEOH &RQQHFWRU )LJXUH 7KUHH LQGLYLGXDO UHTXLUHPHQWV WR FUHDWH ELRGHJUDGDEOH S+VHQVLWLYH VXUIDFWDQWV %36f

PAGE 40

f 7\SH RI O\VRVRPRWURSLF DPLQHV f 3UHVHQFH RI VXEVWLWXHQWV RQ WKH DPLQH KHDG JURXSV 6LQFH LPLGD]ROH DQG PRUSKROLQH ZHUH XVHG DV WKH KHDG JURXSV LQ WKH ILUVW JHQHUDWLRQ O\VRVRPRWURSLF GHWHUJHQWV LQIRUPDWLRQ H[LVWV DERXW WKHLU FKHPLFDO SURSHUWLHV DQG ELRORJLFDO HIIHFWV 'H 'XYH HW DO f 2WKHU KHWHURF\FOLF ULQJ FRPSRXQGV VXFK DV LQGROH PD\ DOVR KDYH O\VRVRPRWURSLF SURSHUWLHV :KHQ FRPSDUHG WR PRUSKROLQH LPLGD]ROH KDV D KLJKHU S.D VLQFH WKH DURPDWLF ULQJ IRUPHG E\ WKH LPLGD]RO\O JURXS GHFUHDVHV WKH QXFOHRSKLOLFLW\ DQG WKH EDVLFLW\ RI WKH DPLQH +RZHYHU FRPSDUHG WR LQGROH LPLGD]ROH KDV KLJKHU EDVLFLW\ GXH WR LWV H[WUD HOHFWURQ ORQH SDLU 7DEOH f 7DEOH 3UHGLFWHG HIIHFW RQ S.D IURP WKH KHDG JURXS RI ELRGHJUDGDEOH S+VHQVLWLYH VXUIDFWDQWV %36f +HDG *URXS (IIHFW RQ S.D 9DOXH ,PLGD]ROH f§! 0RUSKROLQH WW ,QGROH $OO VXEVWLWXHQWV QHDU WKH WLWUDWDEOH DPLQH FDQ DIIHFW S.D (OHFWURQ GRQDWLQJ JURXSV JHQHUDOO\ LQFUHDVH S.D ZKLOH HOHFWURQ ZLWKGUDZLQJ JURXSV JHQHUDOO\ GHFUHDVH WKH S.D RI DQ DPLQH )LJXUH VKRZV WKH RUGHU RI WKH QXFOHRSKLOLFLW\ EDVLFLW\ DQG S.D IRU WKH VXEVWLWXHQWV DW f SRVLWLRQ RI WKH LPLGD]RO\O JURXS

PAGE 41

‘1 ;+ n&+ 1+ 2&+ 2+ DON\O 2 + ; %U &Of &1 &5n 12n )LJXUH 2UGHU RI QXFOHRSKLOLFLW\ EDVLFLW\ DQG S.D IRU GLIIHUHQW VXEVWLWXWHV RQ WKH KHDG JURXS RI ELRGHJUDGDEOH S+VHQVLWLYH VXUIDFWDQWV %36f

PAGE 42

7DLO *URXS 7KH K\GURFDUERQ FKDLQ RI ELRGHJUDGDEOH S+VHQVLWLYH VXUIDFWDQWV %36f SDUWLDOO\ GHWHUPLQHV WKHLU K\GURSKLOLFLWLHV 7KH VWURQJHU WKH LQWHUDFWLRQ EHWZHHQ WKH WDLO JURXSV WKH PRUH OLSRSKLOLF %36 $V WKH OLSRSKLOLFLW\ LQFUHDVHV D ORZHU FULWLFDO PLFHOOH FRQFHQWUDWLRQ &0&f ZLOO EH REVHUYHG )LJXUH JLYHV WKH RUGHU RI WKH H[SHFWHG &0& YDOXHV ZKHQ %36 KDYH WKH VDPH KHDG JURXS E\ FKDQJLQJ WKH WDLO JURXS /LQNDJH %ULGJH 7KH HIIHFWLYHQHVV RI ELRGHJUDGDEOH S+VHQVLWLYH VXUIDFWDQWV %36f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f WKH QXPEHU RI SRWHQWLDO %36 LV QXPHURXV ,Q WKH VXEVHTXHQW VWXGLHV KRZHYHU WKUHH %36 ZHUH VHOHFWHG V\QWKHVL]HG DQG FRPSDUHG

PAGE 43

! 5 Yn9::::::n )LJXUH %LRGHJUDGDEOH S+VHQVLWLYH VXUIDFWDQWV %36f ZLWK WKH VDPH KHDG JURXS EXW GLIIHUHQW WDLO JURXSV LQ RUGHU RI GHFUHDVLQJ K\GURSKLOLFLW\ DQG FULWLFDO PLFHOOH FRQFHQWUDWLRQ &0&f

PAGE 44

f 'RGHF\O O fLPLGD]RO\Of SURSLRQDWH ',3f f 0HWK\O LPLGD]RO\O ODXUHDWH 0,/f f 1GRGHF\O LPLGD]ROH ',f ',3 WKH ILUVW PHPEHU RI WKH %36 IDPLO\ ZDV RULJLQDOO\ GHYLVHG E\ +XJKHV DQG FRZRUNHUV f 0,/ ZDV GHVLJQHG WR FRPSDUH ZLWK ',3 ZKHQ WKH HVWHU OLQNHU EHWZHHQ KHDG DQG WDLO JURXSV LV SRVLWLRQHG LQWR WKH RWKHU GLUHFWLRQ ', ODFNLQJ D ELRGHJUDGDEOH FRQQHFWRU ZDV ORRVHO\ JURXSHG DV D %36 PHPEHU ', ZDV RULJLQDOO\ V\QWKHVL]HG E\ )LUHVWRQH DQG FRZRUNHUV f DQG FRPSDUHG WR RWKHU %36 WR DGGUHVV WKH LPSRUWDQFH RI WKH OLQNHU ZLWK UHVSHFW WR F\WRWR[LFLW\ 6\QWKHVLV RI %LRGHJUDGDEOH S+6HQVLWLYH 6XUIDFWDQWV &KHPLFDOV 11GLPHWK\OIRUPDPLGH '0)f ZDV SXUFKDVHG IURP $OGULFK 0LOZDXNHH :,f 'RGHFDQRO EURPRSURSLRQ\O EURPLGH LPLGD]ROH EURPROGRGHFDQRO WULHWK\ODPLQH ODXULF DFLG DQG 11fGLF\FORKH[\OFDUERGLPLGH ZHUH SXUFKDVHG IURP )OXND 5RQNRQNRPD 1
PAGE 45

EURPLGH PROHf DQG WULHWK\ODPLQH PROHf ZHUH PL[HG DQG VWLUUHG LQ PO RI FKORURIRUP IRU K WR \LHOG FUXGH GRGHF\O EURPRSURSLRQDWH )LJXUH f 7KH FUXGH SURGXFW ZDV ZDVKHG WKUHH WLPHV ZLWK DGHTXDWH ZDWHU WR UHPRYH LPSXULWLHV 7KH RUJDQLF SKDVH ZDV GULHG E\ DGGLQJ DQK\GURXV VRGLXP VXOIDWH DQG GLVWLOOHG XQGHU YDFXXP $IWHU WKLV VLPSOH H[WUDFWLRQ WKH FUXGH SURGXFW GRGHF\O EURPRSURSLRQDWH PROHf ZDV PL[HG ZLWK LPLGD]ROH PROHf LQ FKORURIRUP DQG UHIOX[HG IRU DQRWKHU K )LJXUH f 7KH ILQDO FUXGH ',3 SURGXFW ZDV ZDVKHG ZLWK DQ DGHTXDWH DPRXQW RI ZDWHU WKUHH WLPHV DQG GULHG ZLWK VRGLXP VXOIDWH 7KHQ WKH RLO\ FRPSRXQG ZDV SXULILHG WKURXJK IODVK FKURPDWRJUDSK\ 6WLOO HW DO f XVLQJ VLOLFD JHO PHVK VL]Hf DV WKH DGVRUEHQW DQG PHWKDQROPHWK\OHQH FKORULGH PL[WXUH DV WKH PRELOH SKDVH DW D UDWLR YYf RI WR UHVSHFWLYHO\ 0HWK\O ,PLGD]RO\O /DXUHDWH $ VWDQGDUG HVWHULILFDLWRQ PHWKRG +DVVQHU t $OZ[DQLDQ f ZDV XVHG WR V\QWKHVL]H PHWK\O LPLGD]RO\O ODXUHDWH 0,/f $ PL[WXUH RI LPLGD]RO\O PHWKDQRO PROHf ODXULF DFLG PROHf DQG 11fGLF\FORKH[\OFDUERGLLPLGH PROHf LQ PO RI '0) ZDV VWLUUHG RYHUQLJKW DW r& WR SURGXFH 0,/ )LJXUH f 11 GLF\FORKH[\O XUHD ZDV ILOWHUHG DQG ZDVKHG WKUHH WLPHV ZLWK ZDWHU WKUHH WLPHV ZLWK b DFHWLF DFLG VROXWLRQ DJDLQ WKUHH WLPHV ZLWK ZDWHU DQG WKHQ GULHG ZLWK DQK\GURXV VRGLXP VXOIDWH 3XUH 0,/ ZDV WKHQ REWDLQHG WKURXJK TXLFN IODVK FKURPDWRJUDSK\ ZLWK WKH UDWLR YYf RI PHWKDQRO WR PHWK\OHQH FKORULGH DW WR UHVSHFWLYHO\

PAGE 46

%U EURPRSURSLRQ\O EURPLGH 'RGHFDQRO 2 'RGHF\O EURPRSURSLRQDWH QAQK ,PLGD]ROH $ 'RGHF\O OnLPLGD]RO\Of SURSLRQDWH )LJXUH 6\QWKHWLF SDWKZD\ RI GRGHF\O O fLPLGD]RO\Of SURSLRQDWH ',3f

PAGE 47

AQARK 1Z f§AFK LPLGD]RO\O PHWKDQRO /DXULH DFLG '&& $ A 0HWK\O LPLGD]RO\O ODXUHDWH )LJXUH 6\QWKHWLF SDWKZD\ RI PHWK\O LPLGD]RO\O ODXUHDWH 0,/f

PAGE 48

1'RGHF\O ,PLGD]ROH 1GRGHF\O LPLGD]ROH ',f ZDV V\QWKHVL]HG E\ UHDFWLQJ LPLGD]ROH PROHf DQG EURPROGRGHFDQRO PROHf LQ PO RI '0) DW r& IRU K )LJXUH f &UXGH ', ZDV ZDVKHG ZLWK ZDWHU WKUHH WLPHV b DFHWLF DFLG VROXWLRQ WKUHH WLPHV ZDWHU WKUHH WLPHV DQG WKHQ GULHG ZLWK DQK\GURXV VRGLXP VXOIDWH 3XUH ', ZDV REWDLQHG DIWHU SDVVLQJ WKURXJK TXLFN IODVK FKURPDWRJUDSK\ ZLWK WKH UDWLR YYf RI PHWKDQRO WR PHWK\OHQH FKORULGH DW WR UHVSHFWLYHO\ ,GHQWLILFDWLRQ RI %LRGHJUDGDEOH S+6HQVLWLYH 6XUIDFWDQWV $IWHU SXULI\LQJ WKHVH WKUHH DJHQWV WKHLU VWUXFWXUHV ZHUH LGHQWLILHG WKURXJK D 0+] n+QXFOHDU PDJQHWLF UHVRQDQFH 105f LQ WKH &HQWHU RI 6WUXFWXUDO %LRORJ\ DQG PDVV VSHFWURVFRS\ )$%f LQ WKH 'HSDUWPHQW RI &KHPLVWU\ DW WKH 8QLYHUVLW\ RI )ORULGD 7KH SXULWLHV RI WKH WKUHH ELRGHJUDGDEOH S+VHQVLWLYH VXUIDFWDQWV %36f ZHUH FRQILUPHG E\ HOHPHQWDO DQDO\VLV LQ WKH 'HSDUWPHQW RI &KHPLVWU\ DW WKH 8QLYHUVLW\ RI )ORULGD 'RGHF\O Of,PLGD]RO\Of 3URSLRQDWH 7KH n+105 &'&f VSHFWUXP VKRZHG UHVRQDQFHV RI V +f V +f V +f T +f W +f G +f P EU +f DQG W +f ZKLFK ZDV FRQVLVWHQW ZLWK WKH SURSRVHG VWUXFWXUH )LJXUH f 7KH PDVV VSHFWUXP &,+1 ): f KDG D PROHFXODU LRQ 0Of DW )LJXUH f 7KH HOHPHQWDU\ DQDO\VLV LQGLFDWHG VLPLODU H[SHULPHQWDO SHUFHQWDJHV WR WKH WKHRUHWLFDO YDOXHV 7DEOH f $OO WKHVH DVVD\V ZHUH ZLWKLQ DFFHSWDEOH PDUJLQV RI HUURU PDVV

PAGE 49

,PLGD]ROH EURPR GRGHFDQRO 1GRGHF\O LPLGD]ROH )LJXUH 6\QWKHWLF SDWKZD\ RI 1GRGHF\O LPLGD]ROH ',f

PAGE 50

--/ MU L D -8/ L )LJXUH n+105 VSHFWUXP RI GRGHF\O O fLPLGD]RO\Of SURSLRQDWH ',3f

PAGE 51

IAI U f§Uf Urr7ar L /f§ UaUa f§Uf§ ( )LJXUH 0DVV VSHFWUXP RI GRGHF\O OfLPLGD]RO\Of SURSLRQDWH ',3f

PAGE 52

VSHFWURVFRS\ PPX HOHPHQWDO DQDO\VLV b HDFK HOHPHQWf ZKLFK FRQILUPHG WKH FKHPLFDO VWUXFWXUH DQG SXULW\ RI GRGHF\O O fLPLGD]RO\Of SURSLRQDWH ',3f 7DEOH (OHPHQWDO DQDO\VLV RI GRGHF\O O fLPLGD]RO\Of SURSLRQDWH ',3f &RPSDULVRQ RI H[SHULPHQWDO DQG WKHRUHWLFDO SHUFHQWDJHV RI HDFK HOHPHQW ',3 7KHRUHWLFDO b ([SHULPHQWDO b &DUERQ +\GURJHQ 1LWURJHQ 0HWK\O ,PLGD]RO\O /DXUHDWH 7KH n+105 VSHFWUXP VKRZHG UHVRQDQFHV RI V +f G +f V +f W +f P EU +f DQG W +f )LJXUH f 7KH PDVV VSHFWUXP &,+1 ): f KDG D PROHFXODU LRQ 0Of DW )LJXUH f 7KH FRPEXVWLRQ DQDO\VLV RI H[SHULPHQWDO SHUFHQWDJHV RI HOHPHQWV ZDV LQ DJUHHPHQW ZLWK WKH WKHRUHWLFDO YDOXHV 7DEOH f $OO DVVD\V KDG DFFHSWDEOH PDUJLQV RI HUURU DQG FRQILUPHG WKH FKHPLFDO VWUXFWXUH DQG SXULW\ RI PHWK\O LPLGD]RO\O ODXUHDWH 0,/f

PAGE 53

)LJXUH f+105 VSHFWUXP RI PHWK\O LPLGD]RO\O ODXUHDWH 0,/f

PAGE 54

( )LJXUH 0DVV VSHFWUXP RI PHWK\O LPLGD]RO\O ODXUHDWH 0,/f

PAGE 55

7DEOH (OHPHQWDO DQDO\VLV RI PHWK\O LPLGD]RO\O ODXUHDWH 0,/f &RPSDULVRQ RI H[SHULPHQWDO DQG WKHRUHWLFDO SHUFHQWDJHV RI HDFK HOHPHQW 0,/ 7KHRUHWLFDO b ([SHULPHQWDO b &DUERQ +\GURJHQ 1LWURJHQ 1'RGHF\O ,PLGD]ROH f+105 VSHFWUXP VKRZHG UHVRQDQFHV RI V +f V +f V +f W +f P EU +f DQG W +f )LJXUH f )RU 1GRGHF\O LPLGD]ROH ',f &+1 ): f WKH PDVV VSHFWUXP KDG D PROHFXODU LRQ 0Of DW )LJXUH f 7KH DERYH WZR DVVD\V FRQILUPHG WKH FKHPLFDO VWUXFWXUH RI ', 7KH FRPEXVWLRQ DQDO\VLV RI H[SHULPHQWDO SHUFHQWDJHV RI HOHPHQWV ZDV LQ DJUHHPHQW ZLWK WKH WKHRUHWLFDO YDOXHV LQGLFDWLQJ WKH SXULW\ RI WKLV FRPSRXQG 7DEOH f 7DEOH (OHPHQWDO DQDO\VLV RI 1GRGHF\O LPLGD]ROH ',f &RPSDULVRQ RI H[SHULPHQWDO DQG WKHRUHWLFDO SHUFHQWDJHV RI HDFK HOHPHQW ', 7KHRUHWLFDO b ([SHULPHQWDO b &DUERQ +\GURJHQ 1LWURJHQ

PAGE 56

)LJXUH n+105 VSHFWUXP RI 1GRGHF\O LPLGD]ROH ',f

PAGE 57

L ( )LJXUH 0DVV VSHFWUXP RI 1GRGHF\O LPLGD]ROH ',f

PAGE 58

&+$37(5 3+<6,&2&+(0,&$/ &+$5$&7(5,=$7,21 2) %,2'(*5$'$%/( S+ 6(16,7,9( 685)$&7$176 ,QWURGXFWLRQ ,Q WKLV FKDSWHU ZH KDYH FKDUDFWHUL]HG DQG FRPSDUHG WKUHH PHPEHUV RI WKH %36 IDPLO\ GRGHF\O fOLPLGD]RO\Of SURSLRQDWH ',3f PHWK\O LPLGD]RO\O ODXUHDWH 0,/f DQG 1GRGHF\O LPLGD]ROH ',f )LUVW VXUIDFH DFWLYH SURSHUWLHV LQFOXGLQJ FULWLFDO PLFHOOH FRQFHQWUDWLRQ &0&f DQG HIIHFWLYH UHOHDVH UDWLR 5Hf RI WKH LRQL]HG %36 ZHUH PHDVXUHG DQG YHULILHG 7KH S+ VHQVLWLYLW\ RI %36 WR O\VH OLSRVRPHV IURP WKH H[WHUQDO HQYLURQPHQW DQG WKH EHKDYLRU RI %36 WR GHVWDELOL]H OLSRVRPHV ZKHQ LQFRUSRUDWHG LQ WKHP ZHUH DOVR HYDOXDWHG 7KHQ V\VWHPV ZHUH HVWDEOLVKHG WR GHFLGH WKH FKHPLFDO DQG ELRORJLFDO VWDELOLWLHV RI %36 DQG WKH UHVXOWV FRPSDUHG LQ UHODWLRQ WR WKHLU FKHPLFDO VWUXFWXUHV )LQDOO\ WKH FHOOXODU WR[LFLW\ RI WKHVH DJHQWV ZDV GHWHUPLQHG DQG FRUUHODWHG ZLWK WKHLU ELRGHJUDGDELOLW\ ELRORJLFDO VWDELOLW\f 0DWHULDOV &KHPLFDO &DOFHLQ IHUULF FKORULGH DQG DPPRQLXP WKLRF\DQDWH ZHUH SXUFKDVHG IURP $OGULFK 0LOZDXNHH :,f 'RGHFDQRO LPLGD]ROH DQG ODXULF DFLG ZHUH SXUFKDVHG IURP )OXND

PAGE 59

5RQNRQNRPD 1
PAGE 60

JODFLDO DFHWLF DFLGf S+ P0 .+3 DQG P0 1DA32f S+ P0 .+3 DQG P0 1D+3f S+ P0 .+3 DQG P0 1D+3f S+ P0 .+3 DQG P0 1A+32DQG S+ P0 .+3 DQG P0 VRGLXP K\GUR[LGHf /LSRVRPH 3UHSDUDWLRQ ,QVWHDG RI VHUYLQJ DV D QXFOHLF DFLG GHOLYHU\ V\VWHP OLSRVRPHV /DOHFLWKLQ GLP\ULVWR\OVQJO\FHURSKRVSKRFKROLQH '03&f FKROHVWHURO PRODU UDWLR f ZHUH XVHG DV D PRGHO PHPEUDQH V\VWHP 7R PDLQWDLQ VLPSOLFLW\ RQO\ QHXWUDO OLSLGV ZHUH HPSOR\HG LQ WKH OLSRVRPDO PHPEUDQH V\VWHP :KLOH WKH OLSRVRPHV PD\ QRW IXOO\ UHSUHVHQW HYHQWV RFFXUULQJ LQ ELRORJLFDO VLWXDWLRQV WKH\ VWLOO VHUYHG DV H[FHOOHQW PRGHOV LQ DGGUHVVLQJ SRWHQWLDO PHFKDQLVPV RI OLSLG PHPEUDQH GLVUXSWLRQ &DOFHLQ P0f ZDV HQWUDSSHG ZLWKLQ WKH OLSRVRPHV DV D IOXRUHVFHQW PDUNHU WR PRQLWRU PHPEUDQH O\VLV HYHQWV DQG XQHQWUDSSHG FDOFHLQ ZDV UHPRYHG WKURXJK FHQWULIXJDWLRQ USP PLQf ILYH WLPHV DQG ZDVKHG ZLWK D S+ SKRVSKDWH EXIIHUHG VDOLQH 3%6f HDFK WLPH 5HYHUVHSKDVH HYDSRUDWLRQ YHVLFOH PHWKRG 6]RND t 3DSDKDGMRSRXORV f ZDV XVHG WR SURGXFH XQLODPHOODU YHVLFOHV QPf XVLQJ SRO\FDUERQDWH PHPEUDQHV 3RUHWLFV /LYHUPRUH &$f WKURXJK D KLJK SUHVVXUH H[WUXGHU WKUHH WLPHV /LSH[ %LRPHPEUDQH ,QF 9DQFRXYHU &DQDGDf 7KH VL]H RI WKH OLSRVRPHV YROXPHZHLJKW *DXVVLDQ GLVWULEXWLRQf ZDV PHDVXUHG WR EH s QP VWDQGDUG GHYLDWLRQf E\ D G\QDPLF OLJKW VFDWWHULQJ PHWKRG XVLQJ D 1,&203 0RGHO =/6 =HWD 3RWHQWLDO3DUWLFOH 6L]HU 6DQWD %DUEDUD &$f 7KH FRQFHQWUDWLRQ RI SKRVSKROLSLG LQ HDFK H[SHULPHQW ZDV PHDVXUHG E\ D

PAGE 61

PRGLILFDWLRQ RI D VSHFWURSKRWRPHWULF WHFKQLTXH 6WHZDUW f %ULHIO\ YDU\LQJ DPRXQWV RI /DOHFLWKLQ PJPOf ZHUH DGGHG WR WHVW WXEHV FRQWDLQLQJ PO RI 0 DPPRQLXP IHUURWKLRF\DQDWH DQG PO RI FKORURIRUP 7KH FRQWHQWV ZHUH PL[HG YLJRURXVO\ IRU PLQ DQG FHQWULIXJHG DW USP 6DIHJXDUG &HQWULIXJH &OD\$GDPV ,QFf IRU PLQ WR IXOO\ VHSDUDWH WKH WZR SKDVHV 7KH DTXHRXV SKDVH ZDV UHPRYHG DQG WKH DEVRUEDQFH RI WKH UHPDLQLQJ RUJDQLF SKDVH ZDV PHDVXUHG DW QP ZLWK D VSHFWURSKRWRPHWHU 899LV 3HUNLQ(OPHU VSHFWURSKRWRPHWHU /DPEGD f WR HVWDEOLVK D FDOLEUDWLRQ FXUYH 7KH FRQFHQWUDWLRQV RI XQNQRZQ VDPSOHV ZHUH WKHQ GHWHUPLQHG IURP WKH FDOLEUDWLRQ FXUYH 0HWKRGV &ULWLFDO 0LFHOOH &RQFHQWUDWLRQ 'HWHUPLQDWLRQ 7R GHWHUPLQH WKH FULWLFDO PLFHOOH FRQFHQWUDWLRQ &0&f RI WKH LRQL]HG ELRGHJUDGDEOH S+VHQVLWLYH VXUIDFWDQWV %36f VXUIDFH WHQVLRQ PHDVXUHPHQWV ZHUH SHUIRUPHG XVLQJ D &5&'X1R\ LQWHUIDFLDO WHQVLRPHWHU 0DUWLQ f 7KH S+ ZDV DGMXVWHG WR S+ LQ GLIIHUHQW FRXQWHU LRQ VROXWLRQV +) +& +%U DQG +,f DW D FRQVWDQW URRP WHPSHUDWXUH r&f ,QFUHDVLQJ DPRXQWV RI %36 ZHUH DGGHG LQWR GLIIHUHQW VROXWLRQV DQG VXUIDFH WHQVLRQ PHDVXUHG 7KH FRQFHQWUDWLRQ UHJLRQ LQ ZKLFK VXUIDFH WHQVLRQ VWRSSHG FKDQJLQJ ZDV UHFRUGHG DV WKH &0&

PAGE 62

(IIHFWLYH 5HOHDVH 5DWLR 'HWHUPLQDWLRQ RI %LRGHJUDGDEOH S+6HQVLWLYH 6XUIDFWDQWV 8QLODPHOODU OLSRVRPHV QP QPROPOf FRQWDLQLQJ P0 FDOFHLQ AVHOI TXHQFKLQJ FRQFHQWUDWLRQf ZHUH VXVSHQGHG LQ D S+ DFHWDWH EXIIHU VROXWLRQ ZLWK LQFUHDVLQJ DPRXQWV RI ELRGHJUDGDEOH S+VHQVLWLYH VXUIDFWDQWV %36f (TXLOLEULXP ZDV DOORZHG WR RFFXU IRU PLQ DW URRP WHPSHUDWXUH $ VXEVWDQWLDO SRUWLRQ WR FDXVH PHPEUDQH O\VLV IURP PRVW GHWHUJHQWV KDV WDNHQ SODFH DIWHU PLQ RI LQFXEDWLRQ ZLWK OLSRVRPHV 5XL] HW DO f &RPSOHWH HTXLOLEULXP EHWZHHQ VXUIDFWDQWV DQG OLSLGV FDQ WDNH VHYHUDO KRXUV /LFKWHQEHUJ HW DO f $IWHU WKLV WLPH KRZHYHU VXUIDFWDQW LQGXFHG UHOHDVH RI OLSRVRPDO FRQWHQWV FDQ EH PDVNHG E\ WKH FRQFRPLWDQW VSRQWDQHRXV GLIIXVLRQ RI VROXWHV RXW RI WKH YHVLFOHV 7KHUHIRUH ORQJ WLPH LQFXEDWLRQ ZLWK FRPSOHWH HTXLOLEULXP PD\ QRW EH DSSURSULDWH LQ WKLV VWXG\ 5HOHDVHG FDOFHLQ ZDV H[FLWHG DW QP DQG REVHUYHG DW QP LQ D 3HUNLQ(OPHU OXPLQHVFHQFH VSHFWURSKRWRPHWHU /6% DW URRP WHPSHUDWXUH 7KH SHUFHQWDJH RI UHOHDVHG FDOFHLQ ZDV FDOFXODWHG E\ WKH HTXDWLRQ bf M\f§a r b /LX t 5HJHQ 8 a Kf f ,[ LV WKH b IOXRUHVFHQFH LQWHQVLW\ YDOXH ZKHQ DGGLQJ H[FHVV 7ULWRQ ; P0f ,D DQG ,E DUH WKH IOXRUHVFHQFH LQWHQVLWLHV DIWHU LQFXEDWLRQ ZLWK DQG ZLWKRXW %36 UHVSHFWLYHO\ (IIHFWLYH UHOHDVH UDWLR 5Hf ZDV GHILQHG DW WKH PRODU UDWLR VXUIDFWDQWOLSRVRPHf ZKHQ b FDOFHLQ ZDV UHOHDVHG 'XULQJ WKLV SURFHVV WKH VXUIDFWDQW PXVW FRPH LQ FRQWDFW ZLWK WKH OLSLG ELOD\HU DQG SDUWLWLRQ LQWR WKH K\GURSKRELF HQYLURQPHQW

PAGE 63

S+ 6HQVLWLYLW\ RI %LRGHJUDGDEOH S+6HQVLWLYH 6XUIDFWDQWV RQ /LSRVRPDO &DOFHLQ 5HOHDVH 7R GHWHUPLQH WKH DELOLW\ RI ELRGHJUDGDEOH S+VHQVLWLYH VXUIDFWDQWV %36f WR FDXVH PHPEUDQH O\VLVOHDNDJH DW GLIIHUHQW S+V VWXGLHV ZHUH FRQGXFWHG WKDW YDULHG WKH %36 FRQFHQWUDWLRQ LQ D VROXWLRQ RI FDOFHLQ FRQWDLQLQJ OLSRVRPHV QPROPOf DV GHVFULEHG DERYH ,QFUHDVLQJ %36 FRQFHQWUDWLRQV QPROPO QPROPOf ZHUH DGGHG WR IRXU EXIIHU V\VWHPV S+ DQG f FRQWDLQLQJ OLSRVRPHV ZLWK FDOFHLQ 7KH VXVSHQVLRQV ZHUH LQFXEDWHG IRU PLQ DW URRP WHPSHUDWXUH DQG WKH SHUFHQWDJH RI FDOFHLQ UHOHDVH ZDV FDOFXODWHG E\ WKH HTXDWLRQ bf DKf :}f r b DV SUHYLRXVO\ GHVFULEHG /LX t 5HJHQ f 0HPEUDQH /\VLV 3URILOH RI %LRGHJUDGDEOH S+6HQVLWLYH 6XUIDFWDQWV ZKHQ ,QFRUSRUDWHG LQWR /LSRVRPHV 'LIIHUHQW PRODU UDWLRV 5 5 DQG 5 f RI ELRGHJUDGDEOH S+VHQVLWLYH VXUIDFWDQWV %36f ZHUH LQFRUSRUDWHG LQWR OLSRVRPHV QPROPOf FRQWDLQLQJ P0 FDOFHLQ WR REVHUYH WKH LQGXFHG FDOFHLQ UHOHDVH DW GLIIHUHQW S+V 7KH YDULRXV %36 OLSRVRPHV ZHUH LQFXEDWHG LQ SKRVSKDWH EXIIHU VROXWLRQV S+ f IRU PLQ WR GHWHUPLQH WKH UHOHDVH FKDUDFWHULVWLFV 7KH SHUFHQWDJH RI UHOHDVH ZDV WKHQ UHFRUGHG DQG FRUUHFWHG E\ WKH HTXDWLRQ bf :}f :rf rb DV PHQWLRQHG SUHYLRXVO\

PAGE 64

&KHPLFDO DQG %LRORJLFDO 6WDELOLWLHV RI %LRGHJUDGDEOH S+6HQVLWLYH 6XUIDFWDQWV 7KH DTXHRXV VWDELOLW\ RI ELRGHJUDGDEOH S+VHQVLWLYH VXUIDFWDQWV %36f ZDV GHWHUPLQHG E\ LQFXEDWLQJ GLIIHUHQW FRQFHQWUDWLRQV RI %36 LQ S+ EXIIHUV S+ f ZLWK b GLPHWK\O VXOIR[LGH '062f DV D FRVROYHQW DW r& 6DPSOHV ZHUH UHPRYHG SHULRGLFDOO\ DQG %36 FRQFHQWUDWLRQ ZDV TXDQWLILHG XVLQJ DQ +3/& PHWKRG 7KH +3/& V\VWHP FRQVLVWHG RI D 0LOWRQ 5R\ &0 SXPS DQ /'& $QDO\WLFDO DEVRUEDQFH GHWHFWRU D +HZOHWW 3DFNDUG LQWHJUDWRU DQG D 6SHFWUD 3K\VLFV 63 DXWRVDPSOHU $ r PP & FROXPQ 1RYD3DNf DORQJ ZLWK D PRELOH SKDVH FRQVLVWLQJ RI b DFHWRQLWULOH DQG b P0 S+ 1D+3 VROXWLRQ ZDV XVHG WR VHSDUDWH DQG GHWHUPLQH LQWDFW %36 IURP WKHLU GHJUDGDWLRQ SURGXFWV DW QP %X\XNWLPNLQ HW DO f 7KH IORZ UDWH ZDV VHW DW POPLQ 7KH FKHPLFDO GHJUDGDWLRQ UDWH FRQVWDQW ZDV REWDLQHG DIWHU SORWWLQJ WKH SHDN KHLJKW RI WKH LQWDFW %36 RYHU WLPH DQG XVHG WR FUHDWH %36 S+ K\GURO\VLV UDWH SURILOH 7R GHWHUPLQH WKH K\GURO\VLV UDWH RI %36 LQ ELRORJLFDO V\VWHPV D SRUFLQH HVWHUDVH ZDV XVHG 9DU\LQJ XQLWV RI HVWHUDVH ZHUH LQFXEDWHG DW r& ZLWK FRQVWDQW DPRXQW RI %36 LQ D b '062 S+ EXIIHU VROXWLRQ $OLTXRW ZDV UHPRYHG DW D SHULRGLF LQWHUYDO DQG SHDN KHLJKW RI WKH LQWDFW %36 ZDV PHDVXUHG ZLWK WKH +3/& PHWKRG GHVFULEHG DERYH 7KH ELRORJLFDO UDWH FRQVWDQWV RI WKUHH %36 ZHUH FDOFXODWHG WR FRPSDUH WKHLU ELRGHJUDGDELOLW\

PAGE 65

&HOOXODU 7R[LFLW\ 7HVW RI %LRGHJUDGDEOH S+6HQVLWLYH 6XUIDFWDQWV 7KH FHOOXODU WR[LFLW\ RI ELRGHJUDGDEOH S+VHQVLWLYH VXUIDFWDQWV %36f ZDV PRQLWRUHG LQ DQ 6.Q6+ KXPDQ QHXUREODVWRPD FHOO OLQH DQG LQ D &9 PRQNH\ NLGQH\ ILEUREODVW FHOO OLQH ZLWK D FDOFHLQ$0 DVVD\ /LFKWHQIHOV HW DO f 7R FRQILUP WKH WR[LF HIIHFW RI ',3 DQG 0,/ DV D UHVXOW RI WKHLU PHWDEROLWHV LPLGD]ROH LPLGD]ROH PHWKDQRO GRGHFDQRO DQG ODXULF DFLG ZHUH XVHG WR WHVW WKHLU LQGLYLGXDO F\WRWR[LFLW\ LQ D &9 FHOO OLQH %ULHIO\ IRU WKH 6.Q6+ FHOO OLQH VXEFRQIOXHQW PRQROD\HUHG FXOWXUHV ZHUH LQFXEDWHG LQ D ZHOO SODWH FHOOVZHOOf ZLWK SL RI 530, JURZWK PHGLXP 8PO SHQLFLOOLQ SJPO VWUHSWRP\FLQ DQG b IHWDO ERYLQH VHUXPf DW r& b & DQG b KXPLGLW\ HQYLURQPHQW IRU K )RU WKH &9 FHOO OLQH 0(0 JURZWK PHGLXP 8PO SHQLFLOOLQ SJPO VWUHSWRP\FLQ P0 0(0 VRGLXP S\UXYDWH VROXWLRQ ,; 0(0 DPLQR DFLGV VROXWLRQ DQG b KHDWHG IHWDO ERYLQH VHUXPf ZDV XVHG LQVWHDG RI 530, 7KH JURZWK PHGLXP ZDV WKHQ UHPRYHG DQG %36 DGGHG IURP S0 WR 0 LQ SL RI IUHVK JURZWK PHGLXP 7KH FHOOV ZHUH PDLQWDLQHG IRU DQ DGGLWLRQDO K $IWHU WKH LQFXEDWLRQ FHOOV ZHUH ZDVKHG WKUHH WLPHV ZLWK SKRVSKDWH EXIIHUHG VDOLQH DQG LQFXEDWHG ZLWK SL RI FDOFHLQ$0 S0f IRU PLQ DW URRP WHPSHUDWXUH &DOFHLQ IOXRUHVFHQFH LQWHQVLW\ ZDV WKHQ PHDVXUHG DW DQ H[FLWDWLRQ ZDYHOHQJWK RI QP DQG REVHUYHG DW QP RQ D 3HUNLQ (OPHU /6 % 6SHFWURSKRWRPHWHU 7KH SHUFHQWDJH RI OLYH FHOOV ZDV FDOLEUDWHG DV LQ WKH IROORZLQJ HTXDWLRQ

PAGE 66

6DPSOH 0LQf /LYHbf f§f§ f§f§f§ r b ZKHUH 0D[ LV WKH IOXRUHVFHQFH VLJQDO IURP FHOOV 0D[ 0LQf ZLWKRXW DQ\ WUHDWPHQW 0LQ LV WKH IOXRUHVFHQFH VLJQDO ZLWKRXW FHOOV DQG 6DPSOH LV IOXRUHVFHQFH VLJQDO IURP HDFK VDPSOH 7R FRPSDUH WKH GLIIHUHQFH DPRQJ GLIIHUHQW WUHDWPHQWV D SDUDPHWHU ,' ZDV XVHG ,' ZDV GHILQHG DV WKH GUXJ FRQFHQWUDWLRQ UHTXLUHG WR UHGXFH WKH DEVRUEDQFH RI FDOFHLQ E\ b WKXV LQGLFDWLQJ b FHOO GHDWK 6WDWLVWLFDO $QDO\VLV 6WDWLVWLFDO GLIIHUHQFHV EHWZHHQ WKH WUHDWPHQWV ZHUH GHWHUPLQHG XVLQJ DQDO\VLV RI YDULDQFH ZKHUH DSSURSULDWH 6WDW9LHZ $EDFXV &RQFHSWV ,QF %HUNHOH\ &$f ZLWK S FRQVLGHUHG VWDWLVWLFDOO\ VLJQLILFDQW DQG )LVKHUfV 3/6'f SRVW KRF WWHVW ZDV DSSOLHG 5HVXOWV &ULWLFDO 0LFHOOH &RQFHQWUDWLRQ 'HWHUPLQDWLRQ $Q LPSRUWDQW SDUDPHWHU LQ FKDUDFWHUL]LQJ VXUIDFWDQWV LV WKH FRQFHQWUDWLRQ DW ZKLFK PLFHOOHV IRUP $OO H[SHULPHQWV ZHUH FRQGXFWHG DW S+ WR HQVXUH WKDW DOO ELRGHJUDGDEOH S+VHQVLWLYH VXUIDFWDQWV %36f ZHUH LQ DQ LRQL]HG VWDWH !bf 7KH FULWLFDO PLFHOOH FRQFHQWUDWLRQV &0&Vf RI WKH LRQL]HG %36 ZHUH GHWHUPLQHG E\ XVLQJ VXUIDFH WHQVLRQ PHDVXUHPHQWV LQ GLIIHUHQW FRXQWHU LRQ VROXWLRQ $V %36 FRQFHQWUDWLRQ LQFUHDVHG WKH VXUIDFH WHQVLRQ RI WKH VROXWLRQ VKDUSO\ GHFUHDVHG XQWLO WKH IRUPDWLRQ RI PLFHOOHV RFFXUUHG )LJXUH f ,Q JHQHUDO 1GRGHF\O LPLGD]ROH ',f KDG WKH KLJKHVW &0&V ZKLOH PHWK\O

PAGE 67

)LJXUH &ULWLFDO PLFHOOH FRQFHQWUDWLRQ &0&f PHDVXUHPHQW RI WKUHH LRQL]HG ELRGHJUDGDEOH S+VHQVLWLYH VXUIDFWDQWV %36f 7KH &0& PHDQOVWDQGDUG GHYLDWLRQ 6'ff ZDV FDOFXODWHG LQ D S+ K\GURIOXRULF DFLG VROXWLRQ DW URRP WHPSHUDWXUH ZLWK DQ LQWHUIDFLDO WHQVLRPHWHU Q f 7KH FRQFHQWUDWLRQ UHJLRQ DW ZKLFK VXUIDFH WHQVLRQ VWDELOL]HG ZDV UHFRUGHG DV WKH &0& 7KH &0& ZDV GHWHUPLQHG WR EH P0 IRU LRQL]HG GRGHF\O OfLPLGD]RO\Of SURSLRQDWH ',3f ‘f P0 IRU LRQL]HG PHWK\O LPLGD]RO\O ODXUHDWH 0,/f Af DQG P0 IRU 1GRGHF\O LPLGD]ROH ',f $f

PAGE 68

LPLGD]RO\O ODXUHDWH 0,/f KDG WKH ORZHVW &0&V 7DEOH f LQ DOO IRXU FRXQWHU LRQ VROXWLRQV )RU DOO WKUHH %36 +, +%U +& DQG +) FDXVHG WKH &0& WR GHFUHDVH LQ GHVFHQGLQJ RUGHU 7DEOH 7KH FULWLFDO PLFHOOH FRQFHQWUDWLRQ RI GRGHF\O OfLPLGD]RO\Of SURSLRQDWH ',3f PHWK\O LPLGD]RO\O ODXUHDWH 0,/f DQG 1GRGHF\O LPLGD]ROH ',f LQ IRXU GLIIHUHQW FRXQWHU LRQ VROXWLRQV Q f 6ROXWLRQ %36 P0f ',3 0,/ ', +) +& +%U +, (IIHFWLYH 5HOHDVH 5DWLR 'HWHUPLQDWLRQ RI %LRGHJUDGDEOH S+6HQVLWLYH 6XUIDFWDQWV (IIHFWLYH UHOHDVH UDWLR 5Hf GHVFULEHV WKH PRODU UDWLR RI D VXUIDFWDQW WR WKH WRWDO DPRXQW RI OLSLG UHTXLUHG WR UHOHDVH b OLSRVRPDO FRQWHQWV 5H ZDV GHWHUPLQHG E\ ILWWLQJ D VLJPRLGDO FXUYH RI FDOFHLQ UHOHDVH IURP OLSRVRPHV DW LQFUHDVLQJ PRODU UDWLRV RI ELRGHJUDGDEOH S+VHQVLWLYH VXUIDFWDQWV %36f WR OLSLG XVLQJ WKH 6FLHQWLVW FRPSXWHU SURJUDP 0LFURPDWK 6DOW /DNH &LW\ 8WDKf 5H ZDV GHWHUPLQHG WR EH DQG IRU GRGHF\O OfLPLGD]RO\Of SURSLRQDWH ',3f PHWK\O LPLGD]RO\O ODXUHDWH 0,/f DQG 1GRGHF\O LPLGD]ROH ',f UHVSHFWLYHO\ )LJXUH f

PAGE 69

)LJXUH $ELOLW\ RI YDULRXV ELRGHJUDGDEOH S+VHQVLWLYH VXUIDFWDQWV %36f WR LQGXFH FDOFHLQ UHOHDVH PHDQs6'f DW LQFUHDVLQJ PRODU UDWLRV RI LRQL]HG %36 IURP DQ H[WHUQDO HQYLURQPHQW WR OLSRVRPHV ZKHQ LQFXEDWHG LQ D S+ EXIIHU VROXWLRQ IRU PLQ Q f 7KH HIIHFWLYH UHOHDVH UDWLR ZDV WKHQ GHWHUPLQHG WR EH DQG IRU GRGHF\O O LPLGD]RO\Of SURSLRQDWH ',3f ff PHWK\O LPLGD]RO\O ODXUHDWH 0,/f ‘f DQG 1 GRGHF\O LPLGD]ROH ',f $f UHVSHFWLYHO\

PAGE 70

S+ 6HQVLWLYLW\ RI %LRGHJUDGDEOH S+6HQVLWLYH 6XUIDFWDQWV RQ /LSRVRPDO &DOFHLQ 5HOHDVH 7R GHWHUPLQH ZKHWKHU ELRGHJUDGDEOH S+VHQVLWLYH VXUIDFWDQWV %36f EHFRPH HIIHFWLYH LQ DFLGLF HQYLURQPHQWV EXW KDYH OLPLWHG HIIHFW DW H[WUDFHOOXODU ELRORJLFDO S+ FDOFHLQ FRQWDLQLQJ OLSRVRPHV ZHUH LQFXEDWHG ZLWK LQFUHDVLQJ DPRXQWV RI %36 DW IRXU S+V DQG f $Q LQFUHDVH LQ IOXRUHVFHQFH LQWHQVLW\ LQGLFDWHG FDOFHLQ UHOHDVH WKDW FRUUHODWHG WR PHPEUDQH O\VLV &DOFHLQ ZDV UHOHDVHG VLJPRLGDOO\ DW S+ DV WKH FRQFHQWUDWLRQ RI GRGHF\O O LPLGD]RO\Of SURSLRQDWH ',3f LQFUHDVHG )LJXUH Df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f 6LPLODU SURILOHV ZHUH VHHQ ZLWK PHWK\O LPLGD]RO\O ODXUHDWH 0,/f LQGXFHG FDOFHLQ UHOHDVH )LJXUH Ef 6LJQLILFDQW GLIIHUHQFHV Sf RQ FDOFHLQ UHOHDVH ZHUH REVHUYHG DW GLIIHUHQW S+V ZKHQ WKH PRODU UDWLR RI 0,/ WR OLSLG HTXDOHG WR RU DERYH +RZHYHU FRPSDUHG WR WKH FDOFHLQ UHOHDVH FDXVHG E\ ',3 DW WKH VDPH PRODU UDWLR 0,/ VKRZHG OHVV S+ VHQVLWLYLW\ DQG OHVV FDOFHLQ UHOHDVH DW S+ DQG

PAGE 71

)LJXUH (IIHFW RI GLIIHUHQW ELRGHJUDGDEOH S+VHQVLWLYH VXUIDFWDQWV %36f RQ FDOFHLQ UHOHDVH IURP OLSRVRPHV ZKHQ DGGHG IURP DQ H[WHUQDO HQYLURQPHQW Q f 7KH SHUFHQWDJHV PHDQL6'f RI UHOHDVH ZHUH FDOFXODWHG DIWHU PLQ RI LQFXEDWLRQ LQ IRXU EXIIHU VROXWLRQV S+ Af ;f $f DQG ‘ff Df 'RGHF\O Of LPLGD]RO\Of SURSLRQDWH ',3f Ef 0HWK\O LPLGD]RO\O ODXUHDWH 0,/f Ff 1GRGHF\O LPLGD]ROH ',f

PAGE 72

5HOHDVH )LJXUH f§FRQWLQXHG Ef

PAGE 73

)LJXUH f§FRQWLQXHG Ff

PAGE 74

:KHQ ',3 ZDV UHSODFHG E\ 1GRGHF\O LPLGD]ROH ',f VLPLODU SURILOHV ZHUH VHHQ RQ FDOFHLQ UHOHDVH DW ORZ S+ )LJXUH Ff +RZHYHU QR VLJQLILFDQW GLIIHUHQFH ZDV VHHQ RQ WKH FDOFHLQ UHOHDVH DPRQJ WKH IRXU WHVWHG S+V DW DOO REVHUYHG PRODU UDWLRV 0HPEUDQH /\VLV 3URILOH RI %LRGHJUDGDEOH S+6HQVLWLYH 6XUIDFWDQWV ZKHQ ,QFRUSRUDWHG LQWR /LSRVRPHV 6LQFH WKH XOWLPDWH JRDO ZDV WR LQFRUSRUDWH ELRGHJUDGDEOH S+VHQVLWLYH VXUIDFWDQWV %36f LQWR OLSRVRPHV ZH GHWHUPLQHG WKH DELOLW\ RI XQLRQL]HG %36 LQFRUSRUDWHG LQWR OLSRVRPHV WR EH SURWRQDWHG DW ORZHU S+V WKHUHE\ IDFLOLWDWLQJ WKH UHOHDVH RI HQWUDSSHG PDWHULDOV /LSRVRPHV FRQWDLQLQJ FDOFHLQ ZHUH SUHSDUHG ZLWK LQFUHDVLQJ PRODU UDWLRV 5 5 DQG 5 f RI %36 DQG LQFXEDWHG DW GHFUHDVLQJ S+V 0LQLPDO FDOFHLQ UHOHDVH ZDV REVHUYHG ZKHQ QR GRGHF\O OfLPLGD]RO\Of SURSLRQDWH ',3f ZDV LQFRUSRUDWHG LQWR WKH OLSRVRPHV )LJXUH Df $V WKH S+ GHFUHDVHG FDOFHLQ UHOHDVH LQFUHDVHG JUDGXDOO\ LQ DOO JURXSV $W HDFK UDWLR JURXS FDOFHLQ UHOHDVH LQFUHDVHG VLJQLILFDQWO\ Sf DV S+ GURSSHG IURP WR 6LJQLILFDQW GLIIHUHQFHV Sf RI WKH FDOFHLQ UHOHDVH ZHUH DOVR REVHUYHG DPRQJ DOO PRODU UDWLR JURXSV 5 5 DQG 5 f DW DOO REVHUYHG S+V /LNH ',3 ERWK PHWK\O LPLGD]RO\O ODXUHDWH 0,/f )LJXUH Ef DQG 1GRGHF\O LPLGD]ROH ',f )LJXUH Ff FDXVHG FDOFHLQ UHOHDVH LQ D VLPLODU S+GHSHQGHQW PDQQHU &RPSDUHG WR RWKHU JURXSV WKH V\VWHP DW WKH 5 JURXS ZDV UHODWLYHO\ XQVWDEOH DW SK\VLRORJLFDO S+ SUREDEO\ GXH WR WKH DOWHUQDWLRQV LQ OLSLG SDFNLQJ RI WKH OLSRVRPHV :KHQ FRPSDULQJ WKH FDOFHLQ UHOHDVH FDXVHG E\ WKUHH %36 VLJQLILFDQW GLIIHUHQFHV Sf ZHUH REVHUYHG DW WKH 5 JURXS DV S+ ZDV RU +RZHYHU QR

PAGE 75

S+ Df )LJXUH 0HPEUDQH O\VLV SURILOH RI YDULRXV ELRGHJUDGDEOH S+VHQVLWLYH VXUIDFWDQWV %36f ZKHQ LQFRUSRUDWHG LQWR OLSRVRPHV Q f 7KH HIIHFW RI S+ DQG %36OLSRVRPH PRODU UDWLR 5 Af 5 ‘f DQG 5 $ff RQ %36LQGXFHG FDOFHLQ UHOHDVH IURP OLSRVRPHV ZHUH SORWWHG DIWHU PLQ PHDQs6'f Df 'RGHF\O O fLPLGD]RO\Of SURSLRQDWH ',3f Ef 0HWK\O LPLGD]RO\O ODXUHDWH 0,/f Ff 1GRGHF\O LPLGD]ROH ',f

PAGE 76

D! P HD S+ )LJXUH f§FRQWLQXHG Ef

PAGE 77

S+ Ff )LJXUH f§FRQWLQXHG

PAGE 78

VLJQLILFDQW GLIIHUHQFH RI WKH FDOFHLQ UHOHDVH ZDV VHHQ DW WKH RWKHU PRODU UDWLR JURXSV 5 DQG 5 f DPRQJ WKHVH WKUHH %36 &KHPLFDO DQG %LRORJLFDO 6WDELOLWLHV RI %LRGHJUDGDEOH S+6HQVLWLYH 6XUIDFWDQWV $IWHU UHOHDVLQJ ROLJRQXFOHRWLGHV WR F\WRSODVP DQ LGHDO S+VHQVLWLYH VXUIDFWDQWV PXVW EH GHJUDGHG LQ WKH LQWHUFHOOXODU PLOLHX WKXV OLPLWLQJ LWV SRWHQWLDO WR[LFLW\ %LRGHJUDGDEOH S+VHQVLWLYH VXUIDFWDQWV %36f VKRXOG EH DEOH WR EH GHJUDGHG E\ HVWHU K\GURO\VLV HLWKHU FKHPLFDOO\ RU HQ]\PDWLFDOO\ 7KH K\GURO\WLF VWDELOLW\ RI %36 ZDV DVVHVVHG E\ LQFXEDWLQJ WKH FRPSRXQG LQ S+ EXIIHUV DQG PRQLWRULQJ WKH DPRXQW RI WKH VWDUWLQJ PDWHULDO UHPDLQLQJ LQWDFW )LJXUH f 8VLQJ WKH 6FLHQWLVW SURJUDP WR ILW WKH GHJUDGDWLRQ FXUYH WKH S+GHSHQGHQW SVHXGRILUVW RUGHU GHJUDGDWLRQ UDWH FRQVWDQWV Nf ZDV FDOFXODWHG 'RGHF\O O fLPLGD]RO\Of SURSLRQDWH ',3f ZDV DW LWV PRVW VWDEOH VWDWH N GD\nf DW S+ 7KH GHJUDGDWLRQ UDWH FRQVWDQW UHDFKHG D SODWHDX DIWHU S+ N GD\nf )LJXUH f 6LPLODU WR ',3 PHWK\O LPLGD]RO\O ODXUHDWH 0,/f DQG 1GRGHF\O LPLGD]ROH ',f VKRZHG WKH JUHDWHVW VWDELOLW\ DW S+ N GD\n DQG N GD\ UHVSHFWLYHO\f ', H[KLELWHG WKH ORZHVW UDWH FRQVWDQW DPRQJ WKH WKUHH %36 IURP S+ WR S+ VROXWLRQV 1R GLIIHUHQFH LQ WKH GHJUDGDWLRQ UDWH FRQVWDQW RI %36 ZDV VHHQ ZKHQ WKH S+ ZDV DGMXVWHG WR +RZHYHU VLQFH HDFK S+ EXIIHU VROXWLRQ ZDV FRPSRVHG RI GLIIHUHQW FRPSRXQGV LW PLJKW KDYH LWV LQGLYLGXDO FDWDO\VW HIIHFW RQ WKH FKHPLFDO UDWH FRQVWDQW 7R DVVHVV WKH HQ]\PDWLF VWDELOLW\ RI %36 ZH XVHG YDULRXV DPRXQWV RI SRUFLQH HVWHUDVH DV D PRGHO HQ]\PH 6LPLODU WR WKH GHWHUPLQDWLRQ RI FKHPLFDO GHJUDGDWLRQ UDWH

PAGE 79

7LPH GD\f )LJXUH &KHPLFDO GHJUDGDWLRQ SURILOH RI GRGHF\O O fLPLGD]RO\Of SURSLRQDWH ',3f DW S+ DQG r& RYHU WLPH Q f 7KH SHDN KHLJKWV RI LQWDFW ',3 ZHUH REWDLQHG DW YDULRXV WLPH SRLQWV PHDQs6'f 8VLQJ WKH 6FLHQWLVW SURJUDP WR ILW WKH GHJUDGDWLRQ FXUYH WKH SVHXGRILUVW RUGHU GHJUDGDWLRQ UDWH FRQVWDQW Nf ZDV GHWHUPLQHG WR EH GD\f

PAGE 80

)LJXUH &KHPLFDO GHJUDGDWLRQ S+UDWH SURILOHV RI WKUHH ELRGHJUDGDEOH S+VHQVLWLYH VXUIDFWDQWV %36f 7KH UDWH FRQVWDQW PHDQs6'f PHDVXUHG DW r& RI GRGHF\O O LPLGD]RO\Of SURSLRQDWH ',3f ‘f PHWK\O LPLGD]RO\O ODXUHDWH 0,/f Af DQG 1 GRGHF\O LPLGD]ROH ',f $f ZDV SORWWHG DJDLQVW S+ Q f

PAGE 81

VWDELOLW\ RI %36 ZDV DVVHVVHG E\ PRQLWRULQJ WKH DPRXQW RI WKH VWDUWLQJ PDWHULDO UHPDLQLQJ LQWDFW LQ D S+ EXIIHU VROXWLRQ )LJXUH f )LJXUH VKRZHG WKH ELRORJLFDO GHJUDGDWLRQ UDWH SURILOH RI %36 DW YDULRXV UDWLRV RI HVWHUDVH%36 0,/ ZDV PRUH ELRGHJUDGDEOH WKDQ ',3 WKURXJK RXW WKH WHVWHG UDWLRV )XUWKHUPRUH ', ZDV DOPRVW LQVHQVLWLYH WR WKH DGGLWLRQ RI WKH HVWHUDVH &HOOXODU 7R[LFLW\ 7HVW RI %LRGHJUDGDEOH S+6HQVLWLYH 6XUIDFWDQWV $ PRUH LPSRUWDQW SDUDPHWHU WKDQ ELRGHJUDGDELOLW\ RI ELRGHJUDGDEOH S+VHQVLWLYH VXUIDFWDQWV %36f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f ZDV GHWHUPLQHG WR EH P0 P0 DQG P0 IRU GRGHF\O OfLPLGD]RO\Of SURSLRQDWH ',3f PHWK\O LPLGD]RO\O ODXUHDWH 0,/f DQG 1GRGHF\O LPLGD]ROH ',f UHVSHFWLYHO\ ,Q &9 FHOOV WKH ,' )LJXUH Ef ZDV PHDVXUHG DW P0 P0 DQG P0 IRU ',3 0,/ DQG ', UHVSHFWLYHO\ )RU WKH SRVVLEOH PHWDEROLWHV IURP ',3 DQG 0,/ WKH ,' ZDV P0 IRU LPLGD]ROH P0 IRU GRGHFDQRO P0 IRU LPLGD]ROH PHWKDQRO DQG P0 IRU ODXULF DFLG LQ &9 FHOOV )LJXUH f ', VKRZHG WKH KLJKHVW F\WRWR[LF HIIHFW DQG ZDV RQH DQG WZR RUGHUV RI WKH PDJQLWXGH PRUH WR[LF WKDQ ',3 LQ 6.Q6+ DQG &9 FHOOV UHVSHFWLYHO\ 2Q WKH FRQWUDU\

PAGE 82

)LJXUH %LRORJLFDO GHJUDGDWLRQ SURILOH RI SPROH RI GRGHF\O O fLPLGD]RO\Of SURSLRQDWH ',3f ZKHQ LQFXEDWHG ZLWK 8 RI SRUFLQH HVWHUDVH DW S+ DQG r& RYHU WLPH 7KH SHDN KHLJKWV RI WKH LQWDFW ',3 ZHUH REWDLQHG DW YDULRXV WLPH SRLQWV 8VLQJ WKH 6FLHQWLVW SURJUDP WR ILW WKH GHJUDGDWLRQ FXUYH WKH SVHXGRILUVW RUGHU GHJUDGDWLRQ UDWH FRQVWDQW Nf ZDV GHWHUPLQHG WR EH Kf

PAGE 83

\ [ )LJXUH %LRORJLFDO GHJUDGDWLRQ UDWH SURILOH RI ELRGHJUDGDEOH S+VHQVLWLYH VXUIDFWDQWV %36f 9DULRXV UDWLRV 8SPROHf RI SRUFLQH HVWHUDVH WR WKUHH %36 GRGHF\O O LPLGD]RO\Of SURSLRQDWH ',3f ‘f PHWK\O LPLGD]RO\O ODXUHDWH 0,/f Af DQG 1 GRGHF\O LPLGD]ROH ',f $ff ZHUH SORWWHG DJDLQVW GHJUDGDWLRQ UDWH FRQVWDQWV PHDQs6'f LQ D S+ EXIIHU VROXWLRQ DW r& Q f 7KH OLQHDU UHODWLRQVKLS EHWZHHQ WKH GHJUDGDWLRQ UDWH FRQVWDQW RI HDFK %36 DQG WKH UDWLR RI HVWHUDVH WR %36 ZDV DOVR VKRZQ

PAGE 84

&RQH P0f Df )LJXUH &\WRWR[LF HIIHFW RI ELRGHJUDGDEOH S+VHQVLWLYH VXUIDFWDQWV %36f RQ WZR FHOO OLQHV PHDVXUHG E\ D FDOFHLQ$0 DVVD\ DIWHU K RI LQFXEDWLRQ 'DWD ZHUH H[SUHVVHG DV PHDQs6' Df ,Q 6.Q6+ FHOOV WKH ,'R b OLYH FHOOVf RI PHWK\O LPLGD]RO\O ODXUHDWH 0,/f $f GRGHF\O OfLPLGD]RO\Of SURSLRQDWH ',3f ‘f DQG1GRGHF\O LPLGD]ROH ',f Af ZDV YLVXDOO\ GHWHUPLQHG WR EH P0 P0 DQG P0 UHVSHFWLYHO\ Q f Ef ,Q &9 FHOOV WKH ,' RI 0,/ $f ',3 ‘f DQG ', Af ZDV YLVXDOO\ GHWHUPLQHG WR EH P0 P0 DQG P0 UHVSHFWLYHO\ Q f

PAGE 85

b /LYH &HOOV )LJXUH f§FRQWLQXHG ‘

PAGE 86

&RQH P0f )LJXUH &\WRWR[LF HIIHFW RI SRVVLEOH PHWDEROLWHV IURP ELRGHJUDGDEOH S+VHQVLWLYH VXUIDFWDQWV %36f RQ &9 FHOOV PHDVXUHG E\ D FDOFHLQ$0 DVVD\ DIWHU K RI LQFXEDWLRQ 7KH ,' b OLYH FHOOVf ZDV GHWHUPLQHG WR EH P0 IRU LPLGD]ROH Af P0 IRU GRGHFDQRO ff P0 IRU LPLGD]ROH PHWKDQRO ‘f DQG P0 IRU ODXULF DFLG ;f UHVSHFWLYHO\ Q f 'DWD DUH H[SUHVVHG DV PHDQs6'

PAGE 87

0,/ H[KLELWHG WKH OHDVW F\WRWR[LFLW\ FRPSDUHG WR WKH RWKHU WZR %36 DQG ZDV RQH RUGHU RI WKH PDJQLWXGH OHVV WR[LF WKDQ ',3 LQ ERWK FHOO OLQHV 7KH SRVVLEOH PHWDEROLWHV LPLGD]ROH DQG GRGHFDQRO IURP ',3 ZHUH PRUH WR[LF WKDQ WKRVH PHWDEROLWHV LPLGD]ROH PHWKDQRO DQG ODXULF DFLG IURP 0,/ LQ &9 FHOOV 'LVFXVVLRQ ,PLGD]RO\O EDVHG OLSLGV KDYH EHHQ XVHG VXFFHVVIXOO\ IRU LQ YLWUR GHOLYHU\ RI QXFOHLF DFLGV 6RORGLQ HW DO f HVWDEOLVKLQJ D UDWLRQDO IRU WKH XVH RI LPLGD]ROH 0RVW QRQYLUDO GHOLYHU\ V\VWHPV HQWHU FHOOV WKURXJK HQGRF\WRVLV 7KH PRVW VLJQLILFDQW FKDUDFWHULVWLF RI HQGRVRPHV LV WKH S+ JUDGLHQW IURP LQVLGH WKH HQGRVPH WR WKH LQWUDFHOOXODU VSDFH 'RGHF\O OfLPLGD]RO\Of SURSLRQDWH ',3f PHWK\O LPLGD]RO\O ODXUHDWH 0,/f DQG 1GRGHF\O LPLGD]ROH ',f DUH LPLGD]RO\O EDVHG VXUIDFWDQWV LQ WKH ELRGHJUDGDEOH S+ VHQVLWLYH VXUIDFWDQWV %36f IDPLO\ ZKLFK KDYH EHHQ SURSRVHG WR IDFLOLWDWH WKH WUDQVSRUW RI QXFOHLF DFLG WKURXJK WKH HQGRVRPDO SDWKZD\ %36 WDNH DGYDQWDJH RI WKH DFLGLF HQYLURQPHQW ZLWKLQ HQGRVRPHV WR SURWRQDWH D O\VRVRPRWURSLF DPLQH WKXV LQFUHDVLQJ WKHLU VXUIDFH DFWLYH SURSHUWLHV $IWHU %36 EHFRPH LRQL]HG WKH\ FDQ DVVLVW WKH GHVWDELOL]DWLRQ RI WKH HQGRVRPDO PHPEUDQH 7R OHVVHQ DGYHUVH HIIHFWV RI WKH LRQL]HG %36 DQ HVWHU ERQG ZDV LQWURGXFHG LQWR ',3 DQG 0,/fV VWUXFWXUH PDNLQJ LW ELRGHJUDGDEOH ,Q D SUHOLPLQDU\ VWXG\ +XJKHV HW DO f ',3 KDV EHHQ VKRZQ WR UHGXFH WKH FRQFHQWUDWLRQ RI ROLJRQXFOHRWLGHV UHTXLUHG WR SURGXFH D ELRORJLFDO HIIHFW XVLQJ D WLVVXH FXOWXUH V\VWHP ,Q WKLV UHSRUW WKH SK\VLFRFKHPLFDO SURSHUWLHV RI WKUHH %36 ZHUH V\VWHPDWLFDOO\ FKDUDFWHUL]HG

PAGE 88

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f 7KH PRUH K\GURSKLOLF D VXUIDFWDQW WKH KLJKHU WKH &0& 5RVHQ f )URP WKH FKHPLFDO VWUXFWXUHV ', ',3 DQG 0,/ KDG GHFUHDVLQJ RUGHUV RI K\GURSKLOLFLW\ $V D UHVXOW RI WKLV GLIIHUHQFH ', ',3 DQG 0,/ KDG GHFUHDVLQJ RUGHUV RI &0& LQ JHQHUDO $V WKH VL]H RI WKH FRXQWHU LRQ LQFUHDVHG WKH VXUIDFWDQW EHFDPH PRUH K\GURSKLOLF WKXV REWDLQLQJ WKH KLJKHU &0& &0& LV DQ HVVHQWLDO SDUDPHWHU LQ GHVFULELQJ VXUIDFWDQWV LH K\GURSKLOLFLW\ VXUIDFH H[FHVVf KRZHYHU LW PD\ QRW EH WKH EHVW SDUDPHWHU WR PHDVXUH WKH DELOLW\ RI D VXUIDFWDQW WR FDXVH PHPEUDQH O\VLV /LFKWHQEHUJ 5XL] HW DO f 7KHUHIRUH WKH HIIHFWLYH UHOHDVH UDWLR 5Hf ZDV XWLOL]HG WR GHVFULEH WKH DELOLW\ RI %36 WR O\VH PHPEUDQHV 7KH ORZHU WKH 5H WKH OHVV VXUIDFWDQW UHTXLUHG WR O\VH PHPEUDQHV )RU EDVLF DPLQHV VXFK DV %36 WKH LQWULQVLF LRQL]DWLRQ FRQVWDQW S.D f DQG ORFDO S+ HQYLURQPHQW GHWHUPLQH WKH SHUFHQW LRQL]HG 7KH UHODWLRQVKLS FDQ EH H[SUHVVHG ?%+ LQ +HQGHUVRQ+DVVHOEDFK HTXDWLRQ S.D S+ ORJ ZKHUH >%+@ DQG >%@ >f@ UHSUHVHQWV WKH LRQL]HG DQG XQLRQL]HG EDVHV UHVSHFWLYHO\ )URP WKH SHUVSHFWLYH RI WKHLU

PAGE 89

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f WKDW IDFLOLWDWHG SDUWLWLRQ LQWR OLSRVRPHV PRUH HDVLO\ ', ZDV DOPRVW LQGLVFULPLQDWH WR YDULRXV S+ HQYLURQPHQWV UHVXOWLQJ LQ D VLPLODU FDOFHLQ UHOHDVH SURILOH )RU WKH 0,/ WUHDWHG OLSRVRPH JURXS RQ WKH RWKHU KDQG D OLPLWHG DPRXQW RI FDOFHLQ ZDV GHWHFWHG DW S+ DQG EHFDXVH RI LWV ORZHVW S.D DQG FKHPLFDO KLQGUDQFH HIIHFW LH HVWHU OLQNHUf WKDW PLJKW GHFUHDVH WKH SDUWLWLRQ RI 0,/ LQWR OLSRVRPHV +RZHYHU ZLWK WKH PHGLDQ S.D YDOXH DQG FKHPLFDO KLQGUDQFH LH HVWHU OLQNHU DQG EUDQFKHG PHWK\O JURXSf RI ',3 PRUH FDOFHLQ ZDV UHOHDVHG WKDQ 0,/ DV S+ ZDV GHFUHDVHG IURP WR RU :LWK WKHVH WZR IDFWRUV LH S.D DQG SDUWLWLRQ FRHIILFLHQWf WKH FDOFHLQ UHOHDVH SURILOHV ZHUH WKHUHIRUH HVWDEOLVKHG LQ YDULRXV S+ HQYLURQPHQWV 7KH DELOLW\ RI %36 WR UHOHDVH OLSRVRPHHQWUDSSHG PROHFXOHV ZDV WHVWHG DV D SURRI RI WKH FRPSRXQGfV S+ VHQVLWLYLW\ EDVHG RQ WKH SULQFLSOH H[SHULPHQW $V WKH %36OLSRVRPH PRODU UDWLR ZDV LQFUHDVHG DQGRU S+ GHFUHDVHG PRUH FDOFHLQ ZDV UHOHDVHG IURP WKH OLSRVRPH PRGHO V\VWHP &DOFHLQ UHOHDVH LQGXFHG E\ %36 ZKHQ LQFRUSRUDWHG LQWR WKH OLSRVRPH V\VWHP ZDV VOLJKWO\ KLJKHU WKDQ WKDW FDXVHG E\ %36 ZKHQ DGGHG WR D

PAGE 90

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f DV FRPSDUHG WR WKH DON\O FKDLQ DQDORJ ',f ZKLFK GHPRQVWUDWHG OLWWOH S+ GHSHQGHQF\ 7KLV HIIHFW LV DOVR SRVVLEO\ GXH WR WKH FKDQJH RI S.D DW WKH LQWHUIDFH EHWZHHQ WKH PRQROD\HU RI WKH OLSRVRPH DQG WKH VROXWLRQ +RZHYHU LW LV XQOLNHO\ WKDW WKLV ODFN RI S+ GHSHQGHQF\ LV VROHO\ GXH WR FKDQJHV LQ WKH S.D RI %36 7KH GLIIHUHQW SURILOHV PD\ UHIOHFW WKH HDVH RI %36 WR SDUWLWLRQ LQWR WKH ELOD\HU %36 ZLWK D PRUH SRODU KHDG JURXS HJ HVWHU FRQWDLQLQJf ZRXOG EH H[SHFWHG WR KDYH DGGHG UHVLVWDQFH LQ PHPEUDQH SDUWLWLRQLQJ $IWHU UHOHDVLQJ PHPEUDQH HQWUDSSHG PROHFXOHV LQ D S+VHQVLWLYH PDQQHU WKH ELRGHJUDGDELOLW\ RI %36 ZDV FRQILUPHG 'XH WR LWV SRVVLEOH LQFUHDVHG VWDEOH PHWDEROLWHV DQG ODFN RI FKHPLFDO KLQGUDQFH 0,/ ZDV PRUH ELRGHJUDGDEOH WKDQ ',3 )LJXUH f 2Q WKH RWKHU KDQG ZLWKRXW DQ\ ELRGHJUDGDEOH ERQG HJ HVWHUf ', VKRZHG QR GLIIHUHQFH RI WKH GHJUDGDWLRQ UDWH FRQVWDQWV ZKHQ LQFXEDWHG LQ ELRORJLFDO PHGLD 7KH GHJUDGDWLRQ UDWH RI %36 LQ YLYR ZRXOG EH H[SHFWHG WR EH JUHDWHU GXH WR WKH KLJKHU QXPEHU RI HVWHUDVH HJ

PAGE 91

OLSDVHf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f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f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

PAGE 92

&RQFOXVLRQ 7KH XOWLPDWH JRDO RI WKLV SURMHFW ZDV WR DGMXVW FKHPLFDO FKDUDFWHULVWLFV RI %36 FRPSRQHQWV KHDG DQG WDLO JURXSV LQ RUGHU WR RSWLPL]H %36LQGXFHG UHOHDVH RI ROLJRQXFOHRWLGHV IURP PHPEUDQH FRPSDUWPHQWV )XUWKHUPRUH %36 ZRXOG KDYH PLQLPXP FHOOXODU WR[LFLW\ ZLWK WKH LQWURGXFWLRQ RI D ELRGHJUDGDEOH OLQNHU $W SUHVHQW WKH FULWLFDO PLFHOOH FRQFHQWUDWLRQ &0&f HIIHFWLYH UHOHDVH UDWLR 5Hf ELRGHJUDGDELOLW\ DQG F\WRWR[LFLW\ RI WKHVH WKUHH %36 KDYH OLWWOH ELRORJLFDO UHOHYDQFH $V PRUH GLYHUVH %36 DUH HVWDEOLVKHG DQG FKDUDFWHUL]HG ZLWK WKHVH PHQWLRQHG SDUDPHWHUV LW LV KRSHG WKDW SDUWLFXODU SK\VLFRFKHPLFDO SURSHUWLHV ZLOO EH SUHGLFWLYH RI SURPRWLQJ ROLJRQXFOHRWLGH ELRORJLFDO DFWLYLW\

PAGE 93

&+$37(5 '(/,9(5< 6<67(0 (9$/8$7,21 2) %,2'(*5$'$%/( S+6(16,7,9( 685)$&7$176 ,QWURGXFWLRQ 2OLJRQXFOHRWLGHV PXVW UHDFK WKHLU VLWHV RI DFWLRQ F\WRSODVP RU QXFOHXVf WR K\EULGL]H ZLWK WKHLU WDUJHWV WR H[HUW WKHLU HIIHFW $JUDZDO t ,\HU f +RZHYHU WKH FHOOXODU GHOLYHU\ RI IUHH ROLJRQXFOHRWLGHV LV YHU\ SRRU $NKWDU HW DK 6WHLQ t &KHQJ f 2QH VWUDWHJ\ WR LPSURYH WKH GHOLYHU\ RI WKH ROLJRQXFOHRWLGH LV WR XVH OLSRVRPHV ZKLFK FDQ FDUU\ ROLJRQXFOHRWLGHV ZLWK WKH YHKLFOHV DQG LQFUHDVH WKH LQWUDFHOOXODU DFFXPXODWLRQ YLD HQGRF\WRVLV /LSRVRPHV FDQ GHOLYHU ROLJRQXFOHRWLGHV WR WKH FHOOV EXW WKH REVWDFOH RI HVFDSLQJ IURP HQGRVRPHV VWLOO UHPDLQV $ VHULHV RI ELRGHJUDGDEOH S+VHQVLWLYH VXUIDFWDQWV %36f ZHUH GHYHORSHG WR FRQTXHU WKH SRWHQWLDO SLWIDOO WKDW WKH OLSRVRPH GHOLYHU\ V\VWHP PLJKW KDYH DQG WR IXUWKHU HQKDQFH FHOOXODU GHOLYHU\ RI WKH ROLJRQXFOHRWLGH ,Q SUHYLRXV VWXGLHV WKH SK\VLFRFKHPLFDO SURSHUWLHV RI %36 ZHUH FKDUDFWHUL]HG 7KH\ ZHUH VKRZQ WR EH DEOH WR LQGXFH PHPEUDQH O\VLV LQ D S+ DQG PRODU UDWLR GHSHQGHQW PDQQHU &KDSWHU f 1HYHUWKHOHVV WKH %36OLSRVRPH V\VWHP KDV QRW \HW EHHQ WHVWHG XVLQJ D ELRORJLFDO V\VWHP 7KHUHIRUH ZH IXUWKHU HYDOXDWHG WKH %36OLSRVRPH GHOLYHU\ V\VWHP LQ YLWUR

PAGE 94

7R HYDOXDWH ROLJRQXFOHRWLGH FHOOXODU XSWDNH ZLWK WKH %36OLSRVRPH V\VWHP IORZ F\WRPHWU\ ZDV HPSOR\HG $QRWKHU VFUHHQLQJ PHWKRG WKDW DGGUHVVHG ROLJRQXFOHRWLGH FHOOXODU GHOLYHU\ ZDV WKH XVH RI ODVHU VFDQQLQJ FRQIRFDO PLFURVFRS\ )LVKHU HW DO f :H XVHG WKLV WHFKQLTXH WR LQYHVWLJDWH ROLJRQXFOHRWLGH FHOOXODU XSWDNH DQG GLVWULEXWLRQ WKDW WKH %36OLSRVRPH GHOLYHU\ V\VWHP DIIHFWHG $GGLWLRQDOO\ ZH TXDQWLWDWLYHO\ PRQLWRUHG WKH LQKLELWLRQ HIIHFW RI ROLJRQXFOHRWLGHV LQ %36OLSRVRPHV RQ OXFLIHUDVH HQ]\PH DFWLYLW\ %UDVLHU HW DK f 0DWHULDOV &KHPLFDO 'LK\GURJHQ SRWDVVLXP SKRVSKDWH ('7$ IRUPDOGHK\GH VROXWLRQ DQG 7ULWRQ ; ZHUH ERXJKW IURP )LVKHU 6FLHQWLILF 3LWWVEXUJK 3$f $GHQRVLQH WULSKRVSKDWH GLWKLRWKUHLWRO '77f PDJQHVLXP VXOIDWH DQG WULFLQH ZHUH SXUFKDVHG IURP 6LJPD 6W /RXLV 02f %&$ 3URWHLQ $VVD\ .LW ZDV REWDLQHG IURP 3LHUFH 5RFNIRUG ,/f DQG /DEHO ,7 IOXRUHVFHLQ 1XFOHLF $FLG /DEHOLQJ .LW IURP 0LUDV 0DGLVRQ :,f 7KH S*/ SODVPLG '1$ ZDV REWDLQHG IURP 0U )X[LQJ 7DQJ LQ WKH 'HSDUWPHQW RI 3KDUPDFHXWLFV 8QLYHUVLW\ RI )ORULGD 'OXFLIHULQ ZDV SXUFKDVHG IURP 0ROHFXODU 3UREHV (XJHQH 25f /DOHFLWKLQ DQG 1>OOGLROHROR[\fSURS\O@111WULPHWK\ODPPRQLXP PHWK\OVXOIDWH '27$3f ZDV SXUFKDVHG IURP $YDQWL 3RODU /LSLGV $ODEDVWHU $/f 7KUHH ELRGHJUDGDEOH S+VHQVLWLYH VXUIDFWDQWV %36f GRGHF\O OfLPLGD]RO\Of SURSLRQDWH ',3f PHWK\O LPLGD]RO\O ODXUHDWH 0,/f DQG GRGHF\O LPLGD]ROH ',f ZHUH V\QWKHVL]HG

PAGE 95

DV SUHYLRXVO\ UHSRUWHG &KDSWHU f $OO SXUFKDVHG RU REWDLQHG FKHPLFDOV ZHUH XVHG GLUHFWO\ ZLWKRXW DGGLWLRQDO SXULILFDWLRQ &HOO 7KH &9 OXFLIHUDVH H[SUHVVLQJ FHOO OLQH ZDV D JHQHURXV JLIW IURP 'U 0 & &KR RI WKH 8QLYHUVLW\ RI 1RUWK &DUROLQD 7KH 5$: 7,%f FHOO OLQH ZDV SXUFKDVHG IURP WKH $PHULFDQ 7\SH &XOWXUH &ROOHFWLRQ 5RFNYLOOH 0'f 0HWKRGV 2OLJRQXFOHRWLGH DQG 3ODVPLG '1$ &HOOXODU 8SWDNH (YDOXDWLRQ 8VLQJ )ORZ &\WRPHWU\ 2OLJRQXFOHRWLGH V\QWKHVLV DQG OLSRVRPH SUHSDUDWLRQ 3KRVSKRURWKLRDWH ROLJRQXFOHRWLGHV EDVHV f7** &*7 &77 &&$ 777ff ODEHOHG ZLWK IOXRUHVFHLQ LVRWKLRF\DQDWH ),7&f DW WKH fHQG ZHUH V\QWKHVL]HG LQ WKH '1$ &RUH 6\QWKHVLV /DE DW WKH 8QLYHUVLW\ RI )ORULGD 2OLJRQXFOHRWLGHV ZHUH XVHG GLUHFWO\ ZLWKRXW DGGLWLRQDO SXULILFDWLRQ S*/ SODVPLG '1$ ZLWK D VL]H RI ES DQG SXULW\ RI $$f ZDV ODEHOHG ZLWK IOXRUHVFHLQ XVLQJ D 0LUXV /DEHO ,7 1XFOHLF $FLG ODEHOLQJ .LW 6LPSO\ SJ RI WKH SODVPLG '1$ PJPOf UHDFWHG ZLWK SL RI IOXRUHVFHLQ UHDJHQW DW ZYf UDWLR ZDV GLOXWH WR D ILQDO FRQFHQWUDWLRQ RI PJPO LQ ,; ODEHOLQJ EXIIHU DQG LQFXEDWHG DW r& IRU K 7KH XQUHDFWHG ODEHOLQJ UHDJHQW ZDV UHPRYHG IURP ODEHOHG QXFOHLF DFLG E\ VHSKDGH[ VSLQ FROXPQV DW D IRUFH RIrJ IRU PLQ DQG WKHQ WKH SXULILHG VDPSOH FROOHFWHG

PAGE 96

&DWLRQLF 1> GLROHROR[\fSURS\O@111WULPHWK\ODPPRQLXP PHWK\OVXOIDWH '27$3f OLSRVRPHV ZHUH PDGH DV D FRQWURO YHFWRU IRU QXFOHLF DFLG GHOLYHU\ %LRGHJUDGDEOH S+VHQVLWLYH VXUIDFWDQWV %36f DW YDULRXV PRODU UDWLRV ZHUH FRPELQHG ZLWK '27$3 WR FUHDWH OLSRVRPHV $IWHU OLSRVRPHV ZHUH UHK\GUDWHG D 6RQLF 'LVPHPEUDWRU SUREH ZDV XVHG WR IRUP VPDOO XQLODPHOODU YHVLFOHV E\ DSSO\LQJ :DWWV RI SRZHU IRU V WR WKH OLSRVRPH VXVSHQVLRQ DQG NHHSLQJ RQ LFH IRU V 7KH F\FOH ZDV UHSHDWHG XQWLO D FOHDU VROXWLRQ ZDV VHHQ 7KH VL]H RI WKH OLSRVRPHV YROXPHZHLJKW *DXVVLDQ GLVWULEXWLRQf ZDV GHWHUPLQHG WR EH QP VWDQGDUG GHYLDWLRQf E\ D G\QDPLF OLJKW VFDWWHULQJ PHWKRG XVLQJ D 1,&203 =/6 =HWD 3RWHQWLDO3DUWLFOH 6L]HU 6DQWD %DUEDUD &$f &HOO SUHSDUDWLRQ 0RQROD\HUHG &9 PRQNH\ NLGQH\ ILEUREODVWf FHOOV ZHUH LQFXEDWHG LQ ZHOO SODWHV r FHOOVZHOOf ZLWK PO RI 0(0 JURZWK PHGLXP LQ HDFK ZHOO DW r& b & DQG D b KXPLGLW\ HQYLURQPHQW IRU K 7KH PHGLXP LQFOXGHG 8PO SHQLFLOOLQ SJPO VWUHSWRP\FLQ P0 0(0 VRGLXP S\UXYDWH VROXWLRQ ,; 0(0 DPLQR DFLGV VROXWLRQ DQG b KHDWHG IHWDO ERYLQH VHUXP ([SHULPHQWDO SURFHGXUHV (LWKHU QPROH RI WKH OLSRVRPH '27$3 RU '27$3GRGHF\O OfLPLGD]RO\Of SURSLRQDWH ',3f DW PRODU UDWLR 5 ff ZDV FRPSOH[HG ZLWK QPROH RI WKH ),7&ROLJRQXFOHRWLGH RU SJ RI WKH OLSRVRPH ZLWK SJ RI WKH SODVPLG '1$ LQ HDFK ZHOO IRU PLQ 7KH FKDUJH UDWLR f RI '27$3 WR ROLJRQXFOHRWLGH ZDV VHW WR EH ZKLFK ZDV SURSRVHG DV WKH RSWLPDO UDWLR LQ RWKHU VLPLODU VWXG\ =HOSKDWL t 6]RND

PAGE 97

Df 7KH FKDUJH UDWLR f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f DQG 1GRGHF\O LPLGD]ROH ',ff ZHUH LQFRUSRUDWHG LQWR FDWLRQLF '27$3 OLSRVRPHV DW IRXU PRODU UDWLRV 5 5 5 DQG 5 f $IWHU D PLQ SHULRG RI FRPSOH[DWLRQ HLWKHU QPROH RI WKH OLSRVRPH ZLWK QPROH RI WKH IOXRUHVFHLQ ODEHOHG ROLJRQXFOHRWLGH RU SJ RI WKH OLSRVRPH ZLWK SJ RI WKH IOXRUHVFHLQODEHOHG SODVPLG '1$ ZLWK DW D FKDUJH UDWLR RI f ZDV LQFXEDWHG ZLWK &9 FHOOV LQ SL RI VHUXP IUHH JURZWK PHGLXP $IWHU K WKH FHOOV ZHUH KDUYHVWHG DQG WKHQ DQDO\]HG E\ IORZ F\WRPHWU\ )ORZ F\WRPHWU\ ,Q RUGHU WR PLQLPL]H WKH QXFOHLF DFLGV DGVRUEHG RQWR FHOOV WKH FHOOV ZHUH ZDVKHG WZLFH ZLWK SKRVSKDWH EXIIHUHG VDOLQH 3%6f DQG OLIWHG IURP WKH ZHOOV EHIRUH DQDO\]LQJ WKH VDPSOHV 7KH FHOOV ZHUH WKHQ WUDQVIHUUHG WR WXEHV DQG FHQWULIXJHG DW USP IRU PLQ 7KH VXSHUQDWDQW SLf ZDV GHFDQWHG DQG UHVXVSHQGHG ZLWK SL RI 3%6 7KH DERYH SURFHGXUH ZDV UHSHDWHG WZLFH DQG WKH VDPSOHV NHSW RQ LFH XQWLO DQDO\VLV 7R DGGUHVV

PAGE 98

QXFOHLF DFLG FHOOXODU XSWDNH LQ VRPH VWXGLHV UDWKHU WKDQ XVLQJ 3%6 LQ WKH ILQDO VWHS b IRUPDOGHK\GH ZDV DGGHG LQWR WKH WXEHV IRU PLQ WR IL[ WKH FHOOV 7KH VLJQDOV HPLWWHG IURP WKH ),7&ROLJRQXFOHRWLGH RU IOXRUHVFHLQSODVPLG '1$f ZHUH SHUIRUPHG E\ D %HFWRQ 'LFNLQVRQ )$&6RUW IORZ F\WRPHWHU 6DQ -RVH &$f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f 2OLJRQXFOHRWLGH &HOOXODU 8SWDNH DQG 'LVWULEXWLRQ (YDOXDWLRQ ZLWK &RQIRFDO 0LFURVFRS\ 2OLJRQXFOHRWLGH V\QWKHVLV DQG OLSRVRPH SUHSDUDWLRQ )LIWHHQ EDVHV RI SRO\$ SKRVSKRURWKLRDWH ROLJRQXFOHRWLGHV ODEHOHG ZLWK IOXRUHVFHLQ LVRWKLRF\DQDWH ),7&f DW WKH fHQG ZHUH V\QWKHVL]HG LQ WKH '1$ &RUH 6\QWKHVLV /DE DW WKH 8QLYHUVLW\ RI )ORULGD 7KH\ ZHUH XVHG GLUHFWO\ ZLWKRXW DGGLWLRQDO SXULILFDWLRQ 7ZR GLIIHUHQW OLSLG IRUPXODWLRQV ZHUH XVHG /DOHFLWKLQ DQG /DOHFLWKLQ ZLWK GRGHF\O O fLPLGD]RO\Of SURSLRQDWH ',3f DW PRODU UDWLR 5 f 7KH OLSLG UHK\GUDWLRQ PHWKRG ZDV XVHG WR IRUP QHXWUDO OLSRVRPHV DV ),7&ODEHOHG ROLJRQXFOHRWLGHV GLVVROYHG LQ WKH DTXHRXV VROYHQW 7R LQFUHDVH WKH HQFDSVXODWLRQ HIILFLHQF\ RI

PAGE 99

ROLJRQXFOHRWLGHV LQWR WKH OLSRVRPHV ILYH IUHH]HDQGWKDZ F\FOHV ZHUH HPSOR\HG DIWHU D PLQ SHULRG RI KDQG VKDNLQJ 7KH OLSRVRPHV ZHUH WKHQ SDVVHG WKUHH WLPHV WKURXJK QP SRO\FDUERQDWH PHPEUDQHV 3RUHWLFV /LYHUPRUH &$f XVLQJ D KLJK SUHVVXUH H[WUXGHU /LSH[ %LRPHPEUDQH ,QF 9DQFRXYHU &DQDGDf 7KH FRQFHQWUDWLRQ RI WKH SKRVSKROLSLG ZDV FDOFXODWHG DV SUHYLRXVO\ UHSRUWHG &KDSWHU f 7KH YROXPHZHLJKW *DXVVLDQ GLVWULEXWLRQ RI WKH OLSRVRPH VL]H ZDV GHWHUPLQHG WR EH QP VWDQGDUG GHYLDWLRQf E\ D G\QDPLF OLJKW VFDWWHULQJ PHWKRG XVLQJ D 1,&203 =/6 =HWD 3RWHQWLDO3DUWLFOH 6L]HU 6DQWD %DUEDUD &$f &HOO SUHSDUDWLRQ %HIRUH SODWLQJ 5$: PRXVH PRQRF\WHPDFURSKDJHf FHOOV D FRYHU VOLS ZDV SODFHG LQ HDFK ZHOO RI ZHOO SODWHV IRU ODWHU REVHUYDWLRQ RQ D FRQIRFDO PLFURVFRSH (DFK ZHOO FRQWDLQLQJ D FRYHU VOLS ZDV WUHDWHG ZLWK SL RI FROODJHQ LQ D 0 DFHWLF DFLG VROXWLRQ SJPOf DQG LQFXEDWHG DW URRP WHPSHUDWXUH $IWHU K WKH VROXWLRQ ZDV UHPRYHG E\ ULQVLQJ ZLWK SKRVSKDWH EXIIHUHG VDOLQH 3%6f WKUHH WLPHV 7KH SODWHV ZHUH WKHQ UHDG\ IRU XVH 6XEFRQIOXHQW PRQROD\HUHG 5$: FHOOV ZHUH FXOWXUHG LQ WKH ZHOO SODWHV r FHOOVZHOOf ZLWK PO RI '0(0 JURZWK PHGLXP LQ HDFK ZHOO DW r& b & DQG D b KXPLGLW\ HQYLURQPHQW IRU K 7KH PHGLXP LQFOXGHG 8PO SHQLFLOOLQ SJPO VWUHSWRP\FLQ DQG b IHWDO ERYLQH VHUXP

PAGE 100

([SHULPHQWDO SURFHGXUHV $IWHU WKH LQFXEDWLRQ WKH JURZWK PHGLXP LQ HDFK ZHOO ZDV UHSODFHG ZLWK SL RI VHUXP IUHH PHGLXP FRQWDLQLQJ VDPH DPRXQW RI VWDUWLQJ ),7&ROLJRQXFOHRWLGHV DV WKH IROORZLQJ SUHSDUDWLRQV f ),7&ROLJRQXFOHRWLGH f ),7&ROLJRQXFOHRWLGHOLSRVRPH QPROHf f ),7&ROLJRQXFOHRWLGH',3OLSRVRPH 5 f QPROHf &RQIRFDO PLFURVFRS\ $IWHU K RI LQFXEDWLRQ LQ VHUXP IUHH PHGLXP b IHWDO ERYLQH VHUXP ZDV DGGHG $W WKH HQG RI HDFK VDPSOLQJ WLPH K K DQG Kf WKH ZHOOV ZHUH ZDVKHG ZLWK 3%6 DQG FHOOV IL[HG LQ b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b QHXWUDO GHQVLW\ ILOWHU WR PLQLPL]H SKRWREOHDFKLQJ DQG SKRWRGDPDJH

PAGE 101

4XDQWLWDWLYH (IIHFW (YDOXDWLRQ RI /XFLIHUDVH $FWLYLW\ 2OLJRQXFOHRWLGH V\QWKHVLV DQG OLSRVRPH SUHSDUDWLRQ 3KRVSKRURWKLRDWH ROLJRQXFOHRWLGHV ZLWK DQ DQWLVHQVH VHTXHQFH EDVHV f7** &*7 &77 &&$ 777ff DQG D VHQVH VHTXHQFH EDVHV f777 $&& 77& 7*& *HOn Vff ZHUH V\QWKHVL]HG LQ WKH '1$ &RUH 6\QWKHVLV /DE DW WKH 8QLYHUVLW\ RI )ORULGD 7KH\ ZHUH XVHG GLUHFWO\ ZLWKRXW IXUWKHU SXULILFDWLRQ 6LQFH PRODU UDWLR GLG QRW SOD\ D VLJQLILFDQW UROH LQ FKDUDFWHUL]LQJ ROLJRQXFOHRWLGH FHOOXODU XSWDNH LQGXFHG E\ %36OLSRVRPHV WR PLQLPL]H F\WRWR[LFLW\ DVVRFLDWHG ZLWK ',3 D PRODU UDWLR RI ',3 WR '27$3 5 f ZDV XVHG LQ RXU VXEVHTXHQW ELRORJLFDO HYDOXDWLRQ VWXG\ $ FDWLRQLF OLSLG 1>OOGLROHROR[\fSURS\O@111 WULPHWK\ODPPRQLXP PHWK\OVXOIDWH '27$3f ZLWK RU ZLWKRXW GRGHF\O O fLPLGD]RO\Of SURSLRQDWH ',3f ZHUH PL[HG WR FUHDWH OLSRVRPHV $IWHU OLSRVRPHV ZHUH UHK\GUDWHG DV LQ WKH SUHYLRXV VHW RI H[SHULPHQW D 6RQLF 'LVPHPEUDWRU SUREH ZDV XVHG WR IRUP VPDOO XQLODPHOODU YHVLFOHV QPf DQG VL]H GLVWULEXWLRQ RI WKH OLSRVRPHV FRQILUPHG E\ D 1,&203 =/6 =HWD 3RWHQWLDO3DUWLFOH 6L]HU 6DQWD %DUEDUD &$f &HOO SUHSDUDWLRQ 6XEFRQIOXHQW &9 OXFLIHUDVH H[SUHVVLQJ FHOOV ZHUH JURZQ LQ ZHOO SODWHV r FHOOVZHOOf ZLWK PO RI 0(0 JURZWK PHGLXP LQ HDFK ZHOO DW r& b & DQG D b KXPLGLW\ HQYLURQPHQW IRU K 7KH PHGLXP LQFOXGHG 8PO SHQLFLOOLQ /LJnPO VWUHSWRP\FLQ P0 0(0 VRGLXP S\UXYDWH VROXWLRQ ,; 0(0 DPLQR DFLG VROXWLRQ DQG b KHDWHG IHWDO ERYLQH VHUXP

PAGE 102

([SHULPHQWDO SURFHGXUHV )RU HDFK OLSLG IRUPXODWLRQ '27$3 RU ',3'27$3 5 ff WKHUH ZHUH IRXU WUHDWPHQWV WR LQYHVWLJDWH WKH HIIHFW ZKLFK ',3 PLJKW KDYH RQ LQKLELWLQJ HQ]\PH H[SUHVVLRQ f $QWLVHQVH ROLJRQXFOHRWLGHOLSRVRPH f 6HQVH ROLJRQXFOHRWLGHOLSRVRPH f $QWLVHQVH ROLJRQXOHRWLGH f /LSRVRPH %HIRUH DGGLQJ ROLJRQXFOHRWLGHV LQWR ZHOOV YDULRXV FRQFHQWUDWLRQV RI WKH OLSRVRPH ZHUH FRPSOH[HG ZLWK WKH ROLJRQXFOHRWLGH DW D FKDUJH UDWLR RI f IRU PLQ 7KH JURZWK PHGLXP LQ HDFK ZHOO ZDV UHPRYHG DQG WKH OLSRVRPHROLJRQXFOHRWLGH FRPSOH[ DGGHG LQWR SL RI VHUXP IUHH JURZWK PHGLXP $IWHU K RI LQFXEDWLRQ WKH JURZWK PHGLXP LQFOXGLQJ WKH OLSRVRPHROLJRQXFOHRWLGH FRPSOH[ ZDV GLVFDUGHG DQG UHSODFHG ZLWK QHZ JURZWK PHGLXP FRQWDLQLQJ b IHWDO ERYLQH VHUXP 7KH V\VWHP ZDV LQFXEDWHG IRU DQRWKHU K EHIRUH WKH ILQDO DQDO\VLV RI OXFLIHUDVH DFWLYLW\ /XFLIHUDVH DVVD\ 7KH JURZWK PHGLXP ZDV UHPRYHG DQG WKH FHOOV ZDVKHG ZLWK PO RI S+ SKRVSKDWH EXIIHUHG VDOLQH 3%6f $IWHU UHPRYLQJ 3%6 FHOOV ZHUH O\VHG ZLWK SL RI S+ OXFLIHUDVH O\VLV EXIIHU 0 GLK\GURJHQ SRWDVVLXP SKRVSKDWH P0 ('7$ b 7ULWRQ ; DQG P0 GLWKLRWKUHLWRO '77ff 7KH SODWH ZDV WKHQ VKDNHQ JHQWO\ IRU PLQ 7KH O\VDWHV ZHUH WUDQVIHUUHG LQWR PO WXEHV DQG FHQWULIXJHG DW USP IRU PLQ $ PL[WXUH RI SL RI WKH VXSHUQDWDQW DQG SL RI WKH S+ OXFLIHUDVH DVVD\

PAGE 103

EXIIHU [Q0 WULFLQH P0 DGHQRVLQH WULSKRVSKDWH $73f P0 PDJQHVLXP VXOIDWH DQG P0 '77f ZHUH DGGHG WR D SO LQMHFWLRQ RI P0 'OXFLIHULQ S+ f /XFLIHUDVH DFWLYLW\ RI WKH VDPSOHV ZDV PHDVXUHG ZLWK D 0RQROLJKW /XPLQRPHWHU 7KH OXPLQRPHWHU UHDG OLJKW SURGXFWLRQ IRU V DW URRP WHPSHUDWXUH 3URWHLQ DVVD\ 7R VWDQGDUGL]H FHOO QXPEHUV LQ HDFK ZHOO RI WKH FOXVWHU SODWH WKH WRWDO DPRXQW RI SURWHLQ ZDV TXDQWLILHG XVLQJ D %&$ 3URWHLQ $VVD\ .LW %ULHIO\ SL RI 3%6 ZDV SODFHG LQ HDFK ZHOO RI D ZHOO SODWH $ VWDQGDUG FDOLEUDWLRQ FXUYH ZDV FRQVWUXFWHG IRU HDFK DVVD\ XVLQJ ERYLQH VHUXP DOEXPLQ 7KH VXSHUQDWDQW RI WKH O\VDWH SLf IURP HDFK ZHOO ZDV SODFHG LQ WKH DSSURSULDWH ZHOO RI WKH ZHOO SODWH DQG PL[HG JHQWO\ 7KH %&$ UHDJHQW SLf ZDV WKHQ DGGHG WR HDFK ZHOO 7KH SODWH ZDV LQFXEDWHG DW r& IRU PLQ DQG UHDG DW QP RQ DQ (/ %LR .LQHWLFV 5HDGHU 7KH DPRXQW RI SURWHLQ LQ HDFK ZHOO ZDV WKHQ FDOFXODWHG E\ VXEVWLWXWLQJ WKH DEVRUEHQW WR WKH FDOLEUDWLRQ FXUYH FUHDWHG LQ WKH VDPH SODWH 6WDWLVWLFDO $QDO\VLV 6WDWLVWLFDO GLIIHUHQFHV EHWZHHQ WKH WUHDWPHQWV ZHUH GHWHUPLQHG XVLQJ DQDO\VLV RI YDULDQFH ZKHUH DSSURSULDWH 6WDW9LHZ $EDFXV &RQFHSWV ,QF %HUNHOH\ &$f ZLWK S FRQVLGHUHG VWDWLVWLFDOO\ VLJQLILFDQW DQG )LVKHUfV 3/6'f SRVW KRF WWHVW ZDV DSSOLHG

PAGE 104

5HVXOWV 2OLJRQXFOHRWLGH DQG 3ODVPLG '1$ &HOOXODU 8SWDNH (YDOXDWLRQ 8VLQJ )ORZ &\WRPHWU\ 7R H[DPLQH WKH HIIHFW RI ELRGHJUDGDEOH S+VHQVLWLYH VXUIDFWDQWV %36f LQ WKH FDWLRQLF 1>OOGLROHROR[\fSURS\O@111WULPHWK\ODPPRQLXP PHWK\OVXOIDWH '27$3f OLSRVRPHV RQ ROLJRQXFOHRWLGH RU SODVPLG '1$f FHOOXODU XSWDNH D IORZ F\WRPHWU\ WHFKQLTXH ZDV XVHG )LJXUH f )OXRUHVFHLQ LVRWKLRF\DQDWH ),7&fODEHOHG ROLJRQXFOHRWLGHV RU IOXRUHVFHLQODEHOHG SODVPLG '1$f ZHUH FRPSOH[HG ZLWK FDWLRQLF '27$3 OLSRVRPHV SOXV RU PLQXV GRGHF\O O fLPLGD]RO\Of SURSLRQDWH ',3f DW PRODU UDWLR 5 f IRU PLQ 7KH FRPSOH[ ZDV WKHQ LQFXEDWHG ZLWK &9 FHOOV DQG WKH IOXRUHVFHQFH LQWHQVLW\ HPLWWHG IURP WKH ROLJRQXFOHRWLGHV ZDV UHFRUGHG DW YDULRXV WLPH SRLQWV ZLWK RU ZLWKRXW FHOO IL[DWLRQ 7KH IOXRUHVFHQFH LQWHQVLWLHV LQ ERWK W\SHV RI FHOOV IL[HG RU OLYHf IURP WKH IUHH ROLJRQXFOHRWLGH ZHUH VLJQLILFDQWO\ ORZHU WKDQ WKRVH IURP WKH ROLJRQXFOHRWLGH ZLWK GHOLYHU\ V\VWHPV DW DOO REVHUYHG WLPH SRLQWV )LJXUH f $OVR WKH IOXRUHVFHQFH VLJQDOV IURP ',3 WUHDWHG OLSRVRPHV LQ OLYH FHOOV DW HDUO\ WLPH SRLQWV K DQG Kf ZHUH VLJQLILFDQWO\ Sf KLJKHU WKDQ WKRVH IURP OLSRVRPHV ZLWKRXW ',3 )LJXUH Df :KHQ WKH FHOOV ZHUH IL[HG WR HTXDOL]H WKH LQWUDFHOOXODU FRPSDUWPHQWV ZLWK UHJDUG WR S+ QR GLIIHUHQFH LQ WKH IOXRUHVFHQFH VLJQDO ZDV VHHQ EHWZHHQ WKHVH WZR JURXSV )LJXUH Ef 2OLJRQXFOHRWLGH FHOOXODU XSWDNH FDXVHG E\ WKUHH %36 ',3 PHWK\O LPLGD]RO\O ODXUHDWH 0,/f DQG 1GRGHF\O LPLGD]ROH ',ff DW GLIIHUHQW PRODU UDWLRV 5 5 5 DQG 5 f ZDV IXUWKHU LQYHVWLJDWHG $IWHU K RI LQFXEDWLRQ LQ WKH OLYH &9

PAGE 105

2, 2 D 2 IO R f R O2 &0 L R R L f0 f R A R Zfn 8' r rf r r‘ m mU f rA r ,}r b f r r rr r r L I Pr I r WIrr Mr = r f ‘} n‘r } W r!er Z r L Y_ f -r 99 n n ‹ Ir ` nrn r cU r }W rm } r Yr crr }r 9,r9rr n9 r r L ,0 g ‘r } I rn fL YMt:Y P" ‘‘ 9rn Vr Z Z r7$?-V r -r r 7 r r S:0J}UQLALOLW_}__n\ UXQUU r )/+?)/+HLJKW I:rr I r 9r : \LUILU APQD n )LJXUH 'LVWULEXWLRQ RI WKH OLYH &9 FHOOV YHUVXV WKH IOXRUHVFHQFH LQWHQVLW\ PHDVXUHG E\ IORZ F\WRPHWU\ %HIRUH DQDO\]LQJ WKH IOXRUHVFHQFH VLJQDOV QPROH RI WKH IOXRUHVFHLQODEHOHG ROLJRQXFOHRWLGH FRPSOH[HG ZLWK 1>OOGLROHROR[\fSURS\O@ 111WULPHWK\ODPPRQLXP PHWK\OVXOIDWH '27$3f OLSRVRPHV DW D FKDUJH UDWLR RI f ZHUH LQFXEDWHG LQ WKH ZHOO IRU K 7KH UHODWLYH IOXRUHVFHQFH LQWHQVLW\ RI WKH ILUVW FHOOV ZDV FDOFXODWHG WR EH

PAGE 106

Df )LJXUH 7LPH FRXUVH VWXG\ RI FDWLRQLF OLSRVRPHV RQ IOXRUHVFHLQODEHOHG ROLJRQXFOHRWLGH FHOOXODU XSWDNH LQ &9 FHOOV Q f 2OLJRQXFOHRWLGH QPROHf ZLWKRXW $f DQG ZLWK D GHOLYHU\ V\VWHP LH 1>OOGLROHROR[\fSURS\O@111 WULPHWK\ODPPRQLXP PHWK\OVXOIDWH '27$3f OLSRVRPHV SOXV ‘f RU PLQXV Af GRGHF\O OfLPLGD]RO\Of SURSLRQDWH ',3f ZLWK PRODU UDWLR 5 f DW D FKDUJH UDWLR RI ff ZHUH FRPSDUHG 7KH IOXRUHVFHQFH LQWHQVLWLHV PHDQWVWDQGDUG GHYLDWLRQ 6'ff ZHUH UHFRUGHG E\ IORZ F\WRPHWU\ LQ WZR FHOO FRQGLWLRQV Df /LYH &9 FHOOV Ef )L[HG &9 FHOOV

PAGE 107

Ef )LJXUH f§FRQWLQXHG

PAGE 108

FHOOV VLJQLILFDQW GLIIHUHQFHV Sf LQ WKH IOXRUHVFHQFH LQWHQVLW\ ZHUH REVHUYHG EHWZHHQ WKH ',3 WUHDWHG OLSRVRPH JURXSV 5 5 DQG 5 f DQG EODQN OLSRVRPHV )LJXUH Df +RZHYHU QR GLIIHUHQFH LQ WKH IOXRUHVFHQFH LQWHQVLW\ ZDV VHHQ DPRQJ WKH ',3 WUHDWHG OLSRVRPH JURXSV :KHQ &9 FHOOV ZHUH IL[HG QR GLIIHUHQFH LQ WKH IOXRUHVFHQFH LQWHQVLW\ ZDV REVHUYHG DPRQJ DOO OLSRVRPH JURXSV )LJXUH Df 6LPLODU IOXRUHVFHQFH LQWHQVLW\ SURILOHV ZHUH DOVR VHHQ LQ WKH RWKHU WZR %36 0,/ DQG ',f WUHDWHG OLSRVRPHV DPRQJ DOO PRODU UDWLR JURXSV 5 5 5 DQG 5 f LQ ERWK OLYH DQG IL[HG FHOOV )LJXUH E DQG )LJXUH Ff ,Q DGGLWLRQ QR GLIIHUHQFH RI WKH IOXRUHVFHQFH LQWHQVLW\ ZDV REVHUYHG DPRQJ WKHVH WKUHH %36 DW DQ\ PRODU UDWLR JURXSV :7LHQ ),7&ROLJRQXFOHRWLGHV ZHUH UHSODFHG E\ IOXRUHVFHLQSODVPLG '1$ QR GLIIHUHQFH LQ WKH IOXRUHVFHQFH LQWHQVLW\ EHWZHHQ '27$3 OLSRVRPHV DQG ',3 WUHDWHG '27$3 OLSRVRPHV 5 f ZDV REVHUYHG WKURXJKRXW WKH HQWLUH WLPH FRXUVH LQ WKH OLYH &9 FHOOV )LJXUH Df :KHQ WKH FHOOV ZHUH IL[HG EHIRUH DQDO\VLV QR GLIIHUHQFH RI WKH IOXRUHVFHQFH VLJQDO ZDV VHHQ )LJXUH Ef 3ODVPLG '1$ FHOOXODU XSWDNH FDXVHG E\ WKUHH %36 ',3 0,/ DQG ',f DW GLIIHUHQW PRODU UDWLRV 5 5 5 DQG 5 f ZDV DOVR LQYHVWLJDWHG $IWHU K RI LQFXEDWLRQ LQ ERWK OLYH DQG IL[HG &9 FHOOV QR GLIIHUHQFH LQ WKH IOXRUHVFHQFH VLJQDO ZDV VHHQ DPRQJ DOO ',3OLSRVRPH JURXSV 5 5 5 DQG 5 f )LJXUH Df 6LPLODU IOXRUHVFHQFH LQWHQVLW\ SURILOHV ZHUH DOVR VHHQ LQ WKH RWKHU WZR %36OLSRVRPH JURXSV 0,/ DQG ',f LQ ERWK WKH OLYH DQG IL[HG FHOOV )LJXUH E DQG )LJXUH Ff

PAGE 109

)LJXUH (IIHFW RI FDWLRQLF 1>OOGLROHROR[\fSURS\O@111WULPHWK\ODPPRQLXP PHWK\OVXOIDWH '27$3f OLSRVRPHV FRQWDLQLQJ WKUHH ELRGHJUDGDEOH S+VHQVLWLYH VXUIDFWDQWV %36f DW YDULRXV PRODU UDWLRV 5 RSHQ EDUf 5 VROLG EDUf 5 YHUWLFDO EDUf DQG 5 KRUL]RQWDO EDUff RQ IOXRUHVFHLQODEHOHG ROLJRQXFOHRWLGH FHOOXODU XSWDNH LQ &9 FHOOV Q f 2OLJRQXFOHRWLGHV QPROHf ZHUH FRPSOH[HG ZLWK OLSRVRPHV DW D FKDUJH UDWLR RI f 7KH IOXRUHVFHQFH VLJQDOV PHDQs6'f ZHUH UHFRUGHG E\ IORZ F\WRPHWU\ DIWHU K RI LQFXEDWLRQ ZKLOH WKH FHOOV ZHUH HLWKHU DOLYH RU IL[HG Df 'RGHF\O OfLPLGD]RO\Of SURSLRQDWH ',3f Ef 0HWK\O LPLGD]RO\O ODXUHDWH 0,/f Ff 1GRGHF\O LPLGD]ROH ',f

PAGE 110

)L[HG &HOOV

PAGE 111

)LJXUH f§FRQWLQXHG Ff

PAGE 112

Df )LJXUH 7LPH FRXUVH VWXG\ RI FDWLRQLF OLSRVRPHV RQ IOXRUHVFHLQODEHOHG SODVPLG '1$ FHOOXODU XSWDNH LQ &9 FHOOV Q f 3ODVPLG '1$ SJf ZLWKRXW $f DQG ZLWK D FDUULHU V\VWHP LH 1>OOGLROHROR[\fSURS\O@111WULPHWK\ODPPRQLXP PHWK\OVXOIDWH '27$3f OLSRVRPHV SOXV ‘f RU PLQXV Af GRGHF\O O fLPLGD]RO\Of SURSLRQDWH ',3f ZLWK PRODU UDWLR 5 f DW D FKDUJH UDWLR RI ff ZHUH FRPSDUHG 7KH IOXRUHVFHQFH VLJQDOV PHDQ6'f ZHUH UHFRUGHG E\ WKH IORZ F\WRPHWU\ LQ WZR FHOO FRQGLWLRQV Df /LYH &9 FHOOV Ef )L[HG &9 FHOOV

PAGE 113

Ef )LJXUH f§FRQWLQXHG

PAGE 114

! m6V n IO! L )LJXUH (IIHFW RI FDWLRQLF 1>OOGLROHROR[\fSURS\O@111WULPHWK\ODPPRQLXP PHWK\OVXOIDWH '27$3f OLSRVRPHV FRQWDLQLQJ WKUHH ELRGHJUDGDEOH S+VHQVLWLYH VXUIDFWDQWV %36f DW YDULRXV PRODU UDWLRV 5 RSHQ EDUf 5 VROLG EDUf 5 YHUWLFDO EDUf DQG 5 KRUL]RQWDO EDUff RQ IOXRUHVFHLQODEHOHG SODVPLG '1$ FHOOXODU XSWDNH LQ &9 FHOOV Q f 3ODVPLG '1$ SJf ZHUH FRPSOH[HG ZLWK OLSRVRPHV DW D FKDUJH UDWLR RI f 7KH IOXRUHVFHQFH VLJQDOV ZHUH UHFRUGHG E\ IORZ F\WRPHWU\ DIWHU K RI LQFXEDWLRQ ZLWK OLYH RU IL[HG FHOOV Df 'RGHF\O O fLPLGD]RO\Of SURSLRQDWH ',3f Ef 0HWK\O LPLGD]RO\O ODXUHDWH 0,/f Ff 1GRGHF\O LPLGD]ROH ',f

PAGE 115

9 f )LJXUH f§FRQWLQXHG

PAGE 116

)LJXUH f§FRQWLQXHG Ff

PAGE 117

2OLJRQXFOHRWLGH &HOOXODU 8SWDNH DQG 'LVWULEXWLRQ (YDOXDWLRQ ZLWK &RQIRFDO 0LFURVFRS\ 8VLQJ FRQIRFDO PLFURVFRS\ WKH HIIHFW RI QHXWUDO /DOHFLWKLQ OLSRVRPHV FRQWDLQLQJ GRGHF\O OfLPLGD]RO\Of SURSLRQDWH RQ ROLJRQXFOHRWLGH FHOOXODU GHOLYHU\ ZDV IXUWKHU LQYHVWLJDWHG 7KHUH LV D GLUHFW FRUUHODWLRQ EHWZHHQ FHOOXODU DVVRFLDWHG ROLJRQXFOHRWLGHV DQG WKH LQWHQVLW\ RI SKRWRJUDSK :LWKRXW DQ\ GHOLYHU\ FDUULHU HJ OLSRVRPHVf WKHUH ZDV OLWWOH ROLJRQXFOHRWLGH DVVRFLDWHG ZLWK WKH FHOOV DW WKUHH REVHUYHG WLPH SRLQWV )LJXUH f /LSRVRPHV PLQXV )LJXUH f RU SOXV )LJXUH f ',3 DW PRODU UDWLR 5 f ZHUH XVHG PRUH ROLJRQXFOHRWLGHV ZHUH WDNHQ LQWR WKH FHOOV DV FRPSDUHG WR WKRVH ZLWKRXW GHOLYHU\ V\VWHPV DW DOO REVHUYHG WLPH SRLQWV 7KH FHOOXODU GLVWULEXWLRQV RI WKH ROLJRQXFOHRWLGHV RQFH EURXJKW LQWR WKH FHOOV WKURXJK WZR GHOLYHU\ V\VWHPV LH OLSRVRPHV DQG ',3OLSRVRPHV 5 f OLSRVRPHVf ZHUH VXEVHTXHQWO\ FRPSDUHG )RU WKRVH ROLJRQXFOHRWLGHV WKDW ZHUH WDNHQ LQWR FHOOV WKURXJK /DOHFLWKLQ OLSRVRPHV SHULQXFOHDU ORFDOL]DWLRQ RI WKH ROLJRQXFOHRWLGHV ZDV VHHQ LQ WKH REVHUYHG WLPH SRLQWV )LJXUH f :KHQ ',3 ZDV DGGHG LQWR WKH QHXWUDO OLSRVRPHV 5 f PXFK ZHOO GLVSHUVLRQ RI WKH ROLJRQXFOHRWLGHV LQ WKH FHOOV ZDV REVHUYHG )LJXUH f 4XDQWLWDWLYH (IIHFW (YDOXDWLRQ RI /XFLIHUDVH $FWLYLW\ 7R LQYHVWLJDWH WKH LPSDFW RQ ROLJRQXFOHRWLGH DFWLYLW\ RI GRGHF\O O fLPLGD]RO\Of SURSLRQDWH ',3f LQ FDWLRQLF 1>OOGLROHROR[\fSURS\O@111WULPHWK\ODPPRQLXP PHWK\OVXOIDWH '27$3f OLSRVRPHV D &9 OXFLIHUDVH H[SUHVVLQJ FHOO OLQH ZDV XVHG

PAGE 118

Df )LJXUH &HOOXODU XSWDNHGLVWULEXWLRQ RI IOXRUHVFHLQODEHOHG ROLJRQXFOHRWLGHV LQ 5$: FHOOV &HOOV ZHUH REVHUYHG E\ FRQIRFDO PLFURVFRS\ DW WKUHH WLPH SRLQWV Df K Ef K Ff K

PAGE 121

)LJXUH &HOOXODU XSWDNHGLVWULEXWLRQ RI IOXRUHVFHLQODEHOHG ROLJRQXFOHRWLGHV ZKHQ HQFDSVXODWHG LQ WKH OHFLWKLQ OLSRVRPHV LQ 5$: FHOOV &HOOV ZHUH REVHUYHG E\ FRQIRFDO PLFURVFRS\ DW WKUHH WLPH SRLQWV Df K Ef K Ff K

PAGE 122

)LJXUH f§FRQWLQXHG

PAGE 124

Df )LJXUH &HOOXODU XSWDNHGLVWULEXWLRQ RI IOXRUHVFHLQODEHOHG ROLJRQXFOHRWLGHV ZKHQ HQFDSVXODWHG LQ WKH OHFLWKLQ OLSRVRPHV FRQWDLQLQJ GRGHF\O OfLPLGD]RO\Of SURSLRQDWH ',3f DW PRODU UDWLR 5 f LQ 5$: FHOOV &HOOV ZHUH REVHUYHG E\ FRQIRFDO PLFURVFRS\ DW WKUHH WLPH SRLQWV Df K Ef K Ff KU

PAGE 126

)LJXUH f§FRQWLQXHG

PAGE 127

$IWHU K RI LQFXEDWLRQ WKH SHUFHQWDJHV RI LQKLELWLRQ OXFLIHUDVH DFWLYLW\f ZHUH GHWHUPLQHG 7KH SRVLWLYH FRQWURO b LQKLELWLRQf UHIHUUHG WR QR WUHDWPHQW LQ WKH FHOOV ZKLOH WKH QHJDWLYH FRQWURO b LQKLELWLRQf ZDV WKH EDFNJURXQG LQWHUIHUHQFH 2WKHU DSSURSULDWH FRQWUROV LQFOXGLQJ VHQVH ROLJRQXFOHRWLGHOLSRVRPH DQWLVHQVH ROLJRQXFOHRWLGH DQG OLSRVRPH ZHUH DOVR XVHG 1R LQKLELWLRQ RI OXFLIHUDVH H[SUHVVLRQ ZDV GHWHFWHG DW WKH REVHUYHG FRQFHQWUDWLRQ RI IUHH DQWLVHQVH ROLJRQXFOHRWLGHV )LJXUH Df +RZHYHU ZKHQ DQWLVHQVH RU VHQVHf ROLJRQXFOHRWLGH'27$3 OLSRVRPH FRPSOH[ ZDV XVHG JUHDWHU LQKLELWLRQV RI OXFLIHUDVH DFWLYLW\ ZHUH REVHUYHG )LJXUH Df $V WKH FRQFHQWUDWLRQ RI DQWLVHQVH RU VHQVHf ROLJRQXFOHRWLGH UHDFKHG S0 QPRO POf QR H[WUD LQKLELWLRQ ZDV VHHQ 6LJQLILFDQW GLIIHUHQFHV Sf LQ WKH SHUFHQWDJH RI LQKLELWLRQ EHWZHHQ DQWLVHQVH DQG VHQVH ROLJRQXFOHRWLGHV ZHUH REWDLQHG ZKHQ WKH FRQFHQWUDWLRQ RI ROLJRQXFOHRWLGH ZDV S0 QPRO POf RU KLJKHU :KHQ ',3 DW PRODU UDWLR 5 f ZDV DGGHG WR WKH FDWLRQLF OLSRVRPHV VLPLODU SURILOHV ZHUH QRWLFHG )LJXUH Ef &RPSDULQJ WKH OXFLIHUDVH LQKLELWLRQ HIIHFW WKURXJKRXW WKH HQWLUH REVHUYHG FRQFHQWUDWLRQ UDQJH PRUH LQKLELWLRQ Sf ZDV VHHQ ZKHQ DQWLVHQVH ROLJRQXFOHRWLGHV ZHUH FRPSOH[HG ZLWK WKH ',3 WUHDWHG 5 f '27$3 OLSRVRPHV WKDQ ZKHQ DQWLVHQVH ROLJRQXFOHRWLGHV ZHUH FRPSOH[HG ZLWK WKH '27$3 OLSRVRPHV $ VDPH SKHQRPHQRQ ZDV DOVR REVHUYHG ZKHQ DQWLVHQVH ROLJRQXFOHRWLGHV ZHUH UHSODFHG E\ VHQVH ROLJRQXFOHRWLGHV EHWZHHQ ',3'27$3 OLSRVRPHV DQG '27$3 OLSRVRPHV

PAGE 128

Df )LJXUH (IIHFW RI ROLJRQXFOHRWLGH DFWLYLW\ ZKHQ FRPSOH[HG ZLWK WZR FDWLRQLF OLSRVRPHV RI GLIIHUHQW OLSLG FRPSRVLWLRQV DW D FKDUJH UDWLR RI f RQ &9 OXFLIHUDVH H[SUHVVLQJ FHOOV Q f 7KH SHUFHQWDJH RI LQKLELWLRQ RQ OXFLIHUDVH DFWLYLW\ PHDQL6'f LQ HDFK WUHDWPHQW DQWLVHQVH ROLJRQXFOHRWLGHOLSRVRPH Af VHQVH ROLJRQXFOHRWLGHOLSRVRPH ‘f DQWLVHQVH ROLJRQXFOHRWLGH $f DQG OLSRVRPH ;ff ZDV FDOFXODWHG DQG SORWWHG DJDLQVW ROLJRQXFOHRWLGH FRQFHQWUDWLRQ DIWHU K RI LQFXEDWLRQ ,W VKRXOG EH QRWHG WKDW IRU WKH OLSRVRPH VXEVHW H[SHULPHQW ;f WKH GDWD LQGLFDWHG WKDW WKH LQKLELWLRQ HIIHFW ZDV WKH VDPH IRU WKH DPRXQW RI OLSRVRPH XVHG LQ WKH DQWLVHQVH ROLJRQXFOHRWLGHOLSRVRPH VXEVHW H[SHULPHQW Af EXW QR ROLJRQXFOHRWLGHV ZHUH XVHG Df 1>OOGLROHROR[\fSURS\O@ 111WULPHWK\ODPPRQLXP PHWK\OVXOIDWH '27$3f Ef '27$3 FRQWDLQLQJ GRGHF\O OfLPLGD]RO\Of SURSLRQDWH ',3f DW PRODU UDWLR 5 f

PAGE 129

)LJXUH

PAGE 130

'LVFXVVLRQ $IWHU FKDUDFWHUL]LQJ WKHVH WKUHH ELRGHJUDGDEOH S+VHQVLWLYH VXUIDFWDQWV %36f WKHLU ELRORJLFDO HIIHFWV LQ YLWUR ZHUH IXUWKHU HYDOXDWHG 6WXGLHV ZHUH LQLWLDOO\ FRQGXFWHG WR LQYHVWLJDWH WKH LPSDFW RI %36 LQ FDWLRQLF OLSRVRPHV RQ ROLJRQXFOHRWLGH RU SODVPLG '1$f FHOOXODU XSWDNH LQ WHUPV RI LQFXEDWLRQ WLPH DQG WKH PRODU UDWLR ,QFUHDVHG IOXRUHVFHQFH VLJQDOV ZHUH REVHUYHG GXULQJ WKH HDUO\ WLPH SRLQWV ZKHQ WKH ROLJRQXFOHRWLGHV ZHUH GHOLYHUHG LQ WKH OLYH &9 PRQNH\ NLGQH\ ILEUREODVWf FHOOV E\ GRGHF\O OfLPLGD]RO\Of SURSLRQDWH ',3f WUHDWHG OLSRVRPHV FRPSDUHG WR FRQWURO OLSRVRPHV 6LQFH WKH ROLJRQXFOHRWLGH ODEHOLQJ PDWHULDO IOXRUHVFHLQ LVRWKLRF\DQDWH ),7&f LV D S+VHQVLWLYH IOXRURSKRUH LW GHPRQVWUDWHV KLJKHU IOXRUHVFHQFH LQWHQVLW\ LQ EDVH WKDQ LQ DFLG 7KH UHVXOWV LQGLFDWHG WKDW ZLWK WKH DGGLWLRQ RI ',3 WKH OLSRVRPHV FRXOG LQGXFH HLWKHU KLJKHU ROLJRQXFOHRWLGH FHOOXODU XSWDNH RU GLIIHUHQW ROLJRQXFOHRWLGH LQWUDFHOOXODU GLVWULEXWLRQ LH IURP HQGRVPH WR F\WRSODVPf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f ZLWK OLSRVRPHV LQ WKH DEVHQFH RI %36 DQG D PRUH EDVLF VXUURXQGLQJ HJ HQGRVPH RU F\WRSODVPf ZLWK OLSRVRPHV LQ WKH SUHVHQFH RI %36

PAGE 131

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f FRPSDUHG WR ROLJRQXFOHRWLGH Ef 7KH GRXEOH VWUDQG VWUXFWXUH RI SODVPLG '1$ PD\ DOVR KLQGHU LWV GHOLYHU\ 0RUHRYHU WKH GLIIHUHQW EDFNERQH RI WKH SODVPLG '1$ LH SKRVSKRURGLHVWHUf DQG WKH ROLJRQXFOHRWLGH LH SKRVSKRURWKLRDWHf LQ RXU VWXGLHV UHODWLQJ WR WKHLU UHVSHFWLYH VWDELOLW\ FDQ DOVR FKDQJH WKHLU HIILFLHQF\ +RZHYHU WKH XVH RI %36 WR LQFUHDVH WKH DFWLYLW\ RI SODVPLG '1$ KDV EHHQ VKRZQ LQ D SUHYLRXV VWXG\ /LDQJ t +XJKHV f $V D UHVXOW VRPH RWKHU PHFKDQLVP PD\ EH LQYROYHG LQ %36

PAGE 132

LQGXFHG SODVPLG '1$ GHOLYHU\ ,W LV DOVR SRVVLEOH WKDW RXU H[SHULPHQWV ZHUH QRW VHQVLWLYH HQRXJK WR QRWLFH WKH IXUWLYH GLIIHUHQFH ,Q DGGLWLRQ WKH HIIHFW RI %36 LQ WKH OLSRVRPHV RQ ROLJRQXFOHRWLGH FHOOXODU XSWDNHGLVWULEXWLRQ ZDV DVVHVVHG 7R HOXGH WKH SRVVLEOH FHOO OLQH GHSHQGHQW IDFWRU RQ ROLJRQXFOHRWLGH GHOLYHU\ 5$: PRXVH PRQRF\WHPDFURSKDJHf FHOOV ZHUH XVHG 0RUHRYHU LQVWHDG RI FDWLRQLF OLSRVRPHV ',3 ZDV DGGHG LQWR GLIIHUHQW W\SH RI QHXWUDO OLSRVRPHV ,W ZDV VKRZQ WKDW ROLJRQXFOHRWLGHV WKDW KDG QR GHOLYHU\ FDUULHU ZHUH LQHIILFLHQWO\ LQWHUQDOL]HG LQWR WKH FHOOV :KHQ WKH OLSRVRPHV ZHUH XVHG WR GHOLYHU WKH ROLJRQXFOHRWLGHV PRUH ROLJRQXFOHRWLGHV ZHUH EURXJKW LQWR WKH FHOOV 1HYHUWKHOHVV WKH\ ZHUH ORFDOL]HG LQ WKH SXQFWXDWH F\WRSODVPLF VSRWV SUREDEO\ FRUUHVSRQGLQJ WR WKHLU SUHVHQFH LQ WKH HQGRF\WRWLF FRPSDUWPHQWV HQGRVPH RU O\VRVRPHf DQG LQGLFDWLQJ D SRVVLEOH O\VRVRPDO GHJUDGDWLRQ IDWH 9ODVVRY HW DO f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fSURS\O@ 111WULPHWK\ODPPRQLXP PHWK\OVXOIDWH '27$3f OLSRVRPHV ZHUH XVHG DV DQ ROLJRQXFOHRWLGH GHOLYHU\ V\VWHP DGGLWLRQDO OXFLIHUDVH DFWLYLW\ ZDV UHVWUDLQHG LQ WKH REVHUYHG FRQFHQWUDWLRQ UDQJH WKDQ ROLJRQXFOHRWLGHV ZLWKRXW WKH GHOLYHU\ V\VWHP :KHQ

PAGE 133

GRGHF\O OfLPLGD]RO\Of SURSLRQDWH ',3f ZDV DGGHG DV D SDUW RI WKH OLSRVRPH FRPSRVLWLRQ WR GHOLYHU WKH ROLJRQXFOHRWLGHV DQ HYHQ JUHDWHU DPRXQW RI HQ]\PH H[SUHVVLRQ ZDV LQKLELWHG WKDQ OLSRVRPHV ZLWKRXW ',3 0RUHRYHU QRW RQO\ FRXOG ',3 HQKDQFH WKH HIILFDF\ RI ROLJRQXFOHRWLGHV LQ KDPSHULQJ WKH V\QWKHVLV RI OXFLIHUDVH EXW LW FRXOG DOVR LQFUHDVH ROLJRQXFOHRWLGH SRWHQF\ LH PRUH LQKLELWLRQ DW D IL[HG FRQFHQWUDWLRQ RU VDPH LQKLELWLRQ ZLWK D ORZHU DPRXQW RI ROLJRQXFOHRWLGHf 7KLV IDFW LPSOLHV WKH XVHIXOQHVV RI %36 LQ HQKDQFLQJ WKH LQKLELWLRQ RI OXFLIHUDVH H[SUHVVLRQ +RZHYHU ZKHQ RQO\ WKH GHOLYHU\ V\VWHPV ZHUH XVHG WKH LQKLELWLRQV ZHUH DOVR HOHYDWHG DV PRUH OLSRVRPHV ZHUH HPSOR\HG VXJJHVWLQJ D SRVVLEOH F\WRWR[LF LQWHUIHUHQFH RI OXFLIHUDVH H[SUHVVLRQ IURP WKH FDWLRQLF '27$3 OLSRVRPHV DQG WKH ',3'27$3 OLSRVRPHV ,Q VXPPDU\ ZH KDYH VKRZHG WKDW ZLWK KHOS IURP D FDUULHU FDWLRQLF OLSRVRPHVf JUHDWHU HQ]\PH DFWLYLWLHV ZHUH LQKLELWHG E\ WKH ROLJRQXFOHRWLGHV 7KLV REVHUYDWLRQ ZDV LQ DFFRUGDQFH ZLWK WKH RWKHU FRPSDUDEOH VWXGLHV &DSDFFLROL HW DO 7DNOH HW DO f 6LPLODU HIIHFWV ZHUH DOVR VHHQ ZKHQ RWKHU FDWLRQLF OLSRVRPHV ZHUH XVHG %HQQHWW HW DO .RQRSND HW DO /DSSDODLQHQ HW DO 2OOLNDLQHQ HW DO f :LWK WKH DGGLWLRQ RI ELRGHJUDGDEOH S+VHQVLWLYH VXUIDFWDQWV %36f LQWR WKH FDWLRQLF OLSRVRPHV PRUH GUDPDWLF LQFUHDVH RI WKH LQKLELWLRQ RQ OXFLIHUDVH DFWLYLW\ E\ WKH ROLJRQXFOHRWLGHV ZDV REVHUYHG WKDQ WKH FDWLRQLF OLSRVRPHV ZLWKRXW %36 :H KDYH DOVR LGHQWLILHG WKDW WKH PHFKDQLVP RI %36 WR LQFUHDVH WKH GHOLYHU\ RI ROLJRQXFOHRWLGH ZDV QRW E\ LQFUHDVLQJ ROLJRQXFOHRWLGH FHOOXODU XSWDNH LQ &9 FHOOV 2QFH ROLJRQXFOHRWLGHV ZHUH EURXJKW LQWR WKH LQWUDFHOOXODU FRPSDUWPHQW WKH\ ZHUH UHGLVWULEXWHG E\ SURPRWLQJ WKH IRUPDWLRQ RI D VPDOO PHPEUDQH SRUH 7R EHWWHU

PAGE 134

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t 6]RND Df %LRGHJUDGDEOH S+ VHQVLWLYH VXUIDFWDQWV %36f ZHUH GHYHORSHG DQG XVHG WR HQKDQFH WKH FHOOXODU GHOLYHU\ RI QXFOHLF DFLGV 3UHYLRXV UHSRUWV +XJKHV HW DOV /LDQJ t +XJKHV f DQG FXUUHQW VWXGLHV KDYH EHHQ VKRZQ WKHLU SRWHQWLDO XVH 1HYHUWKHOHVV WKHUH LV SOHQW\ RI VSDFH WR UHILQH WKH HQWLUH %36 GHOLYHU\ V\VWHP 7R IXUWKHU RSWLPL]H WKH XVH RI %36 D QXPEHU RI IDFWRUV QHHG WR EH FRQVLGHUHG 2OLJRQXFOHRWLGH FHOOXODU GHOLYHU\ E\ %36FDWLRQLF OLSRVRPHV UHOLHV RQ D QXPEHU RI YDULDEOHV LQFOXGLQJ WKH OLSRVRPH FRPSRVLWLRQ %HQQHWW HW DO =KRX t +XDQJ f FKDUJH UDWLR $ULPD HW DK -DDNVHODLQHQ HW DK =HOSKDWL t 6]RND Df LQFXEDWLRQ SHULRG =HOSKDWL t 6]RND Df DQG SUHVHQFH RI VHUXP )HLJQHU HW DK f %\ PRGXODWLQJ DQ\ RQH RI WKH DERYH YDULDEOHV WKH FHOOXODU GHOLYHU\ RI WKH ROLJRQXFOHRWLGHV E\ %36OLSRVRPHV FDQ EH VWURQJO\ DIIHFWHG DQG HYHQWXDOO\ EH HQKDQFHG

PAGE 135

&+$37(5 0(&+$1,60 2) $&7,21 ,19(67,*$7,21 2) %,2'(*5$'$%/( S+ 6(16,7,9( 685)$&7$176 ,QWURGXFWLRQ 'RGHF\O OfLPLGD]RO\Of SURSLRQDWH ',3f D PHPEHU RI ELRGHJUDGDEOH S+ VHQVLWLYH VXUIDFWDQWV %36f KDV EHHQ SUHYLRXVO\ GHPRQVWUDWHG WR LQFUHDVH F\WRSODVP GHOLYHU\ RI ROLJRQXFOHRWLGH +XJKHV HW DO f DQG SODVPLG '1$ /LDQJ t +XJKHV f ,W KDV DOUHDG\ EHHQ SURYHG WKDW WKH HOHYDWHG HIIHFW IURP %36OLSRVRPHV ZDV QRW WR LQFUHDVH WKH FHOOXODU XSWDNH RI ROLJRQXFOHRWLGHV EXW WR UHGLVWULEXWLRQ RQFH ROLJRQXFOHRWLGHV ZHUH EURXJKW LQWR FHOOV &KDSWHU f +RZHYHU LW UHPDLQHG XQFOHDU KRZ %36 GHVWDELOL]HG WKH HQGRVRPDO PHPEUDQH DQG UHOHDVHG WKH FRQWHQW RXW RI HQGRVRPHV 7R IXUWKHU XWLOL]H DQG PRGLI\ %36 LQ LQWUDFHOOXODU PDFURPROHFXOH GHOLYHU\ LW LV HVVHQWLDO WR XQGHUVWDQG WKH PHFKDQLVPV UHVSRQVLEOH IRU PHPEUDQH GHVWDELOL]DWLRQ ,Q DGGLWLRQ WR ',3 WZR RWKHU %36 PHWK\O LPLGD]RO\O ODXUHDWH 0,/f DQG 1GRGHF\O LPLGD]ROH ',f ZHUH XVHG WR LQYHVWLJDWH SRVVLEOH PHFKDQLVPV ,QVWHDG RI VHUYLQJ DV D QXFOHLF DFLG GHOLYHU\ V\VWHP OLSRVRPHV ZHUH XVHG DV WKH H[SHULPHQWDO PHPEUDQH PRGHO WR VWXG\ WKH SRVVLEOH PHPEUDQH GHVWDELOL]DWLRQ PHFKDQLVPV ,Q RUGHU WR PDLQWDLQ VLPSOLFLW\ RQO\ QHXWUDO OLSLGV HJ OHFLWKLQf ZHUH XVHG LQ WKH OLSRVRPDO PHPEUDQH V\VWHP :KLOH WKH OLSRVRPHV PD\ QRW IXOO\ UHSUHVHQW HYHQWV RFFXUULQJ LQ ELRORJLFDO VLWXDWLRQV WKH\ VWLOO VHUYHG DV H[FHOOHQW PRGHOV LQ DGGUHVVLQJ

PAGE 136

SRWHQWLDO PHFKDQLVPV RI OLSLG PHPEUDQH GLVUXSWLRQ $Q DTXHRXV VSDFH IXVLRQ DVVD\ 6PRODUVN\ HW DO f DQG D OLSLG PL[LQJ UHVRQDQFH HQHUJ\ WUDQVIHU DVVD\ 6WUXFN HW DO f ZHUH XVHG WR REVHUYH PHPEUDQH GHVWDELOL]DWLRQ WKURXJK IXVLRQ $ OLSRVRPH OHDNDJH DVVD\ RI D PDUNHU FRPSRXQG FDOFHLQ ZDV XVHG WR REVHUYH PHPEUDQH GHVWDELOL]DWLRQ WKURXJK UXSWXUH 0DWHULDOV &KHPLFDO &DOFHLQ ZDV SXUFKDVHG IURP $OGULFK 0LOZDXNHH :,f 1OLVVDPLQH UKRGDPLQH % VXOIRQ\OfSKRVSKDWLG\OHWKDQRODPLQH 5K3(f 1QLWUROEHQ]R[DGLD]RO\Of SKRVSKDWLG\OHWKDQRODPLQH 1%'3(f /DOHFLWKLQ OGLP\ULVWR\OVQJO\FHUR SKRVSKRFKROLQH '03&f GLROHR\OSKRVSKDWLG\OHWKDQRODPLQH '23(f DQG FKROHVWHURO ZHUH SXUFKDVHG IURP $YDQWL 3RODU /LSLGV $ODEDVWHU $/f 1 1fS[\O\OHQHELV S\ULGLQLXP EURPLGHf '3;f DQG ODPLQRQDSKWKDOHQHWULVXOIRQLF DFLG $176f ZHUH SXUFKDVHG IURP 0ROHFXODU 3UREHV (XJHQH 25f 7KUHH ELRGHJUDGDEOH S+VHQVLWLYH VXUIDFWDQWV %36f LQFOXGLQJ GRGHF\O OfLPLGD]RO\Of SURSLRQDWH ',3f PHWK\O LPLGD]RO\O ODXUHDWH 0,/f DQG 1GRGHF\O LPLGD]ROH ',f ZHUH V\QWKHVL]HG DV SUHYLRXVO\ UHSRUWHG &KDSWHU f $OO SXUFKDVHG RU REWDLQHG FKHPLFDOV ZHUH XVHG GLUHFWO\ ZLWKRXW IXUWKHU SXULILFDWLRQ

PAGE 137

%XIIHU 3UHSDUDWLRQ $OO EXIIHUV ZHUH DGMXVWHG ZLWK 1D&O WR DQ HTXDO LRQLF VWUHQJWK 7KH S+ RI WKH EXIIHUV DQG WKHLU FKHPLFDO FRPSRVLWLRQV ZHUH DV IROORZV S+ P0 VRGLXP DFHWDWH DQG P0 JODFLDO DFHWLF DFLGf S+ P0 .+3 DQG P0 1D+3f S+ P0 .+3 DQG P0 1D+3f S+ P0 .+3 DQG P0 1DA32S+ P0 .+3 DQG P0 1A+32DQG S+ P0 .+3 DQG P0 1D2+f /LSRVRPH 3UHSDUDWLRQ /LSRVRPHV /DOHFLWKLQ OGLP\ULVWR\OVQJO\FHURSKRVSKRFKROLQH '03&f FKROHVWHURO PRODU UDWLR f ZHUH SUHSDUHG DQG XVHG LQ PRVW H[SHULPHQWV WR HYDOXDWH IXVLRQ DQG UXSWXUH HYHQWV 7KH OLSLG UHK\GUDWLRQ PHWKRG ZDV XVHG WR SURGXFH YHVLFOHV +XJKHV HW DO f 7KH OLSRVRPHV ZHUH SDVVHG WKURXJK D KLJK SUHVVXUH H[WUXGHU /LSH[ %LRPHPEUDQH ,QF 9DQFRXYHU %&f ZLWK QP SRO\FDUERQDWH PHPEUDQHV WKUHH WLPHV 7KH VL]H RI WKH OLSRVRPHV YROXPHZHLJKW *DXVVLDQ GLVWULEXWLRQf ZDV GHWHUPLQHG WR EH QP VWDQGDUG GHYLDWLRQf ZLWK D 1,&203 =/6 =HWD 3RWHQWLDO3DUWLFOH 6L]HU 6DQWD %DUEDUD &$f 7KH FRQFHQWUDWLRQ RI WRWDO SKRVSKROLSLGV ZDV GHWHUPLQHG E\ D VSHFWURSKRWRPHWULF WHFKQLTXH DV SUHYLRXVO\ GHVFULEHG &KDSWHU f

PAGE 138

0HWKRGV 7LPH &RXUVH )XVLRQ $VVD\ ,QFUHDVLQJ PRODU UDWLRV 5 5 DQG 5 f RI ELRGHJUDGDEOH S+VHQVLWLYH VXUIDFWDQWV %36f WR QPROPO RI OLSRVRPH /DOHFLWKLQ OGLP\ULVWR\OVQJO\FHUR SKRVSKRFKROLQH '03&f FKROHVWHURO PRODU UDWLR f ZHUH XVHG WR PRQLWRU IXVLRQ PHFKDQLVP RYHU WLPH LQ D S+ EXIIHU VROXWLRQ /LSRVRPHOLSRVRPH IXVLRQ ZDV FKDUDFWHUL]HG E\ PHDVXULQJ IOXRUHVFHQFH UHVRQDQFH HQHUJ\ WUDQVIHU EHWZHHQ WZR OLSLG KHDG JURXSV 6WUXFN HW DO f 1QLWUROEHQ]R[DGLD]RO\Of SKRVSKDWLG\OHWKDQRODPLQH 1%'3(f DQG 1OLVVDPLQH UKRGDPLQH % VXOIRQ\Of SKRVSKDWLG\OHWKDQRODPLQH 5K3(f ZHUH LQFRUSRUDWHG LQWR WZR VHSDUDWH SRSXODWLRQV RI YHVLFOHV DW b PROf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bf f r ZKHUH ) DQG ) DUH WKH IOXRUHVFHQFH LQWHQVLWLHV LQ WKH )R SUHVHQFH DQG DEVHQFH RI WKH 5K3( JURXS UHVSHFWLYHO\ 6WUXFN HW DO f

PAGE 139

7R FRUURERUDWH WKH PHPEUDQH IXVLRQ VWXG\ DQ DTXHRXV FRQWHQW PL[LQJ PHWKRG ZDV XVHG 6PRODUVN\ HW DK f ,Q WKLV DVVD\ P0 RI ODPLQRQDSKWKDOHQH WULVXOIRQLF DFLG $176f DQG P0 RI 1 1fS[\O\OHQHELVS\ULGLQLXP EURPLGHf '3;f ZHUH HQFDSVXODWHG LQWR WZR VHSDUDWH OLSRVRPHV /DOHFLWKLQ '03& FKROHVWHURO PRODU UDWLR f 8QHQFDSVXODWHG $176 DQG '3; ZHUH ODWHU UHPRYHG WKURXJK FHQWULIXJDWLRQ USP PLQf ILYH WLPHV DQG ZDVKHG ZLWK D S+ SKRVSKDWH EXIIHUHG VDOLQH 7KHVH WZR OLSRVRPH SRSXODWLRQV QPROPOf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f DIWHU PLQ ,QFUHDVLQJ PRODU UDWLRV 5 5 DQG 5 f RI %36 ZHUH LQFRUSRUDWHG LQWR QPROPO RI OLSRVRPHV /DOHFLWKLQ GLP\ULVWR\OVQ JO\FHURSKRVSKRFKROLQH '03&f FKROHVWHURO PRODU UDWLR f WR PRQLWRU IXVLRQ

PAGE 140

HYHQWV ZLWK FKDQJLQJ S+V f 7KH IOXRUHVFHQFH LQWHQVLWLHV ZHUH WKHQ TXDQWLILHG DIWHU PLQ LQ ERWK DVVD\V %LRGHJUDGDEOH S+6HQVLWLYH 6XUIDFWDQWV,QGXFHG 0HPEUDQH 5XSWXUH /LSRVRPHV /DOHFLWKLQ GLP\ULVWR\OVQJO\FHURSKRVSKRFKROLQH '03&f FKROHVWHURO PRODU UDWLR QPROPOf FRQWDLQLQJ P0 FDOFHLQ VHOITXHQFKLQJ FRQFHQWUDWLRQf ZHUH SUHSDUHG XVLQJ LQFUHDVLQJ PRODU UDWLRV RI ELRGHJUDGDEOH S+VHQVLWLYH VXUIDFWDQWV %36f 5 5 5 DQG 5 f 7KH OLSRVRPHV ZHUH XVHG WR REVHUYH ZKHWKHU PHPEUDQH UXSWXUH RFFXUHG 7KH %36OLSRVRPH SUHSDUDWLRQV ZHUH LQFXEDWHG ZLWK SKRVSKDWH EXIIHUV S+ f IRU PLQ DQG FDOFHLQ UHOHDVH TXDQWLILHG 7KH UHOHDVHG FDOFHLQ ZDV H[FLWHG DW QP DQG REVHUYHG DW QP 7KH SHUFHQWDJH RI UHOHDVHG FDOFHLQ ZDV FDOFXODWHG E\ WKH HTXDWLRQ bf Arb /LX t 5HJHQ Y/F rEf f ,[ LV WKH b IOXRUHVFHQFH LQWHQVLW\ YDOXH ZKHQ DGGLQJ H[FHVV 7ULWRQ ; P0f ,D DQG ,E DUH WKH IOXRUHVFHQFH LQWHQVLWLHV DIWHU LQFXEDWLRQ ZLWK DQG ZLWKRXW %36 UHVSHFWLYHO\ (IIHFWV RI /LSRVRPH &RQFHQWUDWLRQ RQ 0HPEUDQH )XVLRQ DQG 5XSWXUH ,QFUHDVLQJ FRQFHQWUDWLRQV RI OLSRVRPHV /DOHFLWKLQ GLP\ULVWR\OVQJO\FHUR SKRVSKRFKROLQH '03&f FKROHVWHURO PRODU UDWLR f ZLWK ELRGHJUDGDEOH S+ VHQVLWLYH VXUIDFWDQWV %36f DW WZR PRODU UDWLR JURXSV 5 DQG 5 f ZHUH XVHG WR GHWHUPLQH WKHLU GHSHQGHQF\ RQ PHPEUDQH UXSWXUH QPROPO QPROPO DQG

PAGE 141

QPROPOf DQG IXVLRQ QPROPO QPROPO DQG QPROPOf LQ D S+ EXIIHU VROXWLRQ 6LPLODU OLSLG PL[LQJ DVVD\ SURWRFROV ZHUH XVHG DV GHVFULEHG DERYH ,PSDFW RI &KROHVWHURO DQG 'LROHR\OSKRVSKDWLG\OHWKDQRODPLQH RQ 0HPEUDQH )XVLRQ /LSRVRPHV QPROPOf FRQWDLQLQJ /DOHFLWKLQ ZLWK RU ZLWKRXW FKROHVWHURO ZHLJKW UDWLR PRODU UDWLR f DW WZR PRODU UDWLRV 5 DQG 5 f RI GRGHF\O fLPLGD]RO\Of SURSLRQDWH ',3f ZHUH XVHG WR GHWHUPLQH WKH HIIHFW RI WKH DGGLWLRQ RI FKROHVWHURO RQ IXVLRQ $ VLPLODU VWXG\ ZDV FRQGXFWHG ZLWK FKROHVWHURO UHSODFHG E\ GLROHR\OSKRVSKDWLG\OHWKDQRODPLQH '23(f ZHLJKW UDWLR PRODU UDWLR f 7KH SHUFHQWDJH RI IXVLRQ XVLQJ WKH OLSLG PL[LQJ DVVD\ ZDV GHWHUPLQHG LQ GLIIHUHQW S+ EXIIHUV DIWHU PLQ 7R IXUWKHU FKDUDFWHUL]H WKH GLIIHUHQFH RI IXVLRQ HYHQWV FDXVHG E\ FKROHVWHURO DQG '23( OLSRVRPHV QPROPOf ZLWK DQ HTXDO PRODU UDWLR RI FKROHVWHURO RU '23( WR / DOHFLWKLQ ZHUH LQFRUSRUDWHG ZLWK ',3 DW PRODU UDWLR 5 f 7KH SHUFHQWDJH RI IXVLRQ XVLQJ WKH OLSLG PL[LQJ DVVD\ ZDV GHWHUPLQHG LQ GLIIHUHQW S+ EXIIHU VROXWLRQV DIWHU PLQ 6WDWLVWLFDO $QDO\VLV 6WDWLVWLFDO GLIIHUHQFHV EHWZHHQ WKH WUHDWPHQWV ZHUH GHWHUPLQHG XVLQJ DQDO\VLV RI YDULDQFH ZKHUH DSSURSULDWH 6WDW9LHZ $EDFXV &RQFHSWV ,QF %HUNHOH\ &$f ZLWK S FRQVLGHUHG VWDWLVWLFDOO\ VLJQLILFDQW DQG )LVKHUfV 3/6'f SRVW KRF WWHVW ZDV DSSOLHG

PAGE 142

5HVXOWV 7LPH &RXUVH )XVLRQ $VVD\ 7R LGHQWLI\ SRVVLEOH HYHQWV LQ WKH PHPEUDQH GHVWDELOL]DWLRQ VHTXHQFH IXVLRQ DVVD\V ZHUH TXDQWLILHG RYHU WLPH ZLWK DOO WKUHH ELRGHJUDGDEOH S+VHQVLWLYH VXUIDFWDQWV %36fOLSRVRPH V\VWHP XVLQJ D S+ EXIIHU 7KH SHUFHQWDJH RI PHPEUDQH IXVLRQ GHWHUPLQHG E\ WKH OLSLG PL[LQJ DVVD\ GHPRQVWUDWHG D UDSLG LQLWLDO HYHQW IROORZHG E\ D SODWHDX )LJXUH f 2Q WKH RWKHU KDQG IRU WKH DTXHRXV PL[LQJ DVVD\ WKH SHUFHQWDJH RI IXVLRQ LQFUHDVHG VOLJKWO\ LQ WKH HDUO\ SHULRG DQG FRQWLQXHG RYHU WKH HQWLUH WHVW WLPH FRXUVH )LJXUH f )RU ERWK DVVD\V PHPEUDQH IXVLRQ ZDV VLJQLILFDQWO\ GLIIHUHQW S222Of DPRQJ DOO WKH PRODU UDWLR JURXSV LQ HDFK %36 RYHU WLPH 1R VLJQLILFDQW GLIIHUHQFH LQ IXVLRQ DPRQJ WKH WKUHH DJHQWV ZDV REVHUYHG %LRGHJUDGDEOH S+6HQVLWLYH 6XUIDFWDQWV,QGXFHG 0HPEUDQH )XVLRQ 7R LQYHVWLJDWH WKH IXVRJHQLF SURSHUWLHV HOLFLWHG E\ LQFOXVLRQ RI ELRGHJUDGDEOH S+ VHQVLWLYH VXUIDFWDQWV %36f RQ PHPEUDQHV LQFUHDVLQJ DPRXQWV RI WKUHH %36 ZHUH LQFRUSRUDWHG LQWR OLSRVRPHV IROORZHG E\ LQFXEDWLRQ DW GLIIHUHQW S+V )LJXUH f :KHQ IXVLRQ ZDV GHWHUPLQHG ZLWK WKH OLSLG PL[LQJ DVVD\ WKURXJKRXW DOO S+V WHVWHG WKH SHUFHQWDJH RI IXVLRQ ZLWK WKH FRQWURO OLSRVRPHV 5 f LQFUHDVHG VOLJKWO\ DV WKH S+ GHFUHDVHG $IWHU LQFRUSRUDWLQJ %36 LQWR WKH OLSRVRPHV WKH SHUFHQWDJHV RI IXVLRQ ZHUH VWDWLVWLFDOO\ KLJKHU DW DOO REVHUYHG S+V $V WKH PRODU UDWLR RI %36 LQ WKH OLSRVRPHV ZDV UDLVHG IXVLRQ ZDV VLJQLILFDQWO\ LQFUHDVHG S222Of IURP S+ WR S+ )LJXUH f

PAGE 143

/LSLG 0L[LQJ $VVD\ 7LPH PLQf $TXHRXV 0L[LQJ $VVD\ 7 Df )LJXUH 7LPH FRXUVH IXVLRQ VWXG\ RI OLSRVRPH QPROP@f FRQWDLQLQJ GLIIHUHQW ELRGHJUDGDEOH S+VHQVLWLYH VXUIDFWDQWV %36f DW WKUHH PRODU UDWLRV 5 Af 5 ‘f DQG 5 $ff LQ D S+ EXIIHU VROXWLRQ )RU WKH OLSLG PL[LQJ DVVD\ 1%'3( ODEHOHG OLSRVRPHV ZHUH LQFXEDWHG ZLWK 5K3( ODEHOHG OLSRVRPHV ZKLOH IRU WKH DTXHRXV PL[LQJ DVVD\ OLSRVRPHV FRQWDLQLQJ P0 $176 ZHUH LQFXEDWHG ZLWK OLSRVRPHV FRQWDLQLQJ P0 '3; Q f 'DWD ZHUH H[SUHVVHG DV PHDQVWDQGDUG GHYLDWLRQ 6'f Df 'RGHF\O OfLPLGD]RO\Of SURSLRQDWH ',3f Ef 0HWK\O LPLGD]RO\O ODXUHDWH 0,/f Ff 1GRGHF\O LPLGD]ROH ',f

PAGE 144

/LSLG 0L[LQJ $VVD\ $TXHRXV 0L[LQJ $VVD\ 7 Ef )LJXUH f§FRQWLQXHG

PAGE 145

/LSLG 0L[LQJ $VVD\ $TXHRXV 0L[LQJ $VVD\ Ff )LJXUH f§FRQWLQXHG

PAGE 146

/LSLG 0L[LQJ $VVD\ S+ $TXHRXV 0L[LQJ $VVD\ S+ Df )LJXUH )XVLRQ VWXG\ RI OLSRVRPHV QPROPOf ZLWK YDULRXV ELRGHJUDGDEOH S+ VHQVLWLYH VXUIDFWDQWV %36f DW WKUHH PRODU UDWLRV 5 Af 5 ‘f DQG 5 $ff LQ GLIIHUHQW S+V )RU WKH OLSLG PL[LQJ DVVD\ 1%'3( ODEHOHG OLSRVRPHV ZHUH LQFXEDWHG ZLWK 5K3( ODEHOHG RQHV ZKLOH IRU WKH DTXHRXV PL[LQJ DVVD\ OLSRVRPHV FRQWDLQLQJ P0 $176 ZHUH LQFXEDWHG ZLWK WKRVH FRQWDLQLQJ P0 '3; Q f 7KH SHUFHQWDJHV RI IXVLRQ PHDQs6'f ZHUH GHWHUPLQHG DIWHU PLQ Df 'RGHF\O OfLPLGD]RO\Of SURSLRQDWH ',3f Ef 0HWK\O LPLGD]RO\O ODXUHDWH 0,/f Ff 1GRGHF\O LPLGD]ROH ',f

PAGE 147

/LSLG 0L[LQJ $VVD\ S+ $TXHRXV 0L[LQJ $VVD\ S+ Ef )LJXUH f§FRQWLQXHG

PAGE 148

/LSLG 0L[LQJ $VVD\ S+ $TXHRXV 0L[LQJ $VVD\ )LJXUH f§FRQWLQXHG

PAGE 149

$OO WKUHH %36 H[KLELWHG D VLPLODU IXVLRQ SURILOH EXW WKHUH ZDV QR VLJQLILFDQW GLIIHUHQFH EHWZHHQ WKH IXVLRQ HYHQWV DPRQJ WKH WKUHH DJHQWV )RU WKH IOXRUHVFHQFH DTXHRXV PL[LQJ PHWKRG WKH SHUFHQWDJHV RI IXVLRQ E\ FRQWURO OLSRVRPHV 5 f GHPRQVWUDWHG QR VLJQLILFDQW GLIIHUHQFH DW DOO WHVWHG S+V $IWHU WKH DGGLWLRQ RI %36 WKH SHUFHQWDJHV RI IXVLRQ LQFUHDVHG DW DOO S+V $V WKH PRODU UDWLR RI %36 ZDV UDLVHG IXVLRQ LQFUHDVHG VLJQLILFDQWO\ Sf EHORZ S+ )LJXUH f &RPSDUDEOH WR WKH OLSLG PL[LQJ DVVD\ WKHVH WKUHH %36 H[KLELWHG VLPLODU IXVLRQ SURILOHV ZLWK QR VLJQLILFDQW GLIIHUHQFH DPRQJ WKH HYDOXDWHG %36 %LRGHJUDGDEOH S+6HQVLWLYH 6XUIDFWDQWV,QGXFHG 0HPEUDQH 5XSWXUH :H GHWHUPLQHG WKH DELOLW\ RI OLSRVRPH LQFRUSRUDWHG XQLRQL]HG ELRGHJUDGDEOH S+ VHQVLWLYH VXUIDFWDQWV %36f WR IDFLOLWDWH WKH UHOHDVH RI HQWUDSSHG VROXWHV DW DFLGLF S+V /LSRVRPHV FRQWDLQLQJ FDOFHLQ ZHUH SUHSDUHG ZLWK LQFUHDVLQJ DPRXQWV RI %36 DQG LQFXEDWHG DW GHFUHDVLQJ S+V 0LQLPDO FDOFHLQ UHOHDVH ZDV REVHUYHG DW WKH ORZHU %36OLSRVRPH PRODU UDWLR JURXS 5 f EXW DW WKH 5 JURXS FDOFHLQ UHOHDVH LQFUHDVHG VLJQLILFDQWO\ Sf DW DOO S+V )LJXUH f $W S+ DQG VLJQLILFDQW GLIIHUHQFHV Sf LQ WKH SHUFHQWDJH RI UHOHDVHG FDOFHLQ ZDV REVHUYHG DPRQJ DOO PRODU UDWLR JURXSV &RPSDULQJ WKHVH WKUHH %36 WKH SHUFHQWDJH RI UHOHDVH ZDV VLJQLILFDQW GLIIHUHQW Sf DW WKH 5 JURXS LQ DOO REVHUYHG S+V +RZHYHU QR VLJQLILFDQW GLIIHUHQFH ZDV REVHUYHG DW WKH RWKHU WZR UDWLR JURXSV 5 DQG 5 f DPRQJ WKH WKUHH %36OLSRVRPHV

PAGE 150

S+ Df )LJXUH 0HPEUDQH UXSWXUH SURILOH ZLWK WKH LQFRUSRUDWLRQ RI GLIIHUHQW ELRGHJUDGDEOH S+VHQVLWLYH VXUIDFWDQWV %36f DW IRXU PRODU UDWLRV 5 Af 5 ;f 5 ‘f DQG 5 $ff LQ OLSRVRPHV QPROPOf 7KH PHPEUDQH O\VLV HIIHFWV LQ GLIIHUHQW S+V ZHUH GHWHUPLQHG E\ FDOFHLQ UHOHDVH PHDQL6'f DIWHU PLQ Q f Df 'RGHF\O O LPLGD]RO\Of SURSLRQDWH ',3f Ef 0HWK\O LPLGD]RO\O ODXUHDWH 0,/f Ff 1GRGHF\O LPLGD]ROH ',f

PAGE 151

S+ Ef )LJXUH f§FRQWLQXHG

PAGE 152

S+ &f )LJXUH f§FRQWLQXHG

PAGE 153

(IIHFWV RI /LSRVRPH &RQFHQWUDWLRQ RQ 0HPEUDQH )XVLRQ DQG 5XSWXUH 7R TXDQWLI\ WKH OLSRVRPH FRQFHQWUDWLRQ GHSHQGHQF\ RQ PHPEUDQH IXVLRQ DQG UXSWXUH WKH IXVLRQ DQG UXSWXUH HYHQWV RI OLSRVRPHV DW WKUHH FRQFHQWUDWLRQV ZDV FDOFXODWHG 0HDVXUHPHQWV ZHUH PDGH ZLWK OLSRVRPHV FRQWDLQLQJ ELRGHJUDGDEOH S+VHQVLWLYH VXUIDFWDQWV %36f DW WZR PRODU UDWLRV 5 DQG 5 f LQ D S+ EXIIHU VROXWLRQ 'XH WR WKH VHQVLWLYLW\ RI WKH DVVD\V GLIIHUHQW OLSRVRPH FRQFHQWUDWLRQV ZHUH XVHG LQ WKH VWXGLHV SUHVHQWHG 6LJQLILFDQW GLIIHUHQFHV RI PHPEUDQH IXVLRQ Sf ZHUH REVHUYHG DPRQJ WKH WKUHH OLSRVRPH FRQFHQWUDWLRQV ZLWK WKH OLSLG PL[LQJ DVVD\ LQ ERWK UDWLR JURXSV )LJXUH D DQG )LJXUH Df 7KH OLSRVRPHOLSRVRPH IXVLRQ ZDV FRQFHQWUDWLRQ GHSHQGHQW RQ HDFK LQGLYLGXDO %36 +RZHYHU QR VLJQLILFDQW GLIIHUHQFHV LQ UHOHDVHG FDOFHLQ ZHUH REVHUYHG DPRQJ WKH WKUHH OLSRVRPH FRQFHQWUDWLRQV RQ WKH UXSWXUH EHKDYLRU LQ ERWK UDWLR JURXSV )LJXUH E DQG )LJXUH Ef LQGLFDWLQJ WKH FRQFHQWUDWLRQ LQGHSHQGHQF\ RI %36 OLSRVRPH O\VLV SURILOHV ,PSDFW RI &KROHVWHURO DQG 'LROHR\OSKRVSKDWLG\OHWKDQRODPLQH RQ 0HPEUDQH )XVLRQ )RU /DOHFLWKLQ OLSRVRPHV ZLWKRXW DQ\ DGGLWLYH WKH SHUFHQWDJHV RI IXVLRQ LQFUHDVHG VLJQLILFDQWO\ Sf DV WKH S+ GHFUHDVHG WR LQ ERWK UDWLR JURXSV 5 DQG 5 f *UHDWHU IXVLRQ ZDV REVHUYHG DW WKH KLJKHU UDWLR JURXS 5 f WKDQ LQ ORZHU RQH 5 f )LJXUH f 7KH LQFRUSRUDWLRQ RI b ZHLJKWf FKROHVWHURO LQWR WKH OLSRVRPH V\VWHP UHVXOWHG LQ HQKDQFHG IXVLRQ LQ ERWK UDWLR JURXSV +RZHYHU ZKHQ

PAGE 154

Df )LJXUH (IIHFW RI OLSRVRPH FRQFHQWUDWLRQ RQ PHPEUDQH IXVLRQ DQG O\VLV HYHQWV LQ D S+ EXIIHU VROXWLRQ ZLWK ELRGHJUDGDEOH S+VHQVLWLYH VXUIDFWDQWV %36f GRGHF\O O LPLGD]RO\Of SURSLRQDWH ',3f PHWK\O LPLGD]RO\O ODXUHDWH 0,/f DQG 1GRGHF\O LPLGD]ROH ',f LQWR OLSRVRPHV DW D PRODU UDWLR RI 5 f 7KH FRQWURO JURXS UHIHUUHG WR OLSRVRPHV ZLWKRXW %36 'DWD ZHUH H[SUHVVHG DV PHDQs6' Q f Df $ OLSLG PL[LQJ DVVD\ ZDV XVHG WR TXDQWLI\ IXVLRQ DW YDULRXV OLSRVRPH FRQFHQWUDWLRQV QPROPO KRUL]RQWDO OLQH EDUf QPROPO YHUWLFDO OLQH EDUf DQG QPROPO VROLG EDUff Ef $ FDOFHLQ UHOHDVH PHDVXUHPHQW ZDV XVHG WR GHWHUPLQH WKH OLSRVRPH O\VLV EHKDYLRU DW LQFUHDVLQJ OLSRVRPH FRQFHQWUDWLRQV QPROPO RSHQ EDUf QPROPO GRWWHG EDUf DQG QPROPO YHUWLFDO OLQH EDUf

PAGE 155

5HOHDVH Ef )LJXUH f§FRQWLQXHG

PAGE 156

Df )LJXUH (IIHFW RI OLSRVRPH FRQFHQWUDWLRQ RQ PHPEUDQH IXVLRQ DQG O\VLV HYHQWV LQ D S+ EXIIHU VROXWLRQ ZLWK WKUHH ELRGHJUDGDEOH S+VHQVLWLYH VXUIDFWDQWV %36f GRGHF\O OfLPLGD]RO\Of SURSLRQDWH ',3f PHWK\O LPLGD]RO\O ODXUHDWH 0,/f DQG 1GRGHF\O LPLGD]ROH ',f LQWR OLSRVRPHV DW D PRODU UDWLR RI 5 f 7KH FRQWURO JURXS UHIHUUHG WR OLSRVRPHV ZLWKRXW %36 'DWD ZHUH H[SUHVVHG DV PHDQO6' Q f Df $ OLSLG PL[LQJ DVVD\ ZDV XVHG WR TXDQWLI\ IXVLRQ DW YDULRXV OLSRVRPH FRQFHQWUDWLRQV QPROPO KRUL]RQWDO OLQH EDUf QPROPO YHUWLFDO OLQH EDUf DQG QPROPO VROLG EDUff Ef $ FDOFHLQ UHOHDVH PHDVXUHPHQW ZDV XVHG WR GHWHUPLQH WKH OLSRVRPH O\VLV EHKDYLRU DW LQFUHDVLQJ OLSRVRPH FRQFHQWUDWLRQV QPROPO RSHQ EDUf QPROPO GRWWHG EDUf DQG QPROPO YHUWLFDO OLQH EDUff

PAGE 157

)LJXUH f§FRQWLQXHG

PAGE 158

)LJXUH (IIHFW RI FKROHVWHURO DQG GLROHR\OSKRVSKDWLG\OHWKDQRODPLQH '23(f RQ PHPEUDQH IXVLRQ XVLQJ WKH 1%'5K OLSLG PL[LQJ DVVD\ Q f /LSRVRPHV QPROPOf FRQWDLQLQJ GRGHF\O OfLPLGD]RO\Of SURSLRQDWH ',3f DW YDULRXV PRODU UDWLRV 5f ZLWKRXW DQ\ FROLSLG Af ZLWK b ZHLJKWf FKROHVWHURO ‘f RU ZLWK b ZHLJKWf '23( $f ZHUH FRPSDUHG LQ GLIIHUHQW S+ HQYLURQPHQWV 'DWD DUH H[SUHVVHG DV PHDQs6' Df 5 Ef 5

PAGE 159

F R )LJXUH f§FRQWLQXHG

PAGE 160

FKROHVWHURO ZDV UHSODFHG E\ '23( IXVLRQ HYHQWV GHFUHDVHG VLJQLILFDQWO\ LQ ERWK JURXSV S222Of 6LJQLILFDQW GLIIHUHQFHV RI IXVLRQ Sf ZHUH REVHUYHG DPRQJ WKH WKUHH VHSDUDWH IRUPXODWLRQV LQ ERWK UDWLR JURXSV ZKHQ WKH S+ ZDV GURSSHG WR :LWK WKH VDPH PRODU UDWLR WR D QHXWUDO OLSLG GLROHR\OSKRVSKDWLG\OHWKDQRODPLQH '23(f DQG FKROHVWHURO FRQWULEXWHG GLVWLQFWLYHO\ LQ WKH GRGHF\O OfLPLGD]RO\Of SURSLRQDWH 'O3fOLSRVRPH IXVLRQ EHKDYLRU RYHU WKH REVHUYHG S+ )LJXUH f 7KH LQFRUSRUDWLRQ RI FKROHVWHURO LQWR WKH OLSRVRPHV UHVXOWHG LQ DGGLWLRQDO IXVLRQ Sf WKDQ WKH OLSRVRPHV ZLWK /DOHFLWKLQ RQO\ DV S+ GURSSHG WR RU ORZHU :KHQ FKROHVWHURO ZDV UHSODFHG E\ '23( IXVLRQ GHFUHDVHG VLJQLILFDQWO\ Sf DV S+ GHFUHDVHG WR )XVLRQ DIIHFWHG E\ FKROHVWHURO ZDV VLJQLILFDQWO\ KLJKHU WKDQ WKDW DIIHFWHG E\ '23( Sf DW S+ :KHQ WKH S+ ZDV UDLVHG WR RU DERYH IXVLRQ LQIOXHQFHG E\ '23( ZDV VLJQLILFDQWO\ KLJKHU WKDQ WKDW E\ FKROHVWHURO Sf 'LVFXVVLRQ 1XFOHLF DFLG HJ SODVPLG '1$ DQG ROLJRQXFOHRWLGHf WKHUDS\ LV D SURPLVLQJ DSSURDFK IRU WKH WUHDWPHQW RI D YDULHW\ RI GLVRUGHUV 6RNRO t *HZLUW] f 7ZR GHOLYHU\ PHWKRGV YLUDO DQG QRQYLUDO DUH EHLQJ VWXGLHG IRU FHOOXODU GHOLYHU\ RI WKHVH QXFOHLF DFLG DJHQWV 7KH QRQYLUDO V\VWHPV HJ FDWLRQLF OLSRVRPHVf DUH DWWUDFWLYH GXH WR WKH HDVH RI SURGXFWLRQ WKH DELOLW\ WR WUDQVIHFW D YDULHW\ RI FHOO W\SHV DQG WKH ORZHU FKDQFH RI LPPXQH UHDFWLRQV /HH t +XDQJ f :KLOH QRQYLUDO V\VWHPV DUH FXUUHQWO\ VRPHZKDW LQHIILFLHQW WKH\ ZLOO XQGRXEWHGO\ LPSURYH ZLWK WKH SURGXFWLRQ RI QHZ V\VWHPV DQG GHWHUPLQDWLRQ RI UDWHOLPLWLQJ PHFKDQLVPV WKDW JRYHUQ QRQYLUDO QXFOHLF GHOLYHU\

PAGE 161

)LJXUH (IIHFW RI HTXDO PRODU UDWLR RI FKROHVWHURO DQG GLROHR\OSKRVSKDWLG\OHWKDQRODPLQH '23(f WR /DOHFLWKLQ RQ PHPEUDQH IXVLRQ XVLQJ WKH 1%'5K OLSLG PL[LQJ DVVD\ Q f /LSRVRPHV QPROPOf FRQWDLQLQJ GRGHF\O O LPLGD]RO\Of SURSLRQDWH ',3f DW PRODU UDWLR 5 f ZLWKRXW DQ\ FROLSLG Af ZLWK FKROHVWHURO ‘f DQG ZLWK '23( $f ZHUH FRPSDUHG LQ GLIIHUHQW S+ HQYLURQPHQWV 'DWD DUH H[SUHVVHG DV PHDQs6'

PAGE 162

'RGHF\O O fLPLGD]RO\Of SURSLRQDWH ',3f WKH SURWRW\SLFDO PHPEHU RI WKH ELRGHJUDGDEOH S+VHQVLWLYH VXUIDFWDQWV %36f IDPLO\ KDV EHHQ VKRZQ WR LQFUHDVH WKH HIIHFWLYHQHVV RI QXFOHLF DFLG GHOLYHU\ +XJKHV HW DO /LDQJ t +XJKHV f +RZHYHU LW LV XQFOHDU KRZ %36 GHVWDELOL]H WKH HQGRVRPDO PHPEUDQH DQG HOLFLW WKH WUDQVIHU RI WKH ROLJRQXFOHRWLGH DQG SODVPLG '1$ RU LI WKLV LV LWV PDLQ PHFKDQLVP RI DFWLRQ %RWK PHPEUDQH IXVLRQ DQG UXSWXUH HOLFLWHG E\ %36 ZHUH S+ DQG PRODU UDWLR GHSHQGHQW $V WKH S+ EHFDPH DFLGLF RU %36 LQWUDOLSRVRPDO DPRXQW LQFUHDVHG HJ LQFUHDVLQJ WKH DPRXQW RI LRQL]HG %36f WKH IXVLRQ DQG UXSWXUH HYHQWV LQFUHDVHG 7KH H[WHQW RI PHPEUDQH UXSWXUH DQG IXVLRQ ZHUH DOVR HOHYDWHG ZKHQ WKH QRQFKDUJHG %36 ZDV HTXDO WR D PRODU UDWLR RI 7KLV HIIHFW LV SRVVLEO\ GXH WR WKH DOWHUQDWLRQV LQ OLSLG SDFNLQJ 1HZ f :H ZHUH XQDEOH WR IRUP %36OLSRVRPHV DV WKH PRODU UDWLR RI %36 WR WRWDO OLSLGV HTXDOHG WR 5 f RU DERYH :KHQ FRPSDULQJ WZR PHPEUDQH IXVLRQ DVVD\V LW ZDV IRXQG IXVLRQ GHWHUPLQHG E\ WKH OLSLG PL[LQJ DVVD\ ZDV FRQVLVWHQWO\ LH bf KLJKHU WKDQ WKRVH E\ WKH DTXHRXV FRQWHQW PL[LQJ DVVD\ 7KLV GLVFUHSDQF\ ZDV DWWULEXWHG WR WKH VHQVLWLYLW\ RI OLSLG DJJUHJDWLRQ IRU WKH OLSLG PL[LQJ DVVD\ :KHQ PHPEUDQHV DJJUHJDWH WKH IOXRUHVFHQFH HQHUJ\ IURP RQH OLSRVRPH JURXS FDQ DOVR WUDQVIHU WR WKH RWKHU JURXS 'X]JXQHV HW DO
PAGE 163

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f ,W LV WKHUHIRUH DQ LQWHUPHPEUDQH LQWHUDFWLRQ DQG OLSRVRPH FRQFHQWUDWLRQ GHSHQGHQW ,I WKH FDOFHLQ UHOHDVH LV WKH UHVXOW RI OHDNDJH GXULQJ WKH REVHUYHG SHULRG FDXVHG E\ IXVLRQ LW VKRXOG EH FRQFHQWUDWLRQ GHSHQGHQW (OOHQV f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f SKDVH OLSLG ELOD\HU SKDVH DQG PLFHOODU SKDVHf LV WR GHVFULEH WKH SDFNLQJ RI OLSLGV LQWR D ELOD\HU E\ FRQVLGHULQJ WKH $ VKDSH RI WKH PROHFXOH 7KLV FDQ EH UHSUHVHQWHG PRUH TXDQWLWDWLYHO\ E\ 3U ZKHUH 3U $ LV WKH SDFNLQJ UDWLR $K LV WKH HIIHFWLYH FURVVVHFWLRQDO DUHD RI WKH KHDG JURXS DQG $F LV

PAGE 164

WKH HIIHFWLYH FURVVVHFWLRQDO DUHD RI WKH K\GURFDUERQ FKDLQ UHJLRQ
PAGE 165

FRPSRXQGV ZLWKLQ WKH ELOD\HU FDQ WKHQ IDFLOLWDWH PHPEUDQH IXVLRQ DIWHU WKH FRQYHUVLRQ RI WKH SURWRQDWHG %36 :KHQ '23( ZDV SUHVHQW LQ WKH OLSRVRPH IRUPXOD WKH SDFNLQJ UDWLR RI %36 3UOf ZDV VLPLODU WR WKH +Q SKDVH RI '23( 3UOf ZKLFK PDGH WKH OLSRVRPH PRUH IXVRJHQLF WKDQ OLSRVRPHV ZLWKRXW '23( DW DQ DONDOLQH S+ 7KLV HIIHFW ZDV PRUH HYLGHQW LQ WKH SUHVHQFH RI H[WUD b PROf '23( LQ WKH OLSRVRPHV $V WKH S+ GHFUHDVHG WKH SURWRQDWHG %36 3U!Of EHFDPH FRPSOHPHQWDU\ WR '23( 3UOf ,QVWHDG RI FDXVLQJ IXVLRQ WKH OLSRVRPHV ZRXOG WKHQ WHQG WR UHPDLQ DV DQ LQWDFW ELOD\HU VWUXFWXUH DV WKH S+ GHFUHDVHG :KHQ WKH DPRXQW RI %36 ZDV b PROf PRUH WKDQ WKDW RI '23( DW ZKLFK %36 RXWZHLJKHG '23( WKH IXVLRQ ZDV UHODWLYHO\ VWDEOH LQ WKH S+ UDQJH RI DQG LQFUHDVHG DW S+ GDWD QRW VKRZQf +RZHYHU DV H[SHFWHG WKH LQFUHDVHG IXVLRQ ZDV OHVV WKDQ WKH OLSRVRPHV ZLWKRXW '23( %DVHG XSRQ WKH JHQHUDWHG GDWD DQG H[LVWLQJ OLWHUDWXUH UHJDUGLQJ PHPEUDQH IXVLRQ DQG UXSWXUH PHFKDQLVP ZH SURSRVHG D %36LQGXFHG PHPEUDQH GHIHFW PHFKDQLVP )LJXUH f ,W LV VXJJHVWHG WKDW DW DQ DONDOLQH S+ XQLRQL]HG %36 KDYLQJ D UHODWLYHO\ VPDOO KHDG JURXS DV FRPSDUHG WR WKH LRQL]HG VSHFLHV ZLOO UHVLGH ZLWKLQ WKH OLSLG ELOD\HU RU LQQHU OHDIOHW :LWK WKH ORRVH OLSLG SDFNLQJ IURP WKH LQFRUSRUDWLRQ RI XQLRQL]HG %36 WKH VZROOHQ PL[HG ELOD\HUV WHQG WR EH OHVV VWDEOH OHDGLQJ WR HDVLHU IXVLRQ DQG OHDNDJH )LJXUH DQG )LJXUH f FRUUHVSRQGLQJ WR WKH LQFUHDVLQJ DPRXQWV RI %36 SDFNHG ZLWKLQ WKH ELOD\HU $V WKH DPRXQW RI %36 LQ WKH PHPEUDQH LQFUHDVHV WKH VZROOHQ PL[HG ELOD\HU PD\ EH WUDQVIRUPHG LQWR D KXPSEDFNHG PL[HG ELOD\HU DW ZKLFK %36 ZLOO UHVLGH ZLWKLQ UHJLRQV RI KLJK QHJDWLYH FXUYDWXUH 7KH LPLGD]ROH KHDG JURXS ZKLFK LV PRUH K\GURSKRELF WKDQ OHFLWKLQfV KHDG JURXS ZRXOG UHTXLUH OHVV GHK\GUDWLRQ WR IRUP WKH VWDON

PAGE 166

5XSWXUH 3URWRQDWHG %36 : I" A )XVLRQ )LJXUH 3URSRVHG PHFKDQLVP RI OLSRVRPH GHVWDELOL]DWLRQ HOLFLWHG E\ ELRGHJUDGDEOH S+VHQVLWLYH VXUIDFWDQWV %36f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

PAGE 167

WKH ILUVW VWHS LQ WKH PHPEUDQH IXVLRQ SURFHVV WKXV IDYRULQJ WKLV WUDQVLWLRQ DQG UHVXOWLQJ LQ WKH OLSLG PL[LQJ HIIHFWV )LJXUH DQG )LJXUH f :KHQ D SRUWLRQ RI %36 LV SURWRQDWHG 3 !f DV D FRQVHTXHQFH RI WKH DFLGLF S+ SUHVHQW LQ WKH V\VWHP D VWDON SRUH ZLOO EH FUHDWHG WR FRPSOHWH WKH IXVLRQ SURFHVV UHVXOWLQJ WKH FRQWHQW PL[LQJ HIIHFWV )LJXUH DQG )LJXUH f :LWKRXW DQ\ VXEVHTXHQW LQWHUDFWLRQ EHWZHHQ RWKHU KXPSEDFNHG PL[HG ELOD\HUV )LJXUH DQG )LJXUH f WKH SURWRQDWHG %36 PD\ WUDQVIRUP WKH KXPSEDFNHG PL[HG ELOD\HU WR D PL[HG ELOD\HU VKHHW /DVFK f LQ ZKLFK RQO\ D UXSWXUH HYHQW ZLOO EH REVHUYHG )LJXUH f 7KH DERYH K\SRWKHWLFDO PHPEUDQH IXVLRQ GHVFULSWLRQ LV RQO\ RQH RI WKH UHSRUWHG PHFKDQLVPV WKDW KDYH EHHQ SURSRVHG IRU PHPEUDQH IXVLRQ
PAGE 168

WKH UHVXOWV IURP WKLV VWXG\ FDQ EH DSSOLHG WR RXU PRGHO DJHQWV ZKLFK DOWHU WKH LQQHU OHDIOHW PD\ WKHQ LQFUHDVH PHPEUDQH IXVLRQ 'XH WR WKH XQFKDUJHG OLSRSKLOLF QDWXUH RI %36 DW S+ LW ZDV K\SRWKHVL]HG WKDW %36 ZRXOG GLVWULEXWH WR WKH LQQHU OHDIOHW VLPLODU WR RWKHU FRPSRXQGV ZLWK UHODWLYH VPDOO KHDG JURXSV +XDQJ HW DK 0LFKDHOVRQ HW DK f DQG GHPRQVWUDWH D SRUWLRQ RI LWV DFWLYLW\ DW WKLV ORFDWLRQ ,Q WKH FXUUHQW GLVFXVVLRQ WKH LQIOXHQFH RI WKH WUDQVPHPEUDQH S+ JUDGLHQW IRUPHG E\ WKH LQFXEDWLQJ WKH OLSRVRPHV DW DFLGLF S+ ZDV QRW DGGUHVVHG 2QJRLQJ VWXGLHV ZLOO VWXG\ WKLV SKHQRPHQRQ ZKLFK PD\ UHVXOW LQ UHGLVWULEXWLRQ RI %36 ZLWKLQ WKH OLSLG ELOD\HU ,Q WKH SUHFHGLQJ GLVFXVVLRQ ZH KDYH FRQVLGHUHG WKUHH %36 GRGHF\O O LPLGD]RO\O SURSLRQDWH ',3f PHWK\O LPLGD]RO\O ODXUHDWH 0,/f DQG 1GRGHF\O LPLGD]ROH ',ff DV RQH FDWHJRU\ $W WKH RQVHW RI WKH H[SHULPHQWV D JUHDWHU GLIIHUHQFH ZDV H[SHFWHG EHWZHHQ WKH LQGLYLGXDO %36 PHPEHUV ZLWK UHJDUG WR IXVLRQ DQG UXSWXUH ,Q SUHYLRXV VWXGLHV E\ DQ LQWHUIDFLDO WHQVLRPHWHU WKH FULWLFDO PLFHOOH FRQFHQWUDWLRQ &0&f RI WKUHH %36 LQ DQ LRQL]HG VWDWH ZHUH GHWHUPLQHG WR EH P0 P0 DQG P0 IRU ',3 0,/ DQG ', UHVSHFWLYHO\ LQ D S+ K\GURFKORULF DFLG VROXWLRQ S0f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

PAGE 169

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fOf FRPSRXQGV FDQ IDFLOLWDWH WKH IRUPDWLRQ RI WKH VWDON DQG SRVLWLYH FXUYDWXUH 3 !f FRPSRXQGV DUH SUHIHUUHG IRU WKH IRUPDWLRQ RI WKH VWDON SRUH WR FRPSOHWH WKH PHPEUDQH IXVLRQ SURFHVV &KHPRPRUGLN HW DO f 7KH FKHPLFDO VWUXFWXUH RI ELRGHJUDGDEOH S+ VHQVLWLYH VXUIDFWDQWV %36f DOORZV FRH[LVWHQFH RI ERWK XQLRQL]HG 3UOf DQG LRQL]HG %36 3 !f VSHFLHV GXULQJ WKH IXVLRQ SURFHVV ,QVWHDG RI FDXVLQJ PHPEUDQH IXVLRQ ZLWK WKH KH[DJRQDO ,, +Qf SKDVH VLPLODU WR GLROHR\OSKRVSKDWLG\OHWKDQRODPLQH '23(f %36 VHHPHG WR EH DEOH WR SDUWLFLSDWH LQ PHPEUDQH IXVLRQ DW GLIIHUHQW VWDJHV VWDON DQG VWDON SRUHf 7KH DERYH UHVXOWV LPSO\ H[DPSOHV RI KRZ %36 FDQ LQGXFH ERWK PHPEUDQH IXVLRQ DQG UXSWXUH LQ S+ DQG FRQFHQWUDWLRQ GHSHQGHQW PDQQHU 7KHVH ILQGLQJV VXJJHVWHG LPSRUWDQW LPSOLFDWLRQV LQ WKH GHYHORSPHQW RI %36 PHGLDWHG ROLJRQXFOHRWLGH GHOLYHU\ V\VWHP ,W VKRXOG EH QRWHG KRZHYHU WKDW WKHUH ZDV OLWWOH VWUXFWXUH DFWLYLW\ UHODWLRQVKLS EHWZHHQ %36 DQG WKHLU PHPEUDQH GHVWDELOL]DWLRQ HIIHFWV LQ WKH VWXG\ 7R FODULI\ WKH VWUXFWXUH DFWLYLW\

PAGE 170

UHODWLRQVKLS DQG PRUH IXOO\ XQGHUVWDQG WKH PHPEUDQH GHVWDELOL]DWLRQ HIIHFWV RI %36 PRUH ZRUN LQ UHVSHFW WR GLYHUVH KHDG JURXSV OLQNHUV DQG DOLSKDWLF WDLOV LV UHTXLUHG

PAGE 171

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f 0RVW ROLJRQXFOHRWLGHV HLWKHU ZLWK RU ZLWKRXW D GHOLYHU\ V\VWHP DUH WDNHQ LQWR FHOOV WKURXJK HQGRF\WRVLV DQG HOLPLQDWHG E\ O\VRVRPHV WKH ODWHU HQGRF\WRWLF VWDJH +HQFH WKH HQGRVRPDO PHPEUDQH UHSUHVHQWV WKH WKLUG UDWH OLPLWLQJ VWHS 7R LQFUHDVH ROLJRQXFOHRWLGH

PAGE 172

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f ZHUH SURSRVHG DQG GHVLJQHG WR IRFXV RQ GLVUXSWLQJ WKH WKLUG GHOLYHU\ EDUULHU E\ GHVWDELOL]LQJ WKH HQGRVRPDO PHPEUDQH ,QFRUSRUDWLQJ %36 LQWR OLSRVRPHV DV DQ ROLJRQXFOHRWLGH GHOLYHU\ V\VWHP ZRXOG SURWHFW WKH ROLJRQXFOHRWLGHV IURP GHJUDGDWLRQ ILUVW GHOLYHU\ EDUULHUf DQG LQFUHDVH ROLJRQXFOHRWLGH FHOOXODU XSWDNH VHFRQG GHOLYHU\ EDUULHUf $V WKH S+ GHFUHDVHG GXULQJ HQGRF\WRVLV WKH K\GURJHQ LRQ ZRXOG SURWRQDWH %36 DQG DFWLYDWH WKH PHPEUDQH GHVWDELOL]DWLRQ SURFHVV VR WKDW WKH HQGRVRPDO FRQWHQWV LQFOXGLQJ ROLJRQXFOHRWLGHV ZRXOG EH UHOHDVHG WKLUG GHOLYHU\ EDUULHUf $IWHU UHOHDVLQJ ROLJRQXFOHRWLGHV WR WKHLU VLWHV RI DFWLRQ %36 ZRXOG EH GLJHVWHG LQWR OHVV WR[LF PHWDEROLWHV E\ WKH H[LVWLQJ GLJHVWLYH HQ]\PHV LQ WKH F\WRSODVP 7KH DGGLWLRQDO F\WRWR[LF HIIHFWV IURP WKH GHOLYHU\ V\VWHP ZRXOG WKXV EH PLQLPL]HG 7R IXOILOO WKHVH UHTXLUHPHQWV WKUHH DJHQWV GRGHF\O OfLPLGD]RO\Of SURSLRQDWH ',3f PHWK\O OfLPLGD]RO\O ODXUHDWH 0,/f DQG1GRGHF\O LPLGD]ROH ',f ORRVHO\ JURXSHG DV %36 ZHUH V\QWKHVL]HG &ULWLFDO PLFHOOH FRQFHQWUDWLRQ DQG HIIHFWLYH UHOHDVH UDWLR RI WKH SURWRQDWHG %36 ZHUH FDOFXODWHG DV D SURRI RI VXUIDFWDQW FKDUDFWHULVWLFV 7KH TXHVWLRQ RI ZKHWKHU %36 KDYH VXUIDFH DFWLYLWLHV ZKHQ WKH\ DUH SURWRQDWHG DQG DEOH WR LQGXFH OLSRVRPDO FRQWHQW UHOHDVH ZDV LGHQWLILHG DW GLIIHUHQW S+V :KHQ LQFRUSRUDWHG LQWR OLSRVRPHV %36 UHOHDVHG FDOFHLQ LQ D S+GHSHQGHQW PDQQHU $V S+ GHFUHDVHG PRUH

PAGE 173

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f %RWK OLSRVRPH JURXSV H[KLELWHG D VLPLODU FHOOXODU XSWDNH SURILOH +RZHYHU %36OLSRVRPHV SURPRWHG VXEFHOOXODU UHGLVWULEXWLRQ RI ROLJRQXFOHRWLGHV DIWHU WKH\ ZHUH HQGRF\WRVHG LQWR FHOOV WKLUG GHOLYHU\ EDUULHUf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

PAGE 174

JURXS WR WKDW E\ WKH WDLO JURXSf OHVV WKDQ FDQ IRUP WKH KH[DJRQDO ,, +Qf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f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fOLSRVRPHV UHVXOWHG LQ EHWWHU HQ]\PH LQKLELWLRQ WKDQ OLSRVRPHV ZLWKRXW

PAGE 175

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f WKDW DUH QRW LQKLELWHG E\ VSHFLILF ROLJRQXFOHRWLGHV UHSUHVHQW D JRRG FDQGLGDWH WR UHFWLI\ WKH WRWDO QXPEHU RI OLYH FHOOV 6LQFH V\QWKHVL]HG ROLJRQXFOHRWLGHV ZHUH XVHG GLUHFWO\ ZLWKRXW SXULILFDWLRQ WKLV PLJKW DOVR GHFUHDVH WKHLU VSHFLILFLW\ 2UJDQLF VLPSOH H[WUDFWLRQ FDQ EH XVHG WR SXULI\ ROLJRQXFOHRWLGHV DQG SRVVLEO\ LQFUHDVH WKHLU VSHFLILFLW\ $V WKH SRVVLEOH LQWHUIHUHQFHV DUH FOHDUHG HQGRF\WRVLV LQKLELWRUV VXFK DV F\WRFKDODVLQ % FKORURTXLQH 1+& DQG ORZ WHPSHUDWXUH r&f FDQ EH XVHG DV DGGLWLRQDO FRQWUROV WR VWUHQJWKHQ %36 PHGLDWHG ROLJRQXFOHRWLGH DFWLYLW\ &UHDWLRQ RI D 6WUXFWXUH $FWLYLW\ 5HODWLRQVKLS 1RQH RI WKH SURSHUWLHV WKDW ZHUH GHWHUPLQHG LQ WKH SUHYLRXV H[SHULPHQWV FRXOG FRUUHODWH ZLWK WKH VWUXFWXUHV RI WKH WKUHH ELRGHJUDGDEOH S+VHQVLWLYH VXUIDFWDQWV %36f 7KHVH WKUHH DJHQWV ZHUH XVHG WR FRUURERUDWH WKH ILQGLQJV LQ RXU FXUUHQW VWXGLHV ,Q RUGHU WR GUDZ DQ\ FRQFOXVLRQV DERXW WKH VWUXFWXUH DFWLYLW\ UHODWLRQVKLS PRUH %36 LQ WHUPV RI O\VRVRPRWURSLF DPLQH KHDG JURXSV K\GURFDUERQ WDLO JURXSV DQG FRQQHFWLQJ EULGJHV PXVW

PAGE 176

EH V\QWKHVL]HG 7KH SDUDPHWHUV LQFOXGLQJ FULWLFDO PLFHOOH FRQFHQWUDWLRQ &0&f HIIHFWLYH UHOHDVH UDWLR 5Hf S+ VHQVLWLYLW\ FKHPLFDO UDWH FRQVWDQW ELRGHJUDGDELOLW\ F\WRWR[LFLW\ ROLJRQXFOHRWLGH FHOOXODU XSWDNH ROLJRQXFOHRWLGH ELRORJLFDO HIIHFW DQG PHFKDQLVP RI DFWLRQ ZLOO EH DVVHVVHG 1HZO\ V\QWKHVL]HG %36 ZLOO WKHQ EH FRPSDUHG WR DGGUHVV WKHLU LQGLYLGXDO LPSDFW RQ %36 PHGLDWHG ROLJRQXFOHRWLGH FHOOXODU GHOLYHU\ 7KHUHDIWHU WKH ELRORJLFDO HIIHFW RI ROLJRQXFOHRWLGHV FDXVHG E\ %36OLSRVRPHV FDQ EH SUHGLFWHG IURP WKH SHUVSHFWLYH RI WKH HVWDEOLVKHG SDUDPHWHUV

PAGE 177

5()(5(1&(6 $JUDZDO 6 DQG ,\HU 5 3 f 3HUVSHFWLYHV LQ DQWLVHQVH WKHUDSHXWLFV 3KDUPDFRO 7KHU $JUDZDO 6 *RRGFKLOG &LYHLUD 0 3 7KRUQWRQ $ + 6DULQ 3 6 DQG =DPHFQLN 3 & f 2OLJRGHR[\QXFOHRVLGH SKRVSKRUDPLGDWHV DQG SKRVSKRURWKLRDWHV DV LQKLELWLRUV RI KXPDQ LPPXQRGHILFLHQF\ YLUXV 3URF 1DWO $FDG 6FL 86$ f $NKWDU 6 %DVX 6 :LFNVWURP ( DQG -XOLDQR 5 / Df ,QWHUDFWLRQV RI DQWLVHQVH '1$ ROLJRQXFOHRWLGH DQDORJV ZLWK SKRVSKROLSLG PHPEUDQHV OLSRVRPHVf 1XFOHLF $FLGV 5HV $NKWDU 6 .ROH 5 DQG -XOLDQR 5 / f 6WDELOLW\ RI DQWLVHQVH '1$ ROLJRGHR[\QXFOHRWLGH DQDORJV LQ FHOOXODU H[WUDFWV DQG VHUD /LIH 6FL f $NKWDU 6 DQG -XOLDQR 5 / f &HOOXODU XSWDNH DQG LQWUDFHOOXODU IDWH RI DQWLVHQVH ROLJRQXFOHRWLGHV 7UHQGV &HOO %LRO $ODKDUL 6 'HDQ 1 0 )LVKHU 0 )/ 'HORQJ 5 0DQRKDUDQ 0 7LYHO / DQG -XOLDQR 5 / f ,QKLELWLRQ RI H[SUHVVLRQ RI WKH PXOWLGUXJ UHVLVWDQFHDVVRFLDWHG 3 JO\FRSURWHLQ RI E\ SKRVSKRURWKLRDWH DQG n FKROHVWHUROFRQMXJDWHG SKRVSKRURWKLRDWH DQWLVHQVH ROLJRQXFOHRWLGHV 0RO 3KDUPDFRO f $QD]RGR 0 :DLQEHUJ 0 $ )ULHVHQ $ DQG :ULJKW $ f 6HTXHQFH VSHFLILF LQKLELWLRQ RI JHQH H[SUHVVLRQ E\ D QRYHO DQWLVHQVH ROLJRGHR[\QXFOHRWLGH SKRVSKRURWKLRDWH GLUHFWHG DJDLQVW D QRQUHJXODWRU\ UHJLRQ RI WKH KXPDQ LPPXQRGHILFLHQF\ YLUXV W\SH JHQRPH 9LURO $RNL 0 0RULVKLWD 5 +LJDNL 0RULJXFKL $ .LGD +D\DVKL 6 0DWVXVKLWD + .DQHGD < DQG 2JLKDUD 7 f ,Q YLYR WUDQVIHU HIILFLHQF\ RI DQWLVHQVH ROLJRQXFOHRWLGHV LQWR WKH P\RFDUGLXP XVLQJ +9-OLSRVRPH PHWKRG %LRFKHP %LRSKYV 5HV &RPPXQ f $ULPD + $UDPDNL < DQG 7VXFKL\D 6 f (IIHFW RI ROLJRGHR[\QXFOHRWLGHV RQ WKH SK\VLFRFKHPLFDO FKDUDFWHULVWLFV DQG FHOOXODU XSWDNH RI OLSRVRPHV 3KDUP 6FL f

PAGE 178

$URQVRKQ $ / DQG +XJKHV $ f 1XFOHDU ORFDOL]DWLRQ VLJQDO SHSWLGHV HQKDQFH FDWLRQLF OLRVRPHPHGLDWHG JHQH WKHUDS\ 'UXJ 7DUJHWLQJ f %DUU\ ( / *HVHN ) $ DQG )ULHGPDQ 3 $ f ,QWURGXFWLRQ RI DQWLVHQVH ROLJRQXFOHRWLGHV LQWR FHOOV E\ SHUPHDELOL]DWLRQ ZLWK VWUHSWRO\VLQ 2 %LRWHFKQLTXHV f %HORXVRY ( 6 $IRQLQD $ 3RG\PLQRJLQ 0 $ *DPSHU + % 5HHG 0 : :\GUR 5 0 DQG 0H\HU 5 % f 6HTXHQFHVSHFLILF WDUJHWLQJ DQG FRYDOHQW PRGLILFDWLRQ RI KXPDQ JHQRPLF '1$ 1XFOHLF $FLGV 5HV f %HQQHWW & ) &KLDQJ 0 < &KDQ + 6KRHPDNHU ( DQG 0LUDEHOOL & f &DWLRQLF OLSLGV HQKDQFH FHOOXODU XSWDNH DQG DFWLYLW\ RI SKRVSKRURWKLRDWH DQWLVHQVH ROLJRQXFOHRWLGHV 0RO 3KDUPDFRO f %HQQHWW 0 1DQW] 0 + %DODVXEUDPDQLDP 5 3 *UXHQHUW & DQG 0DORQH 5 : f &KROHVWHURO HQKDQFHV FDWLRQLF OLSRVRPHPHGLDWHG '1$ WUDQVIHFWLRQ RI KXPDQ UHVSLUDWRU\ HSLWKHOLDO FHOOV %LRVFL 5HS f %HQW] (OOHQV + /DL 0 = DQG 6]RND ) & -U f 2Q WKH FRUUHODWLRQ EHWZHHQ +,, SKDVH DQG WKH FRQWDFWLQGXFHG GHVWDELOL]DWLRQ RI SKRVSKDWLG\OHWKDQRODPLQH FRQWDLQLQJ PHPEUDQHV 3URF 1DWO $FDG 6FL 86$ f %HUJDQ 5 +DNLP ) 6FKZDUW] 1 .\OH ( &HSDGD 5 6]DER 0 )RZOHU *UHVV 5 DQG 1HFNHUV / f (OHFWURSRUDWLRQ RI V\QWKHWLF ROLJRGHR[\QXFOHRWLGHV D QRYHO WHFKQLTXH IRU H[ YLYR ERQH PDUURZ SXUJLQJ %ORRG f %LHOLQVND $ .XNRZVND /DWDOOR ) -RKQVRQ 7RPDED $ DQG %DNHU 5 -U f 5HJXODWLRQ RI LQ YLWUR JHQH H[SUHVVLRQ XVLQJ DQWLVHQVH ROLJRQXFOHRWLGHV RU DQWLVHQVH H[SUHVVLRQ SODVPLGV WUDQVIHFWHG XVLQJ VWDUEXUVW 3$0$0 GHQGULPHUV 1XFOHLF $FLGV 5HV f %ODNH 0XUDNDPL $ 6SLW] 6 $ *ODYH 6 $ 5HGG\ 0 3 7VnR 3 2 DQG 0LOOHU 3 6 f +\EULGL]DWLRQ DUUHVW RI JORELQ V\QWKHVLV LQ UDEELW UHWLFXORF\WH O\VDWHV DQG FHOOV E\ ROLJRGHR[\ULERQXFOHRVLGH PHWK\OSKRVSKRQDWHV %LRFKHPLVWU\ f %ORQGHO KDUPLVRQ * DQG 6FKXEHUW 0 f 5ROH RI PDWUL[ SURWHLQ LQ F\WRSDWKRJHQHVLV RI YHVLFXODU VWRPDWLWLV YLPV 9LURO %RFN / & *ULIILQ / & /DWKDP $ 9HUPDVV ( + DQG 7RROH f 6HOHFWLRQ RI VLQJOHVWUDQGHG '1$ PROHFXOHV WKDW ELQG DQG LQKLELW KXPDQ WKURPELQ 1DWXUH f

PAGE 179

%RGRU 1 .DPLQVNL DQG 6HON 6 f 6RIW GUXJV /DELOH TXDWHUQDU\ DPPRQLXP VDOWV DV VRIW DQWLPLFURELDOV 0HG &KHP f %RQILOV ( 'HSLHUUHX[ & 0LGRX[ 3 7KXRQJ 1 7 0RQVLJQ\ 0 DQG 5RFKH $ & f 'UXJ WDUJHWLQJ V\QWKHVLV DQG HQGRF\WRVLV RI ROLJRQXFOHRWLGHQHRJO\FRSURWHLQ FRQMXJDWHV 1XFOHLF $FLGV 5HV %RQJDUW] 3 $XEHUWLQ $ 0 0LOKDXG 3 DQG /HEOHX % f ,PSURYHG ELRORJLFDO DFWLYLW\ RI DQWLVHQVH ROLJRQXFOHRWLGHV FRQMXJDWHG WR D IXVRJHQLF SHSWLGH 1XFOHLF $FLGV 5HV f %RXWRULQ $ 6 *XVfNRYD / 9 ,YDQRYD ( 0 .REHW] 1 =DU\WRYD 9 ) 5\WH $ 6
PAGE 180

SURWHFWV WKHP DJDLQVW QXFOHDVHV DQG LQFUHDVHV WKHLU FHOOXODU XSWDNH 3KDUP 5HV f &KHQ :HLWK + / *UHZDO 5 6 :DQJ DQG &XVKPDQ 0 f 6\QWKHVLV RI QRYHO SKRVSKRUDPLGLWH UHDJHQWV IRU WKH DWWDFKPHQW RI DQWLVHQVH ROLJRQXFOHRWLGHV WR YDULRXV UHJLRQV RI WKH EHQ]RSKHQDQWKULGLQH ULQJ V\VWHP %LRFRQMXJ &KHP f &KHPRPRUGLN / .R]ORY 0 0 DQG =LPPHUEHUJ f /LSLGV LQ ELRORJLFDO PHPEUDQH IXVLRQ 0HPEU %LRO &KLQ *UHHQ $ =RQ 6]RND ) & -U DQG 6WUDXELQJHU 5 0 f 5DSLG QXFOHDU DFFXPXODWLRQ RI LQMHFWHG ROLJRGHR[\ULERQXFOHRWLGHV 1HZ %LRO f &KX & 'LMNVWUD /DL 0 = +RQJ DQG 6]RND ) & f (IILFLFHQF\ RI F\WRSODVPLF GHOLYHU\ E\ S+VHQVLWLYH OLSRVRPHV WR FHOOV LQ FXOWXUH 3KDUP 5HV f &LWUR 3HUURWWL &XFFR & $JQDQR 6DFFKL $ =XSL DQG &DODEUHWWD % f ,QKLELWLRQ RI OHXNHPLD FHOO SUROLIHUDWLRQ E\ UHFHSWRUPHGLDWHG XQWDNH RI FP\E DQWLVHQVH ROLJRGHR[\QXFOHRWLGHV 3URF 1DWO $FDG 6FL 86$ f &LWUR 6]F]\OLN 3 *LQREEL 3 =XSL DQG &DODEUHWWD % f ,QKLELWLRQ RI OHXNDHPLD FHOO SUROLIHUDWLRQ E\ IROLF DFLGSRO\O\VLQHPHGLDWHG LQWURGXFWLRQ RI FP\E DQWLVHQVH ROLJRGHR[\QXFOHRWLGHV LQWR +/ FHOOV %U &DQFHU f &RQQROO\ % $ 3RWWHU % 9 (FNVWHLQ ) 3LQJRXG $ DQG *URWMDKQ / f 6\QWKHVLV DQG FKDUDFWHUL]DWLRQ RI DQ RFWDQXFOHRWLGH FRQWDLQLQJ WKH (FR5, UHFRJQLWLRQ VHTXHQFH ZLWK D SKRVSKRURWKLRDWH JURXS DW WKH FOHDYDJH VLWH %LRFKHPLVWU\ f &RQQRU
PAGE 181

&ULVWLQD 'H 2OLYHLUD 0 )DWWDO ( 5RSHUW & 0DOY\ & DQG &RXYUHXU 3 f 'HOLYHU\ RI DQWLVHQVH ROLJRQXFOHRWLGHV E\ PHDQV RI S+VHQVLWLYH OLSRVRPHV &RQWU 5HO &URRNH 6 7 f 3URJUHVV WRZDUG ROLJRQXFOHRWLGH WKHUDSHXWLFV SKDUPDFRG\QDPLF SURSHUWLHV )$6(% f 'H 'XYH & f /\VRVRPHV UHYLVLWHG (XU %LRFKHP f 'H 'XYH & 'H %DUV\ 7 3RROH % 7URXHW $ 7XONHQV 3 DQG 9DQ +RRI ) f /\VRVRPRWURSLF DJHQWV %LRFKHP 3KDUPDFRO f 'HDQ 5 7 -HVVXS : DQG 5REHUWV & 5 f (IIHFWV RI H[RJHQRXV DPLQHV RQ PDPPDOLDQ &HOOV ZLWK SDUWLFXODU UHIHUHQFH WR PHPEUDQH IORZ %LRFKHP f 'HJROV /HRQHWWL 3 *DJQRU & /HPDLWUH 0 DQG /HEOHX % f $QWLYLUDO DFWLYLW\ DQG SRVVLEOH PHFKDQLVPV RI DFWLRQ RI ROLJRQXFOHRWLGHVSRO\/O\VLQHf FRQMXJDWHV WDUJHWHG WR YHVLFXODU VWRPDWLWLV YLUXV P51$ DQG JHQRPLF 51$ 1XFOHLF $FLGV 5HV f 'HJROV /HRQHWWL 3 0HFKWL 1 DQG /HEOHX % f $QWLSUROLIHUDWLYH HIIHFWV RI DQWLVHQVH ROLJRQXFOHRWLGHV GLUHFWHG WR WKH 51$ RI FP\F RQFRJHQH 1XFOHLF $FLGV 5HV f 'HORQJ 5 6WHSKHQVRQ /RIWXV 7 )LVKHU 0 $ODKDUL 6 1ROWLQJ $ DQG -XOLDQR 5 / f &KDUDFWHUL]DWLRQ RI FRPSOH[HV RI ROLJRQXFOHRWLGHV ZLWK SRO\DPLGRDPLQH VWDUEXUVW GHQGULPHUV DQG HIIHFWV RQ LQWUDFHOOXODU GHOLYHU\ 3KDUP 6FL f 'HVKSDQGH 7ROHGR 9HODVTXH] 7KDNNDU /LDQJ : DQG 5RMDQDVDNXO < f (QKDQFHG FHOOXODU XSWDNH RI ROLJRQXFOHRWLGHV E\ (*) UHFHSWRUPHGLDWHG HQGRF\WRVLV LQ $ FHOOV 3KDUP 5HV f 'X]JXQHV 1 6WUDXELQJHU 5 0 %DOGZLQ 3 $ )ULHQG 6 DQG 3DSDKDGMRSRXORV f 3URWRQLQGXFHG IXVLRQ RI ROHLF DFLGSKRVSKDWLG\OHWKDQRODPLQH OLSRVRPHV %LRFKHPLVWU\ f ']DX 9 0DQQ 0 0RULVKLWD 5 DQG .DQHGD < f )XVLJHQLF YLUDO OLSRVRPH IRU JHQH WKHUDS\ LQ FDUGLRYDVFXODU GLVHDVHV 3URF 1DWO $FDG 6FL 86$ f (OOHQV + %HQW] DQG 6]RND ) & f S+LQGXFHG GHVWDELOL]DWLRQ RI SKRVSKDWLG\OHWKDQRODPLQHFRQWDLQLQJ OLSRVRPHV UROH RI ELOD\HU FRQWDFW %LRFKHPLVWU\ f

PAGE 182

(OOLQJWRQ $ f 51$ VHOHFWLRQ $SWDPHUV DFKLHYH WKH GHVLUHG UHFRJQLWLRQ &XUU %LRO f )DWWDO ( 1LU 6 3£UHQWH 5 $ DQG 6]RND ) & -U f 3RUHIRUPLQJ SHSWLGHV LQGXFH UDSLG SKRVSKROLSLG IOLSIORS LQ PHPEUDQHV %LRFKHPLVWU\ f )HLJQHU 3 / *DGHN 7 5 +ROP 0 5RPDQ 5 &KDQ + : :HQ] 3 5LQJROG 0 DQG 'DQLHOVHQ 0 f /LSRIHFWLRQ D KLJKO\ HIILFLHQW OLSLGPHGLDWHG '1$ WUDQVIHFWLRQ SURFHGXUH 3URF 1DWO $FDG 6FL 86$ f )HQVWHU 6 :DJQHU 5 : DQG )URHKOHU % & DQG &KLQ f ,QKLELWLRQ RI KXPDQ LPPXQRGHILFLHQF\ YLUXV W\SH HQY H[SUHVVLRQ E\ & SURS\QH ROLJRQXFOHRWLGHV VSHFLILF IRU 5HYUHVSQVH HOHPHQW VWHPORRS 9 %LRFKHPLVWU\ f )LUHVWRQH 5 $ 3LVDQR 0 %DLOH\ 3 6WXUP $ %RQQH\ 5 :LJKWPDQ 3 'HYOLQ 5 )LQ & 6 .HOOHU / DQG 7ZD\ 3 & Df /\VRVRPRWURSLF DJHQWV FDUEREHQ]R[\JO\F\OSKHQ\ODODQ\O D QHZ SURWHDVHVHQVLWLYH PDVNLQJ JURXS IRU LQWURGXFWLRQ LQWR FHOOV 0HG &KHP f )LUHVWRQH 5 $ 3LVDQR 0 DQG %RQQH\ 5 !f /\VRVRPRWURSLF DJHQWV V\QWKHVLV DQG F\WRWR[LF DFWLRQ RI O\VRVRPRWURSLF GHWHUJHQWV ,Q ,QWHUQDWLRQDO 6\PSRVLXP RQ 6ROXWLRQ %HKDYLRU RI 6XUIDFWDQWV 7KHRUHWLFDO DQG $SSOLHG $VSHFWV 0LWWDO / DQG )HQGOHU ( HGLWRUVf SS 3OHQXP 3UHVV 1HZ
PAGE 183

*DOEUDLWK : 0 +REVRQ : & *LFODV 3 & 6FKHFKWHU 3 DQG $JUDZDO 6 f &RPSOHPHQW DFWLYDWLRQ DQG KHPRG\QDPLF FKDQJHV IROORZLQJ LQWUDYHQRXV DGPLQLVWUDWLRQ RI SKRVSKRURWKLRDWH ROLJRQXFOHRWLGHV LQ WKH PRQNH\ $QWLVHQVH 5HV 'HY f &DPSHU + % 5HHG 0 : &R[ 7 9LURVFR 6 $GDPV $ *DOO $ $ 6FKROOHU DQG 0H\HU 5 % f )DFLOH SUHSDUDWLRQ RI QXFOHDVH UHVLVWDQW n PRGLILHG ROLJRGHR[\QXFOHRWLGHV 1XFOHLF $FLGV 5HV *f *DR : < 6WHLQ & $ &RKHQ 6 'XWVFKPDQ ( DQG &KHQJ < &*DR : < f (IIHFW RI SKRVSKRURWKLRDWH KRPRROLJRGHR[\QXFOHRWLGHV RQ KHUSHV VLPSOH[ YLUXV W\SH LQGXFHG '1$ SRO\PHUDVH %LRO &KHP *HUVKRQ + &KLUODQGR 5 *XWWPDQ 6 % DQG 0LQVN\ $ f 0RGH RI IRUPDWLRQ DQG VWUXFWXUDO IHDWXUHV RI '1$FDWLRQLF OLSRVRPH FRPSOH[HV XVHG IRU WUDQVIHFWLRQ %LRFKHPLVWU\ f *KRVK 0 *KRVK 'DKO 2 DQG &RKHQ 6 f (YDOXDWLRQ RI VRPH SURSHUWLHV RI D SKRVSKRURGLWKLRDWH ROLJRGHR[\ULERQXFOHRWLGH IRU DQWLVHQVH DSSOLFDWLRQ 1XFOHLF $FLGV 5HV f *LQREEL 3 *HLVHU 7 $ 2PEUHV DQG &LWUR f )ROLF DFLGSRO\O\VLQH FDUULHU LPSURYHV HIILFDF\ RI FP\F DQWLVHQVH ROLJRGHR[\QXFOHRWLGHV RQ KXPDQ PHODQRPD 0Of FHOOV $QWLFDQFHU 5HV ODf *LRYDQQDQJHOL & 3HUURXDXOW / (VFXGH & 1JX\HQ 7 DQG +HOHQH & f 6SHFLILF LQKLELWLRQ RI LQ YLWUR WUDQVFULSWLRQ HORQJDWLRQ E\ WULSOH[IRUPLQJ ROLJRQXFOHRWLGH LQWHUFDODWRU FRQMXJDWHV WDUJHWHG WR +,9 SURYLUDO '1$ %LRFKHPLVWU\ f *RGDUG %RXWRULQH $ 6 6DLVRQ%HKPRDUDV ( +HOHQH & f $QWLVHQVH HIIHFWV RI FKROHVWHUROROLJRGHR[\QXFOHRWLGH FRQMXJDWHV DVVRFLDWHG ZLWK SRO\DON\OF\DQRDFU\ODWHf QDQRSDUWLFOHV (XU %LRFKHP f *ULIIH\ 5 + 0RQLD % 3 &XPPLQV / / )UHLHU 6 *UHLJ 0 *XLQRVVR & /HVQLN ( 0DQDOLOL 6 0 0RKDQ 9 2ZHQV 6 5RVV % 5 6DVPRU + :DQFHZLF] ( :HLOHU :KHHOHU 3 DQG &RRN 3 f 2DPLQRSURS\O ULERQXFOHRWLGHV D ]ZLWWHULRQLF PRGLILFDWLRQ WKDW HQKDQFHV WKH H[RQXFOHDVH UHVLVWDQFH DQG ELRORJLFDO DFWLYLW\ RI DQWLVHQVH ROLJRQXFOHRWLGHV 0HG &KHP f *ULJRULHY 0 3UDVHXWK 5RELQ 3 +HPDU $ 6DLVRQ%HKPRDUDV 7 'DXWU\ 9DUVDW $ 7KXRQJ 1 7 +HOHQH & DQG +DUHO %HOODQ $ f $ WULSOH KHOL[IRUPLQJ ROLJRQXFOHRWLGHLQWHUFDODWRU FRQMXJDWH DFWV DV D WUDQVFULSWLRQDO UHSUHVVRU YLD LQKLELWLRQ RI 1) NDSSD % ELQGLQJ WR LQWHUOHXNLQ UHFHSWRU DOSKDUHJXODWRU\ VHTXHQFH %LRO &KHP f

PAGE 184

*XYDNRYD 0 $
PAGE 185

-DDVNHODLQHQ 0RQNNRQHQ DQG 8UWWL $ f 2OLJRQXFOHRWLGHFDWLRQLF OLSRVRPH LQWHUDFWLRQV $ SK\VLFRFKHPLFDO VWXG\ %LRFKLP %LRSK\V $FWD f -XOLDQR 5 / DQG $NKWDU 6 f /LSRVRPHV DV D GUXJ GHOLYHU\ V\VWHP IRU DQWLVHQVH ROLJRQXFOHRWLGHV $QWLVHQVH 5HV 'HY f .DEDQRY $ 9 9LQRJUDGRY 6 9 2YFKDUHQNR $ 9 .ULYRQRV $ 9 0HOLN 1XEDURY 1 6 .LVHOHY 9 DQG 6HYHULQ ( 6 f $ QHZ FODVV RI DQWLYLUDOV DQWLVHQVH ROLJRQXFOHRWLGHV FRPELQHG ZLWK D K\GURSKRELF VXEVWLWXHQW HIIHFWLYHO\ LQKLELW LQIOXHQ]D YLUXV UHSURGXFWLRQ DQG V\QWKHVLV RI YLUXVVSHFLILF SURWHLQV LQ 0'&. FHOOV )(%6 /HWW f .KDOHG = %HQLPHWVND\D / =HOWVHU 5 .KDQ 7 6KDUPD + : 1DUD\DQDQ 5 DQG 6WHLQ & $ f 0XOWLSOH PHFKDQLVPV PD\ FRQWULEXWH WR WKH FHOOXODU DQWLDGKHVLYH HIIHFWV RI SKRVSKRURWKLRDWH ROLJRGHR[\QXFOHRWLGHV 1XFOHLF $FLGV 5HV f .O\VLN .LQVH\ % 0 +XD 3 *ODVV $ DQG 2UVRQ ) 0 f $ EDVH DFULGLQHFRQMXJDWHG ROLJRGHR[\QXFOHRWLGH IRUPV WULSOH[ '1$ ZLWK LWV ,/5 DOSKD SURPRWHU WDUJHW ZLWK JUHDWO\ LPSURYHG DYLGLW\ %LRFRQMXJ &KHP f .RQRSND 3UHW]HU ( )HLJQHU 3 / DQG 'X]JXQHV 1 f +XPDQ LPPXQRGHILFLHQF\ YLUXV W\SH +,9f LQIHFWLRQ LQFUHDVHV WKH VHQVLWLYLW\ RI PDFURSKDJHV DQG 7+3 FHOOV WR F\WRWR[LFLW\ E\ FDWLRQLF OLSRVRPHV %LRFKLP %LRSK\V $FWD f .ULHJ $ 0
PAGE 186

/DSSDODLQHQ 0LHWWLQHQ 5 .HOORNRVNL -DDVNHODLQHQ DQG 6\UMDQHQ 6 f ,QWUDFHOOXODU GLVWULEXWLRQ RI ROLJRQXFOHRWLGHV GHOLYHUHG E\ FDWLRQLF OLSRVRPHV OLJKW DQG HOHFWURQ PLFURVFRSLF VWXG\ +LVWRFKHP &\WRFKHP f /DSSDODLQHQ 3LULOD / -DDVNHODLQHQ 6\UMDQHQ DQG 6\LMDQHQ 6 f (IIHFWV RI OLSRVRPDO DQWLVHQVH ROLJRQXFOHRWLGHV RQ P51$ DQG SURWHLQ OHYHOV RI WKH +39 ( RQFRJHQH $QWLFDQFHU 5HV Df /DSSDODLQHQ 8UWWL $ 6RGHUOLQJ ( -DDVNHODLQHQ 6\UMDQHQ DQG 6\UMDQHQ 6 f &DWLRQLF OLSRVRPHV LPSURYH VWDELOLW\ DQG LQWUDFHOOXODU GHOLYHU\ RI DQWLVHQVH ROLJRQXFOHRWLGHV LQWR &D6NL FHOOV %LRFKLP %LRSKYV $FWD f /DVFK f ,QWHUDFWLRQ RI GHWHUJHQWV ZLWK OLSLG YHVLFOHV %LRFKLP %LRSKYV $FWD f /HH 5 DQG +XDQJ / f /LSLGLH YHFWRU V\VWHPV IRU JHQH WUDQVIHU &ULW 5HY 7KHU 'UXJ &DUULHU 6YVWP f /HIHEYUH Gn+HOOHQFRXUW & 'LDZ / DQG *XHQRXQRX 0 f ,PPXQRPRGXODWLRQ E\ F\WRNLQH DQWLVHQVH ROLJRQXFOHRWLGHV (XU &\WRNLQH 1HWZ f /HPDLWUH 0 %D\DUG % DQG /HEOHX % f 6SHFLILF DQWLYLUDO DFWLYLW\ RI D SRO\ / O\VLQHfFRQMXJDWHG ROLJRGHR[\ULERQXFOHRWLGH VHTXHQFH FRPSOHPHQWDU\ WR YHVLFXODU VWRPDWLWLV YLUXV 1 SURWHLQ P51$ LQLWLDWLRQ VLWH 3URF 1DWO $FDG 6FL 86$ f /HRQHWWL 3 0DFK\ 3 'HJROV /HEOHX % DQG /HVHUPDQ / f $QWLERG\ WDUJHWHG OLSRVRPHV FRQWDLQLQJ ROLJRGHR[\ULERQXFOHRWLGHV FRPSOHPHQWDU\ WR YLUDO 51$ VHOHFWLYHO\ LQKLELW YLUDO UHSOLFDWLRQ 3URF 1DWO $FDG 6FL 86$ f /HRQHWWL 3 0HFKWL 1 'HJROV *DJQRU & DQG /HEOHX % f ,QWUDFHOOXODU GLVWULEXWLRQ RI PLFURLQMHFWHG DQWLVHQVH ROLJRQXFOHRWLGHV 3URF 1DWO $FDG 6FL 86$ f /HRQHWWL 3 5D\QHU % /HPDLWUH 0 *DJQRU & 0LOKDXG 3 ,PEDFK / DQG /HEOHX % f $QWLYLUDO DFWLYLW\ RI FRQMXJDWHV EHWZHHQ SRO\/O\VLQHf DQG V\QWKHWLF ROLJRGHR[\ULERQXFOHRWLGHV *HQH f /HWVLQJHU 5 / =KDQJ 5 6XQ ,NHXFKL 7 DQG 6DULQ 3 6 f &KROHVWHU\OFRQMXJDWHG ROLJRQXFOHRWLGHV V\QWKHVLV SURSHUWLHV DQG DFWLYLW\ DV LQKLELWRUV RI UHSOLFDWLRQ RI KXPDQ LPPXQRGHILFLHQF\ YLUXV LQ FHOO FXOWXUH 3URF 1DWO $FDG 6FL 86$

PAGE 187

/HYLQD $ 6 7DEDWDGVH 5 .KDOLPVND\D / 0 3ULFKRGNR 7 $ 6KLVKNLQ 9 $OH[DQGURYD / $ DQG =DU\WRYD 9 3 f 2OLJRQXFOHRWLGH GHULYDWLYHV EHDULQJ UHDFWLYH DQG VWDELOL]LQJ JURXSV DWWDFKHG WR & RI GHR[\XULGLQH %LRFRQMXJ &KHP f /LDQJ ( DQG +XJKHV $ f &KDUDFWHUL]DWLRQ RI D S+VHQVLWLYH VXUIDFWDQW GRGHF\OULPLGD]RO\Of SURSLRQDWH ',3f DQG SUHOLPLQDU\ VWXGLHV LQ OLSRVRPH PHGLDWHG JHQH WUDQVIHU %LRFKLP %LRSKYV $FWD f /LDQJ : : 6KL ; 'HVKSDQGH 0DODQJD & DQG 5RMDQDVDNXO < f 2OLJRQXFOHRWLGH WDUJHWLQJ WR DOYHRODU PDFURSKDJHV E\ PDQQRVH UHFHSWRUPHGLDWHG HQGRF\WRVLV %LRFKLP %LRSKYV $FWD f /LFKWHQEHUJ f &KDUDFWHUL]DWLRQ RI WKH VROXELOL]DWLRQ RI OLSLG ELOD\HUV E\ VXUIDFWDQWV %LRFKLP %LRSKYV $FWD f /LFKWHQEHUJ =LOEHUPDQ < *UHHQ]DLG 3 DQG =DPLU 6 f 6WUXFWXUDO DQG NLQHWLF VWXGLHV RQ WKH VROXELOL]DWLRQ RI OHFLWKLQ E\ VRGLXP GHR[\FKRODWH %LRFKHPLVWU\ f /LFKWHQIHOV 5 %LGGLVRQ : ( 6FKXO] + 9RJW $ % DQG 0DUWLQ 5 f &$5( /$66 FDOFHLQUHOHDVHDVVD\f $Q LPSURYHG IOXRUHVFHQFHEDVHG WHVW V\VWHP WR PHDVXUH F\WRWR[LF 7 O\PSKRF\WH DFWLYLW\ ,PPXQRO 0HWKRGV f /LW]LQJHU & %URZQ 0 :DOD .DXIPDQ 6 $ 9DQ < )DUUHOO & / DQG &ROOLQV f )DWH RI FDWLRQLF OLSRVRPHV DQG WKHLU FRPSOH[ ZLWK ROLJRQXFOHRWLGH LQ YLYR %LRFKLP %LRSKYV $FWD f /LX < DQG 5HJHQ 6 / f &RQWURO RYHU YHVLFOH UXSWXUH DQG OHDNDJH E\ PHPEUDQH SDFNLQJ DQG E\ WKH DJJUHJDWLRQ VWDWH RI DQ DWWDFNLQJ VXUIDFWDQW $P &KHP 6RF f /RNH 6 / 6WHLQ & =KDQJ ; $YLJDQ 0 &RKHQ DQG 1HFNHUV / 0 f 'HOLYHU\ RI FP\F DQWLVHQVH SKRVSKRURWKLRDWH ROLJRGHR[\QXFOHRWLGHV WR KHPDWRSRLHWLF FHOOV LQ FXOWXUH E\ OLSRVRPH IXVLRQ VSHFLILF UHGXFWLRQ LQ FP\F SURWHLQ H[SUHVVLRQ FRUUHODWHV ZLWK LQKLELWLRQ RI FHOO JURZWK DQG '1$ V\QWKHVLV &XUU 7RS 0LFURELRO ,PPXQRO /RNH 6 / 6WHLQ & $ =KDQJ ; + 0RUL 1DNDQLVKL 0 6XEDVLQJKH & &RKHQ 6 DQG 1HFNHUV / 0 f &KDUDFWHUL]DWLRQ RI ROLJRQXFOHRWLGH WUDQVSRUW LQWR OLYLQJ FHOOV 3URF 1DWO $FDG 6FL 86$ f 0D ' DQG :HL $ 4 f (QKDQFHG GHOLYHU\ RI V\QWKHWLF ROLJRQXFOHRWLGHV WR KXPDQ OHXNDHPLF FHOOV E\ OLSRVRPHV DQG LPPXQROLSRVRPHV /HXN 5HV f

PAGE 188

0DQQ 0 0RULVKLWD 5 *LEERQV + YRQ GHU /H\HQ + ( DQG ']DX 9 f '1$ WUDQVIHU LQWR YDVFXODU VPRRWK PXVFOH XVLQJ IXVLJHQLF 6HQGDL YLUXV +9-f OLSRVRPHV 0RO &HOO %LRFKHP f 0DUFKDQG & %DLOO\ & 1JX\HQ & + %LVDJQL ( *DUHVWLHU 7 +HOHQH & DQG :DULQJ 0 f 6WDELOL]DWLRQ RI WULSOH KHOLFDO '1$ E\ D EHQ]RS\ULGRTXLQR[DOLQH LQWHUFDODWRU %LRFKHPLVWU\ f 0DUVK 0 f 7KH HQWU\ RI HQYHORSHG YLUXVHV LQWR FHOOV E\ HQGRF\WRVLV %LRFKHP f 0DUWLQ $ f 3K\VLFDO 3KDUPDF\ /HD t )HELJHU 3KLODGHOSKLD 0DU]R $ / )LW]SDWULFN 5 5RELQVRQ % : DQG 6FRWW % f $QWLVHQVH ROLJRQXFOHRWLGHV VSHFLILF IRU WUDQVIRUPLQJ JURZWK IDFWRU EHWD LQKLELW WKH JURZWK RI PDOLJQDQW PHVRWKHOLRPD ERWK LQ YLWUR DQG LQ YLYR &DQFHU 5HV f 0D[HOG ) 5 f $FLGLILFDWLRQ RI HQGRF\WLF YHVLFOHV DQG O\VRVRPHV ,Q (QGRF\WRVLV 3DVWDQ DQG :LOOLQJKDP 0 & HGLWRUVf SS 3OHQXP 3UHVV 1HZ
PAGE 189

DQG SUROLIHUDWLQJFHOO QXFOHDU DQWLJHQ ROLJRQXFOHRWLGHV UHVXOWV LQ FKURQLF LQKLELWLRQ RI QHRLQWLPDO K\SHUSODVLD 3URF 1DWO $FDG 6FL 86$ 0RVHU + ( DQG 'HUYDQ 3 % f 6HTXHQFHVSHFLILF FOHDYDJH RI GRXEOH KHOLFDO '1$ E\ WULSOH KHOL[ IRUPDWLRQ 6FLHQFH f 0XUDNDPL $ %ODNH 5 DQG 0LOOHU 3 6 f &KDUDFWHUL]DWLRQ RI VHTXHQFH VSHFLILF ROLJRGHR[\ULERQXFOHRVLGH PHWK\OSKRVSKRQDWHV DQG WKHLU LQWHUDFWLRQ ZLWK UDEELW JORELQ P51$ %LRFKHPLVWU\ f 1HZ 5 5 & f /LSRVRPHV D SUDFWLFDO DSSURDFK 2[IRUG 8QLYHUVLW\ 3UHVV 1HZ
PAGE 190

3HUODN\ / 6DLMR < %XVFK 5 %HQQHWW & ) 0LUDEHOOL & &URRNH 6 7 DQG %XVFK + f *URZWK LQKLELWLRQ RI KXPDQ WXPRU FHOO OLQHV E\ DQWLVHQVH ROLJRQXFOHRWLGHV GHVLJQHG WR LQKLELW SO H[SUHVVLRQ $QWLFDQFHU 'UXJ 'HV f 3KLOOLSV 1 & DQG (PLOL $ f ,PPXQRJHQLFLW\ RI LPPXQROLSRVRPHV ,PPXQRO /HWW 3LQQDGXZDJH 3 6FKPLWW / DQG +XDQJ / f 8VH RI D TXDWHUQDU\ DPPRQLXP GHWHUJHQW LQ OLSRVRPH PHGLDWHG '1$ WUDQVIHFWLRQ RI PRXVH /FHOOV %LRFKLP %LRSK\V $FWD LOO 3R[RQ 6 : 0LWFKHOO 3 0 /LDQJ ( DQG +XJKHV $ f 'HQGULPHU GHOLYHU\ RI 2OLJRQXFOHRWLGHV 'UXJ 'HO f 4XDWWURQH $ 3DSXFFL / 0RUJDQWL 0 &RURQQHOOR 0 0LQL ( 0D]]HL 7 &RORQQD ) 3 *DUEHVL $ DQG &DSDFFLROL 6 f ,QKLELWLRQ RI 0'5 JHQH H[SUHVVLRQ E\ DQWLPHVVHQJHU ROLJRQXFOHRWLGHV ORZHUV PXOWLSOH GUXJ UHVLVWDQFH 2QFRO 5HV f 5HQQHLVHQ /HVHUPDQ / 0DWWKHV ( 6FKURGHU + & DQG 0XOOHU : ( f ,QKLELWLRQ RI H[SUHVVLRQ RI KXPDQ LPPXQRGHILFLHQF\ YLUXV LQ YLWUR E\ DQWLERG\ WDUJHWHG OLSRVRPHV FRQWDLQLQJ DQWLVHQVH 51$ WR WKH HQY UHJLRQ %LRO &KHP f 5RSHUW & /DYLJQRQ 0 'XEHPHW & &RXYUHXU 3 DQG 0DOY\ & f 2OLJRQXFOHRWLGHV HQFDSVXODWHG LQ S+ VHQVLWLYH OLSRVRPHV DUH HIILFLHQW WRZDUG )ULHQG UHWURYLUXV %LRFKHP %LRSK\V 5HV &RPPXQ 5RSHUW & 0DOY\ & DQG &RXYUHXU 3 f ,QKLELWLRQ RI WKH )ULHQG UHWURYLUXV E\ DQWLVHQVH ROLJRQXFOHRWLGHV HQFDSVXODWHG LQ OLSRVRPHV PHFKDQLVP RI DFWLRQ 3KDUP 5HV f 5RSHUW & 0LVKDO = -U 5RGULJXHV 0 0DOY\ & DQG &RXYUHXU 3 f 5HWURYLUXV EXGGLQJ PD\ FRQVWLWXWH D SRUW RI HQWU\ IRU GUXJ FDUULHUV %LRFKLP %LRSK\V $FWD W8 5RVH %XRQRFRUH / DQG :KLWW 0 $ f $ QHZ FDWLRQLF OLSRVRPH UHDJHQW PHGLDWLQJ QHDUO\ TXDQWLWDWLYH WUDQVIHFWLRQ RI DQLPDO FHOOV %LRWHFKQLTXHV f 5RVHQ 0 f 6XUIDFWDQWV DQG ,QWHUIDFLDO 3KHQRPHQD -RKQ :LOH\ t 6RQV 1HZ
PAGE 191

5XL] *RQL ) 0 DQG $ORQVR $ f 6XUIDFWDQWLQGXFHG UHOHDVH RI OLSRVRPDO FRQWHQWV $ VXUYH\ RI PHWKRGV DQG UHVXOWV %LRFKLP %LRSK\V $FWD f 6DJDWD 1 2VNDUVVRQ 0 &RSHODQG 7 %UXPEDXJK DQG 9DQGH:RXGH ) f )XQFWLRQ RI FPRV SURWRRQFRJHQH SURGXFW LQ PHLRWLF PDWXUDWLRQ LQ ;HQRSXV RRF\WHV 1DWXUH f 6DLMR < 3HUODN\ / 9DOGH] % & :DQJ + +HQQLQJ DQG %XVFK + f &HOOXODU SKDUPDFRORJ\ RI SL DQWLVHQVH ROLJRGHR[\QXFOHRWLGH SKRVSKRURWKLRDWH ,6,6 2QFRO 5HV 6DLVRQ%HKPRDUDV 7 7RFTXH % 5H\ &KDVVLJQRO 0 7KXRQJ 1 7 DQG +HOHQH & f 6KRUW PRGLILHG DQWLVHQVH ROLJRQXFOHRWLGHV GLUHFWHG DJDLQVW +DUDV SRLQW PXWDWLRQ LQGXFH VHOHFWLYH FOHDYDJH RI WKH P51$ DQG LQKLELW 7 FHOOV SUROLIHUDWLRQ (0%27 6DUPLHQWR 8 0 3HUH] 5 %HFNHU 0 DQG 1DUD\DQDQ 5 f ,Q YLYR WR[LFRORJLFDO HIIHFWV RI UHO $ DQWLVHQVH SKRVSKRURWKLRDWHV LQ &', PLFH $QWLVHQVH 5HV 'HY f 6FDQORQ 2KWD < ,VKLGD + .LMLPD + 2KNDZD 7 .DPLQVNL $ 7VDL +RPJ DQG .DVKDQL6DEHW 0 f 2OLJRQXFOHRWLGHPHGLDWHG PRGXODWLRQ RI PDPPDOLDQ JHQH H[SUHVVLRQ )$6(% f 6FKDDO + .OHLQ 0 *HKUPDQQ 3 $GDPV 2 DQG 6FKHLG $ f 5HTXLUHPHQW RI 1WHUPLQDO DPLQR DFLG UHVLGXHV RI JS IRU KXPDQ LPPXQRGHILFLHQF\ YLUXV W\SH PHGLDWHG FHOO IXVLRQ 9LURO f 6FKZDE &KDYDQ\ & 'XURX[ *RXELQ /HEHDX +HOHQH & DQG 6DLVRQ %HKPRDUDV 7 f $QWLVHQVH ROLJRQXFOHRWLGHV DGVRUEHG WR SRO\DON\OF\DQRDFU\ODWH QDQRSDUWLFOHV VSHFLILFDOO\ LQKLELW PXWDWHG +DUDVPHGLDWHG FHOO SUROLIHUDWLRQ DQG WXPRULJHQLFLW\ LQ QXGH PLFH 3URF 1DWO $FDG 6FL 86$ f 6HOYDP 0 3 %XFN 6 0 %OD\ 5 $ 0D\QHU 5 ( 0LHG 3 $ DQG (SVWHLQ 6 f ,QKLELWLRQ RI +,9 UHSOLFDWLRQ E\ LPPXQROLSRVRPDO DQWLVHQVH ROLJRQXFOHRWLGH $QWLYLUDO 5HV ,OO 6KDUPD + : DQG 1DUD\DQDQ 5 f 7KH WKHUDSHXWLF SRWHQWLDO RI DQWLVHQVH ROLJRQXFOHRWLGHV %LRHVVDYV f 6KDURQ 1 DQG /LV + f /HFWLQV DV FHOO UHFRJQLWLRQ PROHFXOHV 6FLHQFH f

PAGE 192

6KDZ 3 .HQW %LUG )LVKEDFN DQG )URHKOHU % f 0RGLILHG GHR[\ROLJRQXFOHRWLGHV VWDEOH WR H[RQXFOHDVH GHJUDGDWLRQ LQ VHUXP 1XFOHLF $FLGV 5HV f 6KHD 5 0DUVWHUV & DQG %LVFKRIEHUJHU 1 f 6\QWKHVLV K\EULGL]DWLRQ SURSHUWLHV DQG DQWLYLUDO DFWLYLW\ RI OLSLGROLJRGHR[\QXFOHRWLGH FRQMXJDWHV 1XFOHLF $FLGV 5HV ,% f 6LHJHO 3 f ,QYHUWHG PLFHOODU LQWHUPHGLDWHV DQG WKH WUDQVLWLRQV EHWZHHQ ODPHOODU FXELF DQG LQYHUWHG KH[DJRQDO OLSLG SKDVHV ,, ,PSOLFDWLRQV IRU PHPEUDQHPHPEUDQH LQWHUDFWLRQV DQG PHPEUDQH IXVLRQ %LRSK\V f 6LOYHU & 1JX\HQ & )/ %RXWRULQH $ 6 %LVDJQL ( *DUHVWLHU 7 DQG +HOHQH & f &RQMXJDWHV RI ROLJRQXFOHRWLGHV ZLWK WULSOH[VSHFLILF LQWHUFDODWLQJ DJHQWV 6WDELOL]DWLRQ RI WULSOHKHOLFDO '1$ LQ WKH SURPRWHU UHJLRQ RI WKH JHQH IRU WKH DOSKD VXEXQLW RI LQWHUOHXNLQ ,/5 DOSKDf %LRFRQLXJ &KHP f 6OHSXVKNLQ 9 $ 6LPRHV 6 'D]LQ 3 1HZPDQ 0 6 *XR / 6 3HGURVR 'H /LPD 0 & DQG 'X]JXQHV 1 f 6WHULFDOO\ VWDELOL]HG S+VHQVLWLYH OLSRVRPHV ,QWUDFHOOXODU GHOLYHU\ RI DTXHRXV FRQWHQWV DQG SURORQJHG FLUFXODWLRQ LQ YLYR %LRO &KHP f 6PLWK & & $XUHOLDQ / 5HGG\ 0 3 0LOOHU 3 6 DQG 7VnR 3 f $QWLYLUDO HIIHFW RI DQ ROLJRQXFOHRVLGH PHWK\OSKRVSKRQDWHf FRPSOHPHQWDU\ WR WKH VSOLFH MXQFWLRQ RI KHUSHV VLPSOH[ YLUXV W\SH LPPHGLDWH HDUO\ SUHP51$V DQG 3URF 1DWO $FDG 6FL 86$ f 6PRODUVN\ 0 7HLWHOEDXP 6HOD 0 DQG *LWOHU & f $ VLPSOH IOXRUHVFHQW PHWKRG WR GHWHUPLQH FRPSOHPHQWPHGLDWHG OLSRVRPH LPPXQH O\VLV ,PPXQRO 0HWKRGV f 6RNRO / DQG *HZLUW] $ 0 *HQH WKHUDS\ EDVLF FRQFHSWV DQG UHFHQW DGYDQFHV &ULW 5HY (XNDUYRW *HQH ([SU f 6RORGLQ / %URZQ & 6 %UXQR 0 6 &KRZ & -DQJ ( 'HEV 5 DQG +HDOWK 7 f $ QRYHO VHULHV RI DPSKLSKLOLF LPLGD]ROLQLXP FRPSRXQGV IRU LQ YLWUR DQG LQ YLYR JHQH GHOLYHU\ %LRFKHPLVWU\ 6WHLQ & $ DQG &KHQJ < & f $QWLVHQVH ROLJRQXFOHRWLGHV DV WKHUDSHXWLF DJHQWV LV WKH EXOOHW UHDOO\ PDJLFDO" 6FLHQFH f 6WHLQ & $ DQG &RKHQ 6 f 2OLJRGHR[\QXFOHRWLGHV DV LQKLELWRUV RI JHQH H[SUHVVLRQ D UHYLHZ &DQFHU 5HV f

PAGE 193

6WHLQ & $ 0RUL /RNH 6 / 6XEDVLQJKH & 6KLQR]XND FRKQ 6 DQG 1HFNHUV / 0 f 3KRVSKRURWKLRDWH DQG QRUPDO ROLJRGHR[\ULERQXFOHRWLGHV ZLWK f OLQNHG DFULGLQH FKDUDFWHUL]DWLRQ DQG SUHOLPLQDU\ NLQHWLFV RI FHOOXODU XSWDNH *HQH f 6WHYHQVRQ 0 DQG ,YHUVHQ 3 / f ,QKLELWLRQ RI KXPDQ LPPXQRGHILFLHQF\ YLUXV W\SH PHGLDWHG F\WRSDWKLF HIIHFWV E\ SRO\/O\VLQHfFRQMXJDWHG V\QWKHWLF DQWLVHQVH ROLJRGHR[\ULERQXFOHRWLGHV *HQ 9LURO 3W f 6WHZDUW $ 3LFKQ & 0HXQLHU / 0LGRX[ 3 0RQVLJQ\ 0 DQG 5RFKH $ & f (QKDQFHG ELRORJLFDO DFWLYLW\ RI DQWLVHQVH ROLJRQXFOHRWLGHV FRPSOH[HG ZLWK JO\FRV\ODWHG SRO\/O\VLQH 0RO 3KDUPDFRO 6WHZDUW & f &RORULPHWULF GHWHUPLQDWLRQ RI SKRVSKROLSLGV ZLWK DPPRQLXP IHUURWKLRF\DQDWH $QDO %LRFKHP WLO 6WLOO : & .DKQ 0 DQG 0LWUD $ f 5DSLG FKURPDWRJUDSKLF WHFKQLTXH IRU SUHSDUDWLYH VHSDUDWLRQV ZLWK PRGHUDWH UHVROXWLRQ 2UJ &KHP f 6WUXFN 3ORHNVWUD DQG 3DJDQR 5 ( f 8VH RI UHVRQDQFH HQHUJ\ WUDQVIHU WR PRQLWRU PHPEUDQH IXVLRQ %LRFKHPLVWU\ f 6]RND ) DQG 3DSDKDGMRSRXORV f 3URFHGXUH IRU SUHSDUDWLRQ RI OLSRVRPHV ZLWK ODUJH LQWHUQDO DTXHRXV VSDFH DQG KLJK FDSWXUH E\ UHYHUVHSKDVH HYDSRUDWLRQ 3URF 1DWO $FDG 6FL 86$ f 7DNOH % 7KLHUU\ $ 5 )O\QQ 6 0 3HQJ % :KLWH / 'HYRQLVK : *DOEUDLWK 5 $ *ROGEHUJ $ 5 DQG *HRUJH 6 7 f 'HOLYHU\ RI ROLJRULERQXFOHRWLGHV WR KXPDQ KHSDWRPD FHOOV XVLQJ FDWLRQLF OLSLG SDUWLFOHV FRQMXJDWHG WR IHUULF SURWRSRUSK\ULQ ,; KHPHf $QWLVHQVH 1XFOHLF $FLG 'UXJ 'HY 7KLHUU\ $ 5 DQG 'ULWVFKLOR $ f ,QWUDFHOOXODU DYDLODELOLW\ RI XQPRGLILHG SKRVSKRURWKLRDWHG DQG OLSRVRPDOO\ HQFDSVXODWHG ROLJRGHR[\QXFOHRWLGHV IRU DQWLVHQVH DFWLYLW\ 1XFOHLF $FLGV 5HV 7RXOPH .ULVFK + 0 /RUHDX 1 7KXRQJ 1 7 DQG +HOHQH & f 6SHFLILF LQKLELWLRQ RI P51$ WUDQVODWLRQ E\ FRPSOHPHQWDU\ ROLJRQXFOHRWLGHV FRYDOHQWO\ OLQNHG WR LQWHUFDODWLQJ DJHQWV 3URF 1DWO $FDG 6FL 86$ 7URXHW $ 'HSUH]'H &DPSDQHHUH DQG 'H 'XYH & f &KHPRWKHUDS\ WKURXJK O\VRVRPHV ZLWK D '1$GDXQRUXELFLQ FRPSOH[ 1DW 1HZ %LRO 8KOPDQ ( DQG 3H\PDQ $ f $QWLVHQVH ROLJRQXFOHRWLGHV D QHZ WKHUDSHXWLF SULQFLSOH &KHP 5HY

PAGE 194

8KOPDQ ( 3H\PDQ $ DQG :LOO : f $QWLVHQVH FKHPLFDO PRGLILFDWLRQ ,Q (QF\FORSHGLD RI &DQFHU %HUWLQR 5 HGLWRUf SS $FDGHPLF 3UHVV 6DQ 'LHJR 9HUVSLHUHQ 3 &RPHOLVVHQ $ : 7KXRQJ 1 7 +HOHQH & DQG 7RXOPH f $Q DFULGLQHOLQNHG ROLJRGHR[\QXFOHRWLGH WDUJHWHG WR WKH FRPPRQ n HQG RI WU\SDQRVRPH P51$V NLOOV FXOWXUHG SDUDVLWHV *HQH f 9ODVVRY 9 9 %DODNLUHYD / $ DQG
PAGE 195


PAGE 196

=KDQJ 5 /X = =KDR + =KDQJ ; 'LDVLR 5 % +DEXV / -LDQJ = ,\HU 5 3
PAGE 197

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

PAGE 198

, FHUWLI\ WKDW KDYH UHDG WKLV VWXG\ DQG WKDW LQ P\ RSLQLRQ LW FRQIRUPV WR DFFHSWDEOH VWDQGDUGV RI VFKRODUO\ SUHVHQWDWLRQ DQG LV IXOO\ DGHTXDWH LQ VFRSH DQG TXDOLW\ DV D GLVVHUWDWLRQ IRU WKH GHJUHH RI 'RFWRU RI 3KLORVRSK\ cI M M -HIIUH\ A n+XJKHV &KDLU $VVLVWDQW 3URIHVVRU RI 3KDUPDFHXWLFV FHUWLI\ WKDW KDYH UHDG WKLV VWXG\ DQG WKDW LQ P\ RSLQLRQ LW FRQIRUPV WR DFFHSWDEOH VWDQGDUGV RI VFKRODUO\ SUHVHQWDWLRQ DQG LV IXOO\ DGHTXDWH LQ VFRSH DQG TXDOLW\ DV D GLVVHUWDWLRQ IRU WKH GHJUHH RI 'RFWRU RI 3KLORVRSK\ 9"R [ L *D\OH ƒ %UD]HDX $VVRFLDWH 3URIHVVRU RI 3KDUPDFHXWLFV FHUWLI\ WKDW KDYH UHDG WKLV VWXG\ DQG WKDW LQ P\ RSLQLRQ LW FRQIRUPV WR DFFHSWDEOH VWDQGDUGV RI VFKRODUO\ SUHVHQWDWLRQ DQG LV IXOO\ DGHTXDWH LQ VFRSH DQG TXDOLW\ DV D GLVVHUWDWLRQ IRU WKH GHJUHH RI 'RFWRU RI 3KLORVRSK\ 6L FIR S +DUWPXW 'HUHQGRUI 3URIHVVRU RI 3KDUPDFHXWLFV FHUWLI\ WKDW KDYH UHDG WKLV VWXG\ DQG WKDW LQ P\ RSLQLRQ LW FRQIRUPV WR DFFHSWDEOH VWDQGDUGV RI VFKRODUO\ SUHVHQWDWLRQ DQG LV IXOO\ DGHTXDWH LQ VFRSH DQG TXDOLW\ DV D GLVVHUWDWLRQ IRU WKH GHJUHH RI 'RFWRU RI 3KLORVRSK\ /DV]OR 3URNDL $VVRFLDWH 3URIHVVRU RI 3KDUPDFHXWLFV FHUWLI\ WKDW KDYH UHDG WKLV VWXG\ DQG WKDW LQ P\ RSLQLRQ LW FRQIRUPV WR DFFHSWDEOH VWDQGDUGV RI VFKRODUO\ SUHVHQWDWLRQ DQG LV IXOO\ DGHTXDWH KL VFRSH DQG TXDOLW\ DV D GLVVHUWDWLRQ IRU WKH GHJUHH RI 'RFWRU RI 3KLORVRSK\ M_ LRQ 6FKXVIHU 3URIHVVRU RI %LRFKHPLVWU\ DQG 0ROHFXODU %LRORJ\

PAGE 199

, FHUWLI\ WKDW KDYH UHDG WKLV VWXG\ DQG WKDW LQ P\ RSLQLRQ LW FRQIRUPV WR DFFHSWDEOH VWDQGDUGV RI VFKRODUO\ SUHVHQWDWLRQ DQG LV IXOO\ DGHTXDWH LQ VFRSH DQG TXDOLW\ DV D GLVVHUWDWLRQ IRU WKH GHJUHH RI 'RFWRU RI 3KLORVRSK\ $r!4 ,DQ 5 7HEEHWW 3URIHVVRU RI 0HGLFLQDO &KHPLVWU\ 7KLV GLVVHUWDWLRQ ZDV VXEPLWWHG WR WKH *UDGXDWH )DFXOW\ RI WKH &ROOHJH RI 3KDUPDF\ DQG WR WKH *UDGXDWH 6FKRRO DQG ZDV DFFHSWHG IDV SDUWLDKIXOILOOPHQW SI WKH UHTXLUHPHQWV IRU WKH GHJUHH RI 'RFWRU RI 3KLORVRSK\ -M cO $ IInO $XJXVW 'HDQ &RO KDUPDF\ 'HDQ *UDGXDWH 6FKRRO


xml version 1.0 encoding UTF-8
REPORT xmlns http:www.fcla.edudlsmddaitss xmlns:xsi http:www.w3.org2001XMLSchema-instance xsi:schemaLocation http:www.fcla.edudlsmddaitssdaitssReport.xsd
INGEST IEID ETLR0HV6V_5LY91N INGEST_TIME 2014-08-22T18:50:14Z PACKAGE AA00024971_00001
AGREEMENT_INFO ACCOUNT UF PROJECT UFDC
FILES


USE OF BIODEGRADABLE pH-SENSITIVE SURFACTANTS IN LIPOSOME
MEDIATED OLIGONUCLEOTIDE DELIVERY
By
CHIH-WEI EARVIN LIANG
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1998

Dedicated to my parents Tu-Jow and Chow-Son Liang.

ACKNOWLEDGMENTS
I would like to express my sincere appreciation to Dr. Jeffrey A. Hughes,
chairman of my committee. Without his patience, understanding, guidance, and
encouragement, I could not receive my Ph.D. degree. I extend my gratitude to my other
committee members, Dr. Hartmut Derendorf, Dr. Laszlo Prokai, Dr. Sheldon Schuster,
and Dr. Ian R. Tebbett, for their suggestions and criticisms, and especially Dr. Gayle A.
Brazeau for her consistent care and concern.
I also give my acknowledgment to all the personnel in the Department of
Pharmaceutics including the secretaries, graduate students, and in particular the Brazeau
& Hughes group who helped me throughout my entire graduate life and research at the
University of Florida. Finally, I want to thank my parents and siblings for their unselfish
love and continuous support during both my good and bad times in the past 26 years.
m

TABLE OF CONTENTS
page
ACKNOWLEDGMENTS iii
KEY TO ABBREVIATIONS vi
ABSTRACT viii
CHAPTERS
1 INTRODUCTION 1
Objective 2
Hypothesis 2
2 BACKGROUND AND SIGNIFICANCE 3
Oligonucleotide Therapy Overview 3
Toxic Effects of Oligonucleotide 6
Barriers to Oligonucleotide Transfer and Activity 6
Strategies Available to Deliver Oligonucleotides 10
Origin of Biodegradable pH-Sensitive Surfactants-Lysosomotropic
Detergents 21
Significance of Biodegradable pH-Sensitive Surfactants 22
Specific Aims 23
3 DESIGN AND SYNTHESIS OF BIODEGRADABLE pH-
SENSmVE SURFACTANTS 28
Introduction 28
Rationale 28
Selection of Biodegradable pH-Sensitive Surfactants 33
Syntheis of Biodegradable pH-Sensitive Surfactants 35
Identification of Biodegradable pH-Sensitive Surfactants 39
4 PHYSICOCHEMICAL CHARACTERIZATION OF
BIODEGRADABLE pH-SENSITIVE SURFACTANTS 49
Introduction 49
Materials 49
iv

Methods 52
Results 57
Discussion 78
Conclusion 83
5 DELIVERY SYSTEM EVALUATION OF BIODEGRADABLE pH-
SENSITIVE SURFACTANTS 84
Introduction 84
Materials 85
Methods 86
Results 95
Discussion 121
Conclusion 125
6 MECHANISM OF ACTION INVESTIGATION OF
BIODEGRADABLE pH-SENSITIVE SURFACTANTS 126
Introduction 126
Materials 127
Methods 129
Results 133
Discussion 151
Conclusion 160
7 CONCLUSION AND FUTURE PROSPECT 162
Conclusion 162
Future Aims 165
REFERENCES 168
BIOGRAPHICAL SKETCH 188
v

KEY TO ABBREVIATIONS
ANTS l-aminonaphthalene-3,6,8-trisulfonic acid
BPS biodegradable pH-sensitive surfactant(s)
CMC critical micelle concentration
DDAB dimethyldioctadecylammonium bromide
DC-Chol 3p-[N-(N\N’-dimethylaminoethane)carbamoyl]cholesterol
DI N-dodecyl imidazole
DIP dodecyl 2-(l ’-imidazolyl) propionate
DMF N,N-dimethylformamide
DMRIE l,2-dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide
DMPC 1,2-dimyristoyl-sn-glycero-3-phosphocholine
DMSO dimethyl sulfoxide
DOPE dioleoylphosphatidylethanolamine
DOSPA 2,3-dioleyloxy-sperminecarboxamido-N,N-dimethyl-l-propanaminium
DOTAP N-[l-(l-2,3-dioleoloxy)propyl]-N,N,N-trimethylammonium methylsulfate
DOTMA N-[l-(2,3-dioleoloxy)-propyl]-N,N,N-trimethylammonium chloride
DPX N, N’-p-xylylenebis-(pyridinium bromide)
FITC fluorescein isothiocyanate
HVJ hemagglutinating virus of Japan
MIL methyl 1-imidazolyl laureate
vi

NBD-PE N-(7-nitro-2,l,3-benzoxadiazol-4-yl)-phosphatidylethanolamine
NMR nuclear magnetic resonance
PBS phosphate buffered saline
PE phosphatidylethanolamine
R molar ratio of the biodegradable pEl-sensitive surfactants to the other lipids
Re effective release ratio
Rh-PE N-(lissamine rhodamine B sulfonyl)-phosphatidylethanolamine
vii

Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
USE OF BIODEGRADABLE pH-SENSITIVE SURFACTANTS IN LIPOSOME
MEDIATED OLIGONUCLEOTIDE DELIVERY
By
Chih-Wei Earvin Liang
August, 1998
Chairman: Jeffrey A. Hughes, Ph.D.
Major Department: Pharmaceutics
Oligonucleotide therapy is a promising approach for the treatment of a variety of
disorders. A factor limiting the therapeutic effect of oligonucleotides is their inefficient
transport to the cytoplasm or nucleus. Most oligonucleotides with or without a delivery
system enter cells by endocytosis. Initially they accumulate in endosomes which are
intracellular compartments with acidic intraluminal pH. In time, the endosomes mature
into lysosomes and oligonucleotides are eliminated. Consequently, the development of a
delivery system that increases oligonucleotide transfer from endosóme to cytoplasm is
essential. Biodegradable pH-sensitive surfactants (BPS) were therefore designed to
enhance liposome mediated oligonucleotide delivery. BPS are a unique family of easily
metabolized compounds that demonstrate pH-dependent surface activity. Through
simple and fast chemical reactions, three BPS, dodecyl 2-(l’-imidazolyl) propionate
(DIP), methyl 1-imidazolyl laureate (MIL), and N-dodecyl imidazole (DI), were

synthesized for use in subsequent studies. Critical micelle concentration and effective
release ratio of the ionized BPS were determined to identify their surface active
properties. Surfactant like behaviors of BPS in a pH-dependent manner were then
assessed. Also, several physicochemical parameters of BPS were measured and
compared including chemical stability, biodegradability, and cytotoxicity. Utilizing an in
vitro model and confocal microscopy, liposomes combined with BPS demonstrated better
quantitative and qualitative effects in enhancing oligonucleotide cellular delivery than
liposomes without BPS. The enhanced effect due to the presence of BPS, as shown by
flow cytometry, was not due to increased cellular uptake of oligonucleotides, but rather
their redistribution upon entering the cells. A liposomal model was employed to better
understand the membrane defect mechanisms elicited by BPS that enabled the
intracellular redistribution of oligonucleotides. This system suggests that depending
upon the pH and molar ratio of BPS to membrane lipids, BPS can induce both membrane
fusion and rupture which are different from other surfactants and fusogenic compounds.
They appear to contribute and participate in the membrane fusion at different stages. In
comparison to other similar delivery systems, BPS are more economical, easily produced,
and less toxic. Consequently, they provide a potential excellent choice for
oligonucleotide delivery.
IX

CHAPTER 1
INTRODUCTION
Oligonucleotides have been used as a gene therapy approach since the late 1970s
(Zamecnik & Stephenson, 1978). The theoretical approach of oligonucleotides is very
attractive since it allows for the inhibition of a specific protein. Oligonucleotides with or
without a carrier are transported into cells mostly by endocytosis (Akhtar & Juliano,
1992; Loke et ah, 1988) and accumulate in endosomes, intracellular compartments with
an acidic intraluminal pH (Maxfield, 1985; McGraw & Maxfield, 1991). A factor
limiting the pharmacological effectiveness of oligonucleotides is their inefficient
transport to their sites of action in the cytoplasm or nucleus. The rate and extent of
movement from endosomes appear to be important in determining oligonucleotide
effects.
Consequently, the development of accessory compounds that enhance endosóme
to cytoplasm transfer may be vital to oligonucleotide therapy. By taking advantage of the
characteristics of surfactants and exploiting a naturally occurring event, the pH gradient,
during oligonucleotide transport, we have proposed a delivery system that may increase
oligonucleotide cytoplasmic delivery and biological activity.
1

2
Objective
The main objective of the project was to characterize biodegradable pH-sensitive
surfactants (BPS) both physicochemically and biochemically which can serve as
adjuvants for improved transfer of oligonucleotides from the endosóme to the cytoplasm
without associated cellular toxicity.
Hypothesis
The overall hypothesis was that the endosomal membrane presents a major barrier
to oligonucleotide delivery. By the addition of biodegradable pH-sensitive surfactants
(BPS) to a liposomal delivery system, the low endosomal pH can activate the pH-
sensitive surfactants and facilitate the transfer of endosomal contents (i.e.,
oligonucleotide) to the cytoplasm or nucleus. The biodegradable linker can be digested
into less toxic metabolites by the endogenous digestive enzymes.

CHAPTER 2
BACKGROUND AND SIGNIFICANCE
Oligonucleotide Therapy Overview
The ability of short, synthetic, and single-stranded DNA or RNA oligonucleotides
to interfere with individual gene expression in a sequence-specific manner is the
foundation for oligonucleotide-based therapy. The first clear exploration of
oligonucleotides was reported by Zamecnik and Stephenson (1978). However, due to a
number of impediments (e.g., understanding of the sequence and topology of the nucleic
acid target, synthesis of research quantities of oligonucleotides, and modification of
stabilized oligonucleotides), research in using oligonucleotides for biological studies was
limited until the late 1980s. Following advances in oligonucleotide chemistry and initial
biological studies (Agrawal et al., 1988; Gao et al., 1989; Smith et al., 1986), interest in
oligonucleotide therapy began to increase.
Oligonucleotides, short nucleotide polymer in length (12 to 40 mer) of a synthetic
single-stranded nucleic acid, are designed to a specific gene (Cooney et al., 1988; Moser
& Dervan, 1987), mRNA (Stein & Cohen, 1988), or protein (Bock et al., 1992; Ellington
1994). After binding to their target in cells, oligonucleotides can prevent the production
of a specific protein product (Figure 2-1). Detailed mechanisms are explained in
3

4
Oligonucleotide
><
Site of Action
Figure 2-1: A depiction of protein synthesis and possible sites of action for designed
oligonucleotides. There are three potential sites where oligonucleotides can have actions.
First, oligonucleotides (antigenes) can be used to inhibit the transcription process from
double stranded DNA to single stranded mRNA through Hoogsteen base pairing
interactions. Second, complimentary oligonucleotides (antisense oligonucleotides) can
be designed to bind with mRNA to restrain the translation process through Watson-Crick
hydrogen bond interactions. Finally, oligonucleotides (aptamers) can interact with a
synthesized protein to interfere with its activity via hydrogen bondings.

5
excellent review articles (Crooke, 1993; Helene & Toulme, 1990; Scanlon et al., 1995;
Sharma & Narayanan, 1995; Uhlman & Peyman, 1990).
Simply, a triplex forming oligonucleotide (antigene oligonucleotide) is capable of
binding to the major groove in double-stranded DNA via Hoogsteen base interactions,
thereby causing a triple helical structure and further resulting in sequence-specific
inhibition of transcription. On the contrary, an antisense oligonucleotide complementary
to a specific sequence of mRNA can hybridize to a given mRNA through Watson-Crick
hydrogen bonds (Zamecnik, 1996). It can inhibit translation by several proposed
mechanisms including activation of RNase H and blockade of ribosomal reading. RNase
H is an endogenous cellular enzyme which can recognize a hybrid duplex between DNA
and RNA (Ghosh et al., 1993). RNase H leads to RNA cleavage and release of the DNA-
oligonucleotide. The freed DNA-oligonucleotide is then able to hybridize to another
RNA strand and repeat the RNase H dependent degradation, thus forming the basis for a
catalytic effect.
Oligonucleotide therapy is most often directed at inhibiting production of disease
causing proteins. The fact that very distinct interactions of oligonucleotides to target
sequences occur suggests that oligonucleotide therapy have the potential to be orders of
magnitude more specific than conventional drug therapy. Therefore, it may yield a
greater therapeutic effect and is an exciting technology for manipulating gene expression
in the treatment of human gene diseases.

6
Toxic Effects of Oligonucleotide
High doses of oligonucleotides have been reported harmful in animal studies and
the toxicity of oligonucleotides appears to be species dependent. Administration of 100
mg/kg i.p. three times weekly for two weeks in mice and rats resulted in significant
toxicity including acute renal failure, liver damage, spleen damage, immune stimulation,
severe thrombocytopaenia, and death (Krieg et al., 1995; Sarmiento et ah, 1994). Bolus
i.v. administration of oligonucleotides in monkeys produced a transient decrease in
peripheral total white blood cell, neutrophil counts, prolongation of acitvated partial-
thromboplastin time, hypotension, and death (Galbraith et ah, 1994). As a result of the
relatively long retention time in the reticuloendothelial system organ, accumulation of
oligonucleotides and their metabolites may be responsible for these toxicities (Zhang et
ah, 1995).
Barriers to Oligonucleotide Transfer and Activity
The therapeutic promise of specific oligonucleotide interaction is great. However,
several technical problems including stability and delivery must be overcome before
oligonucleotides can be useful drugs.
In Vitro/In Vivo Stability
Of all the possible obstacles, rapid degradation of unmodified DNA and RNA
phosphorodiester oligonucleotides in the biological milieu is the first problem
encountered (Akhtar et ah, 19916; Shaw et ah, 1991). Enzymes, non-specific endo- and

7
exo-nucleases, limit phosphorodiester oligonucleotides’ physiological half-life to a few
minutes (Akhtar et al., 19916). This short biological half-life makes the therapeutic use
of phosphodiester oligonucleotides unlikely. Biologically stable oligonucleotides are
achievable by chemically altering the phosphorodiester backbone. To maximize the
effect, the modified oligonucleotides should be stable in both serum and inside the cell,
able to reach their site of action, and form stable Watson-Crick or Hoogsteen complexes
with target sequences.
These modifications summarily occur in three locations (for detailed review see
Uhlman et ah, 1997):
• Intemucleotide phosphodiester bridge
• Base group
• Sugar group
Based upon the above criteria, a number of structural analogues with nuclease
resistance have been developed including phosphorothioate (Connolly et ah, 1984;
Cowsert et ah, 1993) and methyl phosphonate (Blake et ah, 1985; Murakami et ah, 1985).
Of these modified oligonucleotides, phosphorothioate oligonucleotides are possibly the
most potent because they are highly resistant to nucleases, retain a net charge, are soluble
in water, and can act as substrates for RNase hh. However, phosphorothioate
oligonucleotides may also cause a variety of non-sequence dependent effects (Guvakova
et ah, 1995; Khaled et ah, 1996; Perez et ah, 1994).

8
Cellular Transport
Another major encumbrance to the therapeutic use of oligonucleotides is the
inefficient delivery of oligonucleotides to the cytoplasm or nucleus. There are two
transport aspects that need to be distinguished:
• Cellular uptake
• Entry into the cytoplasm/nucleus
Cellular uptake refers to both oligonucleotide membrane binding and general
internalization within the cell. Entry into the cytoplasm/nucleus concerns the amount of
oligonucleotides that reach a pharmacological active compartment. Oligonucleotides
internalization by cultured cells is inefficient (Akhtar et al., 19916; Stein & Cheng, 1993).
Only a small fraction of added oligonucleotides can actually gain entry into cells and it is
commonly assumed that most oligonucleotides can be brought into cells through
(receptor mediated, adsorptive, or fluid phase) endocytosis (Akhtar & Juliano, 1992;
Loke et ah, 1989).
After entry into cells, oligonucleotides must penetrate the endosomal membrane
to exert their effects in the nucleus or cytoplasm. Not all of the internalized
oligonucleotides are necessarily available to interact with intended subcellular targets.
Indeed, most of them are eliminated by lysosomes, the later endocytotic stage (Figure 2-
2). Unlike gene delivery, however, following cellular entry and escape from endosomal
compartments with an effective nuclear pore size of approximately 10 nm in diameter
(Aronsohn & Hughes, 1997), oligonucleotides are able to migrate to the nucleus without
difficulty (Chin et ah, 1990; Leonetti et ah, 1991). An issue that needs to be addressed is

9
5.0
Lysosome
Oligonucleotide Elimination
Oligonucleotide Liposome+Oligonucleotide
Figure 2-2: Possible oligonucleotide fates in a cell for two delivery systems, a) Limited
amount of oligonucleotides can be taken into cells when not used with any delivery
system. For those oligonucleotides that can be brought into cells, the mechanism is
mostly through endocytosis along with subcellular compartments (i.e., endosóme and
lysosome) of a pH-gradient profile. Most of the endocytosed oligonucleotides would
then be eliminated, b) When using a delivery system (e.g., liposome), more
oligonucleotides can be brought into cells, thereby increasing their probability of
escaping from lysosomes. Still, most of the oligonucleotides would be eliminated
through the entire endocytosis process.

10
that, like other drugs, oligonucleotides may bind to intracellular proteins which can cause
side effects and limit free fraction. Only free unbound oligonucleotides can interact with
targets at the sites of action and demonstrate biological effects.
Oligonucleotides traveled to sites of action face several barriers. By optimizing
oligonucleotide transfer at each stage of the delivery process, the amount of
oligonucleotides with or without their carrier to achieve the same biological effect can be
minimized in comparison to umnodified oligonucleotides. Increasing the amount of
cellular uptake and/or escape of oligonucleotides from the endosomes may be of
considerable value in improving the extent of oligonucleotides at their sites of action and
the inhibition of certain protein expression. Hence, the optimization can decrease the
cytotoxicity associated with a large amount of oligonucleotides and delivery systems.
Strategies Available to Deliver Oligonucleotides
A number of strategies have been pursued to facilitate the entry of
oligonucleotides into the cytoplasm. The strategies are used either alone or in
combination with others to optimize the effect. Each system has its own advantages and
drawbacks. According to the two mentioned oligonucleotide transport aspects, these
strategies can generally be separated into two parts.
The first group renders delivery systems that increase the amount of
oligonucleotides that associate with target cells. They include conjugation of molecules
to oligonucleotides (i.e., conjugating agents), complexation of oligonucleotides with
cationic molecules (i.e., complexing agents), encapsulation of oligonucleotides into
vesicles (i.e., encapsulating agents), and labeling targets to either oligonucleotides or their

11
delivery carriers (i.e., targeting agents). These systems increase the probability of
oligonucleotides escaping endocytotic degradation and reaching the cytoplasm or
nucleus.
Irrespective of the above methods, the underlying principle is to increase uptake
of oligonucleotides. Thus, the increased oligonucleotide concentration in endosomes
enhances the chance of oligonucleotides to reach cytoplasms. These delivery systems
therefore exhibit superior effects compared to plain oligonucleotides in tissue culture
systems. However, the majority of oligonucleotides that are brought into cells would still
be eliminated during endocytosis (Figure 2-2). The biological activity able to be
observed results from a diminutive amount of oligonucleotides that escape from the
endosomal compartments.
To further optimize oligonucleotide delivery, endosóme destabilizing (escaping)
systems have been developed. This group applies devices (i.e., oligonucleotide
cytoplasmic transfer techniques) or offers delivery systems (i.e., membrane destabilizing
agents) that improve oligonucleotide efflux to the cytoplasm.
Conjugating Agents
Emphasis on the ability of oligonucleotides to penetrate biological membranes is
one of the major elements in making oligonucleotide therapy possible. An strategy is to
conjugate hydrophobic anchor groups at either end of the oligonucleotide through
chemical reactions to extend their hydrophobicity and/or exo-nuclease resistance, thereby
increasing the interaction with target cells.

12
Cholesterol is a typical conjugating agent that has been used as a hydrophobic
anchor group at either the 3’- or 5’- terminus of oligonucleotides (Alahari et al., 1996;
Boutorin et ah, 1989; Godard et ah, 1995; Letsinger et ah, 1989). Alkyl side chains are
another commonly used conjugating agent. Examples include hexadecyl moieties affixed
to the 5’-end (Shea et ah, 1990), dodecyl moieties to the 3’-end (Saison-Behmoaras et ah,
1991), hexanol to the 3’-end (Gamper et ah, 1993), aminohexyl to the 3’-end (Gamper et
ah, 1993), and an undecyl derivative to the 5’-end of oligonucleotides (Kabanov et ah,
1990).
Poly(L-lysine) is another type of conjugating agent. By attaching
oligonucleotides to poly(L-lysine) at the 3’-end (Degols et ah, 1991; Degols et ah, 1989;
Lemaitre et ah, 1987; Leonetti et al., 1988; Stevenson & Iversen, 1989), cellular uptake is
increased most likely due to a better interaction with the negative charge cellular
membrane. In addition to the possible permeability mechanism, the biological effect
improved by poly(L-lysine) conjugates can also be a consequence of better protective
properties against nucleases.
As mentioned above, one major advantage of using conjugating agents is to
increase the initial membrane interaction which leads to greater cellular accumulation of
oligonucleotides. However, there are also a number of disadvantages that hinder the use
of conjugating agents such as the chemical synthesis of the connector between the
oligonucleotides agents. This process is both time consuming and expensive.
Furthermore, the manipulation of the conjugating agents (e.g., poly(L-lysine)) can
account for increased cytotoxic effects.

13
Complexing Agents
Unlike conjugating agents, the basic principle behind the use of complexing
agents is to bind oligonucleotides to their carrier in a strong but non-covalent manner
based upon an electrostatic attraction. This system carries more oligonucleotides into
cells through endocytosis and hence increases their probability of reaching the cytoplasm.
Cationic polymers such as poly(L-lysine) (Deshpande et al., 1996; Ginobbi et al.,
1997; Stewart et al., 1996), polyethylenimine (Boussif et al., 1995), polyamidoamine
PAMAM starburst dendrimers (Bielinska et al., 1996; Delong et al., 1997; Hughes et al.,
1996; Kukowska-Latallo et al., 1996; Poxon et al., 1996), avidin (Pardridge & Boado,
1991), polyisohexylcyanoacrylate nanoparticles (Chavany et al., 1994), and
polyalkylcyanoacrylate nanoparticles (Chavany et al., 1992; Godard et al., 1995; Schwab
et al., 1994) are some comlexing agents that have been used in oligonucleotide delivery.
Cationic liposomes are other complexing agents that have been investigated. Liposomes
are vesicles comprised of lipid bilayer(s) similar in structure to biological membranes.
Utilizing their versatility (e.g., size, charge, and composition) and several advantages
(e.g., economical, ability to attach chemicals to their surface, and easily produced),
different systems involving liposomes can be applied to increase the delivery of
oligonucleotides to their sites of action. Cationic liposomes are among one of these
strategies. Cationic liposomes that have improved oligonucleotide cellular delivery
include N-[l-(2,3-dioleoloxy)-propyl]-N,N,N-trimethylammonium chloride (DOTMA)
(Bennett et al., 1992; Hughes et al., 1996; Konopka et al., 1996; Perlaky et al., 1993;
Saijo et al., 1993), N-[l-(l-2,3-dioleoloxy)propyl]-N,N,N-trimethylammonium

14
methylsulfate (DOTAP) (Capaccioli et al., 1993; Lappalainen et al., 1994; Liang &
Hughes, 1998; Quattrone et al., 1994; Takle et al., 1997; Zelphati & Szoka, 1996a), 3(3-
[N-(N’,N,-dimethylaminoethane)carbamoyl]cholesterol (DC-Chol) (Litzinger et al.,
1996), spermine-cholesterol (Guy Caffey et al., 1995), spermidine-cholesterol (Guy
Caffey et al., 1995), 2,3-dioleoyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-l-
propanaminum trifluoroacetate (DOSPA) (Lappalainen et al., 1997; Lappalainen et al.,
1996), l,2-dimyristyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide (DMRIE)
(Konopka et al., 1996), and dimethyldioctadecylammonium bromide (DDAB)
(Jaaskelainen et al., 1994; Ollikainen et al., 1996; Rose et al., 1991).
In contrast to conjugating agents, the ease of production of the complexing agents
is the biggest advantage. No chemical linkage between oligonucleotides and complexing
agents is required. In addition, they provide high capacity to retain oligonucleotides.
Complexing agents may also prevent oligonucleotides from enzymatic degradation by
forming poor substrates (Gershon et al., 1993). However, a major concern in using
complexing agents is their possible toxic effects. Cationic polymers and cationic
liposomes eventually are more toxic to cells than neutral counterparts as their
concentrations are increased (Barry et al., 1993; Clarenc et al., 1993; Wagner et al., 1993;
Yeoman et al., 1992). In addition, the intrinsic properties of the carriers such as
liposomes or nanoparticles lead to increased immunological problems with the
oligonucleotide complex (Chavany et al., 1994; Phillips & Emili, 1991).

15
Encapsulating Agents
Encapsulating and complexing agents are possibly the most popular systems used
in the delivery of oligonucleotides. Not only do both methods protect oligonucleotides
from degradation (Leonetti et al., 1990; Schwab et al., 1994; Thierry & Dritschilo, 1992),
but they also increase cellular uptake (Juliano & Akhtar, 1992). However, the
preparation behind the similar outcomes is quite different. Complexing agents bind to
oligonucleotides through an electrostatic attraction while encapsulating agents entrap
oligonucleotides within vesicles.
The most popular encapsulating approach currently being investigated is through
the use of liposomes. In addition to the above mentioned advantages, liposomes offer the
potential to reach specific targets via attached ligands. They may control or sustain
oligonucleotide release (Akhtar et al., 19916). Abundant examples using liposomes
illustrate the improved effect of oligonucleotides (Akhtar et al., 19916; Anazodo et al.,
1995; Hatta et al., 1997; Hatta et al., 1996; Marzo et al., 1997; Ogo et al., 1994; Wielbo et
al., 1996). Furthermore, cyclodextrin analogs including 2-hydroxypropyl beta-
cyclodextrin (Habus et al., 1995; Zhao et al., 1995), hydroxyethyl beta-cyclodextrin
(Zhao et al., 1995), and encapsin (Zhao et al., 1995) have also been demonstrated as
possible carrier candidates for oligonucleotide delivery.
Similar to complexing agents, the biggest advantage of encapsulating agents in
oligonucleotide delivery is their ease of production. Unlike complexing agents,
encapsulating agents are believed to be less cytotoxic. However, compared to

16
complexing agents, they have a lower capacity to bring oligonucleotides into cells which
may reduce the efficiency of the encapsulating agent system.
Targeting Agents
Targeting agents may be categorized into two groups. The first group targeting at
the nucleic acid level is known as intercalating agents. These agents are mostly often
attached at the 3’- or 5’-end of oligonucleotides. The moieties linked to oligonucleotides
interact strongly and nonspecifically with nucleic acids. After entering into cells and
interacting with target nucleic acids, the hybrids are stabilized by the intercalation of the
agents in the RNA-DNA duplex. Hence, they increase the affinity of oligonucleotides to
their targets.
Of all the intercalating agents, acridine is most widely used and investigated as a
possible means to increase the effect of oligonucleotides (Fukui & Tanaka, 1996;
Grigoriev et al., 1992; Klysik et al., 1997; Lacoste et ah, 1997; McConnaughie & Jenkins,
1995; Stein et ah, 1988; Toulme et ah, 1986; Verspieren et ah, 1987; Walter, 1995).
Other examined intercalators are chlorambucil (Belousov et ah, 1997),
benzopyridoquinoxaline (Marchand et ah, 1996; Silver et ah, 1997), benzopyridoindole
(Giovannangeli et ah, 1996; Silver et ah, 1997), benzophenanthridine (Chen et ah, 1995),
and phenazinium (Levina et ah, 1993).
The second group of targeting agents is accessed by utilizing moieties that can
selectively and specifically transport oligonucleotides to a target cell population.
Therefore, their accumulation in intracellular compartments is increased. The moieties

17
can be either conjugated to oligonucleotides or attached to a carrier system (e.g., poly(L-
lysine) or liposomes) linked to the oligonucleotides.
For cells that express the characteristics of receptor mediated endocytosis, ligands
represent good candidates as targeting agents to initiate cellular uptake of
oligonucleotides. Glycoproteins and neoglycoproteins bearing an appropriate sugar
residue specifically attach to sugar binding receptors (Sharon & Lis, 1989). By labeling
oligonucleotides at the 3’-end to the neoglycoprotein (6-phosphomannosylated
glycoprotein), an improved effect was observed (Bonfils et al., 1992). Similarly,
asialoorosomucoid (Bunnell et al., 1992; Wu & Wu, 1992) or mannosylated glycoprotein
(Liang et al., 1996) conjugated to poly(L-lysine) has been employed to target and
enhance cellular uptake of oligonucleotides.
Since malignant cells are correlated with an increased need for essential nutrients
(e.g., folic acid and transferrin) relative to benign cells, these nutrients can be used as
potential candidates to target oligonucleotides in the inhibition of cancerous cell growth.
Further improved oligonucleotide cellular uptake is seen when folic acid (Citro et al.,
1994; Ginobbi et al., 1997), epidermal growth factor (Deshpande et al., 1996), and
transferrin (Citro et al., 1992) is linked to poly(L-lysine). Liposomes coated with
maleylated bovine serum albumin (Chaudhuri, 1997), folic acid (Wang et al., 1995), or
ferric protoporphyrin IX (Talke et al., 1997) have been shown to increase the cellular
uptake of oligonucleotides.
In order to increase the specificity of oligonucleotides, liposomes can also be
attached to antibodies to reach the desired targets. Several monoclonal antibody-targeted
liposomes, immunoliposomes, have been developed and used for mediating

18
oligonucleotides to specific receptors on targeted cells (Lefebvre-d’Hellencourt et al.,
1995; Leonetti et al., 1990; Loke et al., 1989; Ma & Wei, 1996; Renneisen et al., 1990;
Selvam et al., 1996; Zelphati et al., 1994; Zelphati et al., 1993).
The major advantage of targeting agents is to enhance oligonucleotide cellular
uptake specifically. The targeting strategy can be incorporated with other systems to
further increase the cellular biological activity of oligonucleotides. Similar to
conjugating agents, a disadvantages that may hamper the development of targeting agents
is the synthetic linking process. Furthermore, targeting particular routes of endocytosis is
strongly dependent upon receptor subtype thereby limiting the use of targeting agents.
Oligonucleotide Cytoplasmic Transfer Techniques
Even if cellular uptake of oligonucleotides through a delivery system was
increased, escape from endosóme must still be accomplished. One way to avoid this
barrier is to transfer them directly into cytoplasm or nucleus. This has been accomplished
through electroporation (Bergan et al., 1996; Flanagan & Wagner, 1997; Griffey et al.,
1996; Schaal et al., 1995) and microinjection (Blondel et al., 1990; Fenster et al., 1994;
Lamprecht et al., 1997; O’Keefe et al., 1994; Sagata et al., 1988).
Electroporation involves delivering a high-voltage pulse of a defined magnitude
and length to the oligonucleotide-cell system. The membrane structures of the cells are
loosened and oligonucleotides can be introduced directly into the cell’s cytoplasm. On
the other hand, microinjection was performed by injecting oligonucleotides directly into
the nucleus.

19
The above methods prevent lysosomal elimination without falling into the trap of
the endocytosis pathway. However, these techniques have limited use from the
standpoint of clinical therapy. Therefore, it is necessary to develop more practical
delivery systems to improve oligonucleotide therapy.
Membrane Destabilizing Agents
Membrane destabilizing agents provide a means to disrupt endosomal
membranes. Some agents are conjugated directly to oligonucleotides through chemical
reactions while other agents may be a part of liposome composition to which
oligonucleotides are either complexed or encapsulated.
Fusogenic and pH-sensitive lipids
Fusogenic and pH-sensitive lipids have been used together as liposomes (i.e.,
encapsulating agents) to promote efflux of oligonucleotides from the endosomal
compartment (Bentz et al., 1985; Connor et ah, 1984; Duzgunes et ah, 1985). Fusogenic
lipids include phosphatidylethanolamine (PE) derivatives while pH-sensitive lipids that
have titratable carboxylic acids contain oleic acid (Cristina De Oliveira et ah, 1997; Ma &
Wei, 1996; Ropert et ah, 1996; Ropert et ah, 1993; Ropert et ah, 1992) and cholesteryl
hemisuccinate (Chu et ah, 1990; Slepushkin et ah, 1997).
A fusogenic lipid is able to form hexagonal II phase that influences membrane
fusion and oligonucleotide release. Before the disruption of the endosomal membrane
occurs inside the cells, however, liposomes must maintain their integrity to encapsulate
oligonucleotides. A pH-sensitive lipid is therefore introduced into the liposomal matrix.
With a chemical structure complementary to the hexagonal II phase (e.g.,

20
dioleoylphosphatidylethanolamine (DOPE)), the pH-sensitive lipid will assist in retaining
the bilayer vesicle structure of the liposomes at an alkaline pH. When the pH decreases
as a result of the acidification of the endosóme, the titratable head group of the pH-
sensitive lipid is protonated. Hence, it destabilizes the bilayer structure and PE promotes
membrane fusion (Duzgunes et al., 1985). Eventually, oligonucleotides are released out
of the endosomes. Also, cationic liposomes (i.e., complexing agents) usually comprise a
fusogenic lipid (e.g., DOPE) and a cationic lipid (e.g., 2,3 dioleyloxy-N-
[2(sperminecarboxamido)ethyl]-N,N-dimethyl propanaminium (DOSPA), N-[l-(l-2,3-
dioleoloxy)propyl]-N,N,N-trimethylammonium methylsulfate (DOTAP), N-[l-(2,3-
dioleoloxy)-propyl]-N,N,N-trimethylammonium chloride (DOTMA),
dimethyldioctadecylammonium bromide (DDAB), and 3p-[N-(N’,N’-
dimethylaminoethane)carbamoyl]cholesterol (DC-Chol)) to improve oligonucleotide
delivery (Guy Caffey et al., 1995; Lappalainen et ah, 1997; Lappalainen et al., 1996;
Litzinger et al., 1996; Ollikainen et al., 1996; Takle et al., 1997; Zalphati & Szoka,
19966).
In addition to increasing cellular uptake of oligonucleotides, pH-sensitive
liposomes further increase the entry of oligonucleotides to the cytoplasm. However,
since both pH-sensitive anionic lipids and nucleic acids have a negative charge, they may
have limited capacity to entrap nucleic acids.
Viral peptides
Oligonucleotides incubated with or coupled to viral peptides derived from the
hemagglutinin envelop protein of the Influenza virus (Bongartz et al., 1994; Hughes et

21
al., 1996; Yu et al., 1994) provide a route to improve their cytoplasmic delivery. These
peptides are able to form a transmembrane channel through a conformational change
induced by the acidification following endocytosis (Carrasco, 1994; Fattal et al., 1994;
Marsh, 1984; Párente et ah, 1990). Viral peptides can therefore help to transfer
oligonucleotides into the cytoplasmic compartment.
When liposomes are decorated with viral peptides from the Sendai virus
(hemagglutinating virus of Japan (HVJ)), oligonucleotide delivery can also be improved
(Aoki et ah, 1997; Dzau et al., 1996; Kumar et ah, 1997; Mann et ah, 1997; Morishita et
ah, 1993; Yonemitsu et ah, 1997). Unlike the system from the Influenza virus, most
HVJ-liposomes appear to employ a cell-membrane fusion mechanism (Okada et ah,
1975), thus averting oligonucleotides from the endocytotic pathway and releasing them
directly into the cytoplasm.
The advantage of fusogenic peptides is the ability to follow the natural virus entry
pathway. However, fusogenic peptides are expensive to produce and could also pose the
problem of immunogenicity on repeat administration.
Origin of Biodegradable pH-Sensitive Surfactants-Lysosomotropic Detergents
The use of detergents to disrupt phospholipid bilayers (e.g., endosomal
membranes) is efficient (Pinnaduwage et ah, 1989) and provide a rationale approach to
enhance oligonucleotide release from endosomes, but most detergents are indiscriminate
of membrane type and attack the first cellular membrane they contact. In order to provide
selectivity, a trigger is required to activate detergents in specific subcellular locations. A
lysosomotropic amine whose pKa value is between approximately 5 and 7 (De Duve,

22
1983; De Duve et al., 1974) bearing a hydrophobic tail group is classified as a
lysosomotropic detergent (Firestone et al., 1979). At an alkaline environment, the
molecule is predominated by its hydrophobic tail making it simply an oily substance with
limited surface active properties. The non-charged lysosomotropic detergent can be
passively diffused across cellular membranes. Due to the low intralysosomal pH usually
between 4 and 6 (McGraw & Maxfield, 1991), the detergent is protonated and trapped
once inside lysosomes, allowing a continuous gradient for drug entry (Dean et al., 1984;
Forster et al., 1987; Wilson, 1989). When accumulation of the protonated form of the
compound progresses to a certain concentration, the material disrupts the lysosomal
membrane, releasing a variety of lysosomal enzymes into the cytoplasm. Once released
within the cells, these digestive enzymes are able to degrade cellular structures resulting
in cell death (Miller et al., 1983; Wilson et al., 1987).
Consequently, a series of lysosomotropic detergents were synthesized and tested
(Firestone et al., 1982a; Firestone et al., 19826; Firestone et al., 1979). The development
of lysosomotropic detergents originated as a potential way to destroy tumor cells under
the assumption that the malignant cells carry more lysosomes than benign cells (Trouet et
al., 1972). Although later abandoned due to problems with nonspecific lysosomal
cellular destruction, the theory behind this approach has provided the basis for the
development of biodegradable pH-sensitive surfactants (BPS).
Significance of Biodegradable pH-Sensitive Surfactants
Due to the possible pitfalls in nucleic acid delivery, the lysosomotropic detergents
were modified and biodegradable pH-sensitive surfactants (BPS) were developed to

23
induce endosomal membrane defects. The novelty of the delivery system stems from the
two following reasons:
• Exploitation of a naturally occurring oligonucleotide-liposome transport mechanism
(endocytosis) with the incorporation of BPS into the delivery system (e.g., liposomes)
• Biodegradable drug approach to decrease toxicity
Developing BPS that can be activated at the endosóme (early lysosome) stage will
enable the destabilization of the endosomal membranes and liberate oligonucleotides to
their sites of action in the cytoplasm or nucleus (Figure 2-3). However, unlike
lysosomotropic detergents, BPS may be cleaved into less toxic metabolites by the
endogenous digestive enzymes after being released into the cytoplasm due to their
biodegradability. Furthermore, BPS can be quickly synthesized by a one to two-step
standard chemical reaction (e.g., esterification and substitution) with commercially
available inexpensive starting materials (e.g., dodecanol and imidazole). Thus, compared
to other delivery agents, BPS may be more economical, easily produced, and less toxic.
Specific Aims
The inefficiency of nucleic acid delivery systems is likely due, in part, to the
failure of endosomes to release oligonucleotides before reaching degradative lysosomes.
A solution is to incorporate compounds in a delivery vector that will selectively increase
the release of encapsulated nucleic acids from the endosóme. To meet the above criteria,
we developed a group of biodegradable pH-sensitive surfactants (BPS).

24
Oligonucleotide Release
BPS-Liposome
Figure 2-3: Proposed mechanism of the biodegradable pH-sensitive surfactants (BPS)-
liposome system. Like the regular liposome system, similar amount of oligonucleotides
will be brought into cells. After being activated during endocytosis, protonated BPS can
destabilize the endosomal membrane which would release the oligonucleotide into the
cytoplasm.

25
The proposed studies focused on four parts. First, BPS were designed and
synthesized to provide an alternative to oligonucleotide delivery. Second, the
physicochemical properties of three synthesized BPS were evaluated and compared.
Third, the oligonucleotide biological effect derived from the BPS-liposome delivery
system was assessed. Finally, the mechanisms behind how BPS destabilize the
endosomal membrane were investigated.
Specific Aim 1: Design and Synthesis of Biodegradable pH-Sensitive Surfactants
Objective
We designed biodegradable pH-sensitive surfactants (BPS) by varying the head
groups, linkage bridges, and tail groups to investigate the impact on the physicochemical
characteristics. By changing the combinations of different linkage bridges and
hydrocarbon chains with a lysosomotropic amine, imidazole, on the surfactant molecules,
three BPS were synthesized.
Specific Aim 2: Physicochemical Characterization of Biodegradable pH-Sensitive
Surfactants
Objective
The objective was to acquire the surface active properties of the ionized
biodegradable pH-sensitive surfactants (BPS), investigate the membrane destabilization
ability of BPS at varied pHs, determine their stability, and screen their cytotoxicity.

26
Hypothesis
The first hypothesis was that as the pH of the environment decreased, the surface
activity of BPS would increase, thereby destabilizing the liposomal membrane. The
second hypothesis was that the linkage connector of BPS was mostly responsible for their
stability which would further determine their cytotoxicity.
Specific Aim 3: Delivery System Evaluation of Biodegradable pH-Sensitive
Surfactants
Objective
The objective was to quantitatively and qualitatively evaluate the cellular delivery
of oligonucleotides using a biodegradable pH-sensitive surfactants (BPS)-liposomal
delivery system.
Hypothesis
The hypothesis was that when using liposomes to deliver oligonucleotides in
vitro, the effect would be further enhanced in the presence of BPS as a component of the
liposome composition.
Specific Aim 4: Mechanism of Action Investigation of Biodegradable pH-Sensitive
Surfactants
Objective
With the different chemical structures from other fusogenic compounds and
surfactants, biodegradable pH-sensitive surfactants (BPS) were expected to have
disparate membrane activities. The possible mechanisms of how BPS caused membrane
defects were therefore investigated.

27
Hypothesis
The hypothesis was that BPS could induce both membrane fusion and rupture and
eventually release liposomal contents in a pH-dependent manner.

CHAPTER 3
DESIGN AND SYNTHESIS OF BIODEGRADABLE pH-SENSITIVE
SURFACTANTS
Introduction
The idea of using lysosomotropic detergents to gain access to cells exploits the
fact that surfactants lyse lysosomal membranes. Biodegradable pH-sensitive surfactants
(BPS) expand this concept by providing a mechanism to control the lytic properties of the
surfactants. By gathering information of the potential effect on which each individual
component of BPS has impact, a rational approach may be under taken. It is
hypothesized that using the correct design and synthesis of a series of original BPS, the
potency of BPS can be predicted. To begin this project, three BPS, dodecyl 2-(l
imidazolyl) propionate (DIP), methyl 1-imidazolyl laureate (MIL), N-dodecyl imidazole
(DI), were synthesized using standard well understood reactions (esterification and
substitution).
Rationale
A surfactant (surface active agent) is a substance that adsorbs onto the surfaces or
interfaces of a system and alters the free energies of those surfaces or interfaces to a
marked degree (Rosen, 1989). Surface active agents have a characteristic molecular
structure consisting of a head group that is hydrophilic and a tail group that is
28

29
hydrophobic thus making the compounds amphoteric. Depending on the number and
nature of the polar and nonpolar groups present, the agents can be designed to be more
hydrophilic or lipophilic.
In order to design biodegradable pH-sensitive surfactants (BPS), there are three
critical requirements or structural components (Figure 3-1). 1.) The first one is a
lysosomotropic amine as the head group of BPS. 2.) To make the lysosomotropic agent
amphoteric, it is necessary to attach a hydrocarbon chain to the amine as the tail group.
At an alkaline environment, the pH-sensitive surfactant will be un-ionized and lipophilic.
When incorporating the surfactant into liposomes as a delivery system, the lipophilicity
of the surfactant will enhance its chance to remain within the lipid bilayers. After the
amine is protonated due to a pH gradient (e.g., endocytosis), the pH-sensitive surfactant
will increase its surface activity significantly. This change is strong enough to induce
membrane destabilization. 3.) The pH-sensitive surfactant would then be degraded by the
endogenous enzymes into less toxic metabolites with the introduction of a biodegradable
connector. By understanding the relationship among the specific characteristic of each
individual BPS component (head group, tail group, and linkage bridge), it should be
possible to optimize the design of BPS.
Head Group
The head group of biodegradable pH-sensitive surfactants (BPS) is the major
factor determining its ionization constant (pKa) controlling the amount of activated
surfactant at endosomal pH. Two important criteria influence the pKa:

A Lipophilic
Hydrocarbon Chain
A pH Sensitive
Lysosomotropic Amine
An Enzymatically
Cleavable Connector
Figure 3-1: Three individual requirements to create biodegradable pH-sensitive
surfactants (BPS).

31
© Type of lysosomotropic amines
® Presence of substituents on the amine head groups
Since imidazole and morpholine were used as the head groups in the first
generation lysosomotropic detergents, information exists about their chemical properties
and biological effects (De Duve et al., 1974). Other heterocyclic ring compounds, such
as indole, may also have lysosomotropic properties. When compared to morpholine,
imidazole has a higher pKa since the aromatic ring formed by the imidazolyl group
decreases the nucleophilicity and the basicity of the amine. However, compared to
indole, imidazole has higher basicity due to its extra electron lone pair (Table 3-1).
Table 3-1: Predicted effect on pKa from the head group of biodegradable pH-sensitive
surfactants (BPS).
Head Group
Effect on pKa Value
Imidazole
<—>
Morpholine
tt
Indole
1
All substituents near the titratable amine can affect pKa. Electron donating
groups generally increase pKa while electron withdrawing groups generally decrease the
pKa of an amine. Figure 3-2 shows the order of the nucleophilicity, basicity, and pKa for
the substituents at 2’ position of the imidazolyl group.

32
â– N
XH3
'CH3
> NH 2 > OCH 3 > OH > alkyl
O
> H > X (I, Br, Cl) > CN > C-R' > NO'
Figure 3-2: Order of nucleophilicity, basicity, and pKa for different substitutes on the
head group of biodegradable pH-sensitive surfactants (BPS).

33
Tail Group
The hydrocarbon chain of biodegradable pH-sensitive surfactants (BPS) partially
determines their hydrophilicities. The stronger the interaction between the tail groups,
the more lipophilic BPS. As the lipophilicity increases, a lower critical micelle
concentration (CMC) will be observed. Figure 3-3 gives the order of the expected CMC
values when BPS have the same head group by changing the tail group.
Linkage Bridge
The effectiveness of biodegradable pH-sensitive surfactants (BPS) is related to its
rate of hydrolysis which is controlled by the linkage bridge of the molecule. An ester
bond is subject to hydrolysis with the rate dependent upon the extent of steric hindrance
caused by the substituents. An amide bond, however, is stable and unlikely to undergo
hydrolysis quickly in aqueous solutions. For a drug to be effective, an optimum
hydrolysis rate is regulated which must allow the molecule to stay intact long enough to
have an effect while also allowing it to break down after releasing oligonucleotides to
their sites of action.
Selection of Biodegradable pH-Sensitive Surfactants
With the different combinations of head groups, tail groups, and linkers possible
for biodegradable pH-sensitive surfactants (BPS), the number of potential BPS is
numerous. In the subsequent studies, however, three BPS were selected, synthesized, and
compared.

>
R
>
v'VWWWWWW'
Figure 3-3: Biodegradable pH-sensitive surfactants (BPS) with the same head group but
different tail groups in order of decreasing hydrophilicity and critical micelle
concentration (CMC).

35
• Dodecyl 2-(l ’-imidazolyl) propionate (DIP)
• Methyl 1-imidazolyl laureate (MIL)
• N-dodecyl imidazole (DI)
DIP, the first member of the BPS family, was originally devised by Hughes and
co-workers (1996). MIL was designed to compare with DIP when the ester linker
between head and tail groups is positioned into the other direction. DI, lacking a
biodegradable connector, was loosely grouped as a BPS member. DI was originally
synthesized by Firestone and co-workers (1979) and compared to other BPS to address
the importance of the linker with respect to cytotoxicity.
Synthesis of Biodegradable pH-Sensitive Surfactants
Chemicals
N,N-dimethylformamide (DMF) was purchased from Aldrich (Milwaukee, WI).
Dodecanol, 2-bromopropionyl bromide, imidazole, 12-bromo-l-dodecanol, triethylamine,
lauric acid, and N,N’-dicyclohexylcarbodimide were purchased from Fluka
(Ronkonkoma, NY). 1-imidazolyl methanol was a gift from Dr. Kenneth Sloan
(Department of Medicinal Chemistry, University of Florida). All chemicals were used
directly without additional purification.
Dodecyl 2-(l’-Imidazolyl) Propionate
Dodecyl 2-(l ’-imidazolyl) propionate (DIP) was synthesized as modified from a
previous report (Hughes et al., 1996). Briefly, dodecanol (0.05 mole), 2-bromopropionyl

36
bromide (0.025 mole), and triethylamine (0.025 mole) were mixed and stirred in 50 ml of
chloroform for 24 h to yield crude dodecyl 2-bromopropionate (Figure 3-4). The crude
product was washed three times with adequate water to remove impurities. The organic
phase was dried by adding anhydrous sodium sulfate and distilled under vacuum. After
this simple extraction, the crude product, dodecyl 2-bromopropionate (0.015 mole), was
mixed with imidazole (0.03 mole) in chloroform and refluxed for another 24 h (Figure 3-
4).
The final crude DIP product was washed with an adequate amount of water three
times and dried with sodium sulfate. Then, the oily compound was purified through flash
chromatography (Still et al., 1978) using silica gel (235-400 mesh size) as the adsorbent
and methanol-methylene chloride mixture as the mobile phase at a ratio (v/v) of 3.5 to
96.5, respectively.
Methyl 1-Imidazolyl Laureate
A standard esterificaiton method (Hassner & Alwxanian, 1978) was used to
synthesize methyl 1-imidazolyl laureate (MIL). A mixture of 1-imidazolyl methanol
(0.05 mole), lauric acid (0.025 mole), and N,N’-dicyclohexylcarbodiimide (0.025 mole)
in 50 ml of DMF was stirred overnight at 75°C to produce MIL (Figure 3-5). N,N-
dicyclohexyl urea was filtered and washed three times with water, three times with 5%
acetic acid solution, again three times with water, and then dried with anhydrous sodium
sulfate. Pure MIL was then obtained through quick flash chromatography with the ratio
(v/v) of methanol to methylene chloride at 2.5 to 97.5, respectively.

37
Br
2-bromopropionyl bromide Dodecanol
O
Dodecyl 2-bromopropionate
N
^NH
w
Imidazole
A
Dodecyl 2-(l'-imidazolyl) propionate
Figure 3-4: Synthetic pathway of dodecyl 2-(l ’-imidazolyl) propionate (DIP).

38
^n^oh
Nw
0
+ —^ch3
1 -imidazolyl methanol Laurie acid
DCC
0
A *
Methyl 1 -imidazolyl laureate
Figure 3-5: Synthetic pathway of methyl 1-imidazolyl laureate (MIL).

39
N-Dodecyl Imidazole
N-dodecyl imidazole (DI) was synthesized by reacting imidazole (0.05 mole) and
12-bromo-l-dodecanol (0.025 mole) in 50 ml of DMF at 75°C for 24 h (Figure 3-6).
Crude DI was washed with water three times, 5% acetic acid solution three times, water
three times, and then dried with anhydrous sodium sulfate. Pure DI was obtained after
passing through quick flash chromatography with the ratio (v/v) of methanol to
methylene chloride at 3.5 to 96.5, respectively.
Identification of Biodegradable pH-Sensitive Surfactants
After purifying these three agents, their structures were identified through a 300-
MHz 'H-nuclear magnetic resonance (NMR) in the Center of Structural Biology and mass
spectroscopy (FAB) in the Department of Chemistry at the University of Florida. The
purities of the three biodegradable pH-sensitive surfactants (BPS) were confirmed by
elemental analysis in the Department of Chemistry at the University of Florida.
Dodecyl 2-(l’-Imidazolyl) Propionate
The 'H-NMR (CDC13) spectrum showed resonances of 7.60 (s, 1H), 7.05 (s, 1H),
7.00 (s, 1H), 4.85 (q, 1H), 4.15 (t, 2H), 1.75 (d, 3H), 1.20-1.40 (m br, 20H), and 0.85 (t,
3H) which was consistent with the proposed structure (Figure 3-7). The mass spectrum
(CI8H32N202, F.W.: 308.2464) had a molecular ion (M+l) at 309.2537 (Figure 3-8). The
elementary analysis indicated similar experimental percentages to the theoretical values
(Table 3-2). All these assays were within acceptable margins of error (mass

Imidazole
12-bromo-1 -dodecanol
N-dodecyl imidazole
Figure 3-6: Synthetic pathway of N-dodecyl imidazole (DI).

41
JUÃœL
jr
i
J
a JUL
1.00
19.62 3JO.
Figure 3-7: 'H-NMR spectrum of dodecyl 2-(l ’-imidazolyl) propionate (DIP).

42
1001
80-
303.2530
60
40
20
96.0757
141.0698
!
f-^f-
100
154.9694
r
209.1249
I
—r“
150
r**T~*
200
237.2050
1
.L,—
r~T
300
I
1—r—
350
250
E+ 06
1.56
Figure 3-8: Mass spectrum of dodecyl 2-(l’-imidazolyl) propionate (DIP).

43
spectroscopy: 15 mmu; elemental analysis: 0.4% each element) which confirmed the
chemical structure and purity of dodecyl 2-(l ’-imidazolyl) propionate (DIP).
Table 3-2: Elemental analysis of dodecyl 2-(l ’-imidazolyl) propionate (DIP).
Comparison of experimental and theoretical percentages of each element.
DIP
Theoretical % Experimental %
Carbon
Hydrogen
Nitrogen
70.1 69.8
10.9 10.9
9.1 8.7
Methyl 1-Imidazolyl Laureate
The ’H-NMR spectrum showed resonances of 7.50 (s, 1H), 7.00 (d, 2H), 5.90 (s,
2H), 2.35 (t, 2H), 1.20-1.40 (m br, 18H), and 0.90 (t, 3H) (Figure 3-9). The mass
spectrum (CI6H28N,02, F.W.: 280.2151) had a molecular ion (M+l) at 281.2231 (Figure
3-10). The combustion analysis of experimental percentages of elements was in
agreement with the theoretical values (Table 3-3). All assays had acceptable margins of
error and confirmed the chemical structure and purity of methyl 1-imidazolyl laureate
(MIL).

44
Figure 3-9: ‘H-NMR spectrum of methyl 1-imidazolyl laureate (MIL).

45
281.2223
E+ 06
4.70
Figure 3-10: Mass spectrum of methyl 1-imidazolyl laureate (MIL).

46
Table 3-3: Elemental analysis of methyl 1-imidazolyl laureate (MIL). Comparison of
experimental and theoretical percentages of each element.
MIL
Theoretical % Experimental %
Carbon
Hydrogen
Nitrogen
68.6 68.7
11.0 11.0
10.0 9.6
N-Dodecyl Imidazole
’H-NMR spectrum showed resonances of 7.45 (s, 1H), 7.05 (s, 1H), 6.90 (s, 1H),
3.95 (t, 2H), 1.20-1.40 (mbr, 20H), and 0.85 (t, 3H) (Figure 3-11). ForN-dodecyl
imidazole (DI) (C15H28N2, F.W.: 236.2252), the mass spectrum had a molecular ion
(M+l) at 237.2324 (Figure 3-12). The above two assays confirmed the chemical
structure of DI. The combustion analysis of experimental percentages of elements was in
agreement with the theoretical values indicating the purity of this compound (Table 3-4).
Table 3-4: Elemental analysis of N-dodecyl imidazole (DI). Comparison of experimental
and theoretical percentages of each element.
DI
Theoretical % Experimental %
Carbon
Hydrogen
Nitrogen
76.3 76.5
11.9 11.5
11.9 12.2

47
Figure 3-11: 'H-NMR spectrum of N-dodecyl imidazole (DI).

48
100 i
80-
60-
40-
20-
237.2324
82.0868
235.2149
137.1053
535.3390
249.2260
E+ 05
8.04
100
200
300
400
500
600
Figure 3-12: Mass spectrum of N-dodecyl imidazole (DI).

CHAPTER 4
PHYSICOCHEMICAL CHARACTERIZATION OF BIODEGRADABLE pH-
SENSITIVE SURFACTANTS
Introduction
In this chapter, we have characterized and compared three members of the BPS
family, dodecyl 2’-(l-imidazolyl) propionate (DIP), methyl 1-imidazolyl laureate (MIL)
and N-dodecyl imidazole (DI). First, surface active properties including critical micelle
concentration (CMC) and effective release ratio (Re) of the ionized BPS were measured
and verified. The pH sensitivity of BPS to lyse liposomes from the external environment
and the behavior of BPS to destabilize liposomes when incorporated in them were also
evaluated. Then, systems were established to decide the chemical and biological
stabilities of BPS and the results compared in relation to their chemical structures.
Finally, the cellular toxicity of these agents was determined and correlated with their
biodegradability (biological stability).
Materials
Chemical
Calcein, ferric chloride, and ammonium thiocyanate were purchased from Aldrich
(Milwaukee, WI). Dodecanol, imidazole, and lauric acid were purchased from Fluka
49

50
(Ronkonkoma, NY). 1-imidazole methanol was a gift from Dr. Kenneth Sloan
(Department of Medicinal Chemistry, University of Florida). Calcein-AM was purchased
from Molecular Probes (Eugene, OR). L-a-lecithin, dioleoylphosphatidylethanolamine
(DOPE), and l,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) were purchased
from Avanti Polar Lipids (Alabaster, AL). Porcine esterase (300 U/mg protein) and
cholesterol were purchased from Sigma (St. Louis, MO). All purchased or obtained
chemicals were used directly without additional purification.
Ammonium ferrothiocyanate (0.1 M) was prepared by dissolving 8.1 g of ferric
chloride and 15.2 g of ammonium thiocyanate in 500 ml of distilled water. Dodecyl 2-
(l’-imidazolyl) propionate (DIP), Methyl 1-imidazolyl laureate (MIL), and N-dodecyl
imidazole (DI) were synthesized as previously reported (Chapter 3).
Cell
The SKnSH (HTB-11) cell line was purchased from American Type Culture
Collection (Rockville, MD). The CV-1 cell line was a generous gift from Dr. M. C. Cho
(Department of Pharmaceutics, University of North Carolina).
Buffer Preparation
All pH buffers were adjusted with NaCl to an equal ionic strength (Bodor et ah,
1980). The pHs of the buffers (and their chemical compositions) used were as follows:
pH 1.4 (420 mM potassium chloride and 80 mM hydrochloric acid), pH 2.8 (123 mM
citric acid and 60 mM sodium hydroxide), pH 4.2 (150 mM sodium acetate and 350 mM

51
glacial acetic acid), pH 5.0 (300 mM KH2P04 and 50 mM Na^PO,,), pH 6.0 (150 mM
KH2P04 and 100 mM Na2HP04), pH 6.5 (120 mM KH2P04 and 100 mM Na2HP04), pH
7.0 (80 mM KH2P04 and 120 mM N^HPOJ, and pH 8.0 (200 mM KH2P04 and 188 mM
sodium hydroxide).
Liposome Preparation
Instead of serving as a nucleic acid delivery system, liposomes (L-a-lecithin: 1,2-
dimyristoyl-sn-glycero-3-phosphocholine (DMPC): cholesterol; molar ratio 6:1:8) were
used as a model membrane system. To maintain simplicity, only neutral lipids were
employed in the liposomal membrane system. While the liposomes may not fully
represent events occurring in biological situations they still served as excellent models in
addressing potential mechanisms of lipid membrane disruption. Calcein (100 mM) was
entrapped within the liposomes as a fluorescent marker to monitor membrane lysis events
and unentrapped calcein was removed through centrifugation (14,000 rpm, 5 min) five
times and washed with a pH 7.4 phosphate buffered saline (PBS) each time.
Reverse-phase evaporation vesicle method (Szoka & Papahadjopoulos, 1978) was
used to produce unilamellar vesicles (600 nm) using polycarbonate membranes (Poretics;
Livermore, CA) through a high pressure extruder three times (Lipex Biomembrane Inc.;
Vancouver, Canada). The size of the liposomes (volume-weight Gaussian distribution)
was measured to be 609±158 nm (standard deviation) by a dynamic light scattering
method using a NICOMP Model 380 ZLS Zeta Potential/Particle Sizer (Santa Barbara,
CA). The concentration of phospholipid in each experiment was measured by a

52
modification of a spectrophotometric technique (Stewart, 1980). Briefly, varying
amounts of L-a-lecithin (0-50 mg/ml) were added to test tubes containing 2 ml of 0.1 M
ammonium ferrothiocyanate and 2 ml of chloroform. The contents were mixed
vigorously for 1 min and centrifuged at 6,000 rpm (Safeguard Centrifuge, Clay-Adams
Inc.) for 5 min to fully separate the two phases. The aqueous phase was removed and the
absorbance of the remaining organic phase was measured at 488 nm with a
spectrophotometer (UV/Vis Perkin-Elmer spectrophotometer Lambda 3) to establish a
calibration curve. The concentrations of unknown samples were then determined from
the calibration curve.
Methods
Critical Micelle Concentration Determination
To determine the critical micelle concentration (CMC) of the ionized
biodegradable pH-sensitive surfactants (BPS), surface tension measurements were
performed using a CRC-DuNoüy interfacial tensiometer (Martin, 1993). The pH was
adjusted to pH 3.0 in different counter ion solutions (HF, HC1, HBr, and HI) at a constant
room temperature (22°C). Increasing amounts of BPS were added into different solutions
and surface tension measured. The concentration region in which surface tension stopped
changing was recorded as the CMC.

53
Effective Release Ratio Determination of Biodegradable pH-Sensitive Surfactants
Unilamellar liposomes (600 nm; 10 nmol/ml) containing 100 mM calcein ^self-
quenching concentration) were suspended in a pH 4.2 acetate buffer solution with
increasing amounts of biodegradable pH-sensitive surfactants (BPS). Equilibrium was
allowed to occur for 30 min at room temperature. A substantial portion to cause
membrane lysis from most detergents has taken place after 30 min of incubation with
liposomes (Ruiz et al., 1988). Complete equilibrium between surfactants and lipids can
take several hours (Lichtenberg et al., 1979). After this time, however, surfactant induced
release of liposomal contents can be masked by the concomitant spontaneous diffusion of
solutes out of the vesicles. Therefore, long time incubation with complete equilibrium
may not be appropriate in this study.
Released calcein was excited at 496 nm and observed at 517 nm in a Perkin-Elmer
luminescence spectrophotometer LS-50B at room temperature. The percentage of
released calcein was calculated by the equation /(%) =
jy—~ * 100% (Liu & Regen,
(U ~ h)
1993). Ix is the 100% fluorescence intensity value when adding excess Triton X-100 (10
mM); Ia and Ib are the fluorescence intensities after incubation with and without BPS,
respectively. Effective release ratio (Re) was defined at the molar ratio
(surfactant/liposome) when 50% calcein was released. During this process, the surfactant
must come in contact with the lipid bilayer and partition into the hydrophobic
environment.

54
pH Sensitivity of Biodegradable pH-Sensitive Surfactants on Liposomal Calcein
Release
To determine the ability of biodegradable pH-sensitive surfactants (BPS) to cause
membrane lysis/leakage at different pHs, studies were conducted that varied the BPS
concentration in a solution of calcein containing liposomes (10 nmol/ml) as described
above. Increasing BPS concentrations (0.5 nmol/ml-500 nmol/ml) were added to four
buffer systems (pH 4.2, 6.0, 6.5, and 8.0) containing liposomes with calcein. The
suspensions were incubated for 30 min at room temperature and the percentage of calcein
release was calculated by the equation /(%) =
a.-h)
(W»)
* 100% as previously described
(Liu & Regen, 1993).
Membrane Lysis Profile of Biodegradable pH-Sensitive Surfactants when
Incorporated into Liposomes
Different molar ratios (R=0, R=0.2, and R=0.4) of biodegradable pH-sensitive
surfactants (BPS) were incorporated into liposomes (10 nmol/ml) containing 100 mM
calcein to observe the induced calcein release at different pHs. The various BPS-
liposomes were incubated in phosphate buffer solutions (pH 5-8) for 30 min to determine
the release characteristics. The percentage of release was then recorded and corrected by
the equation /(%) =
(W»)
(W*)
*100% as mentioned previously.

55
Chemical and Biological Stabilities of Biodegradable pH-Sensitive Surfactants
The aqueous stability of biodegradable pH-sensitive surfactants (BPS) was
determined by incubating different concentrations of BPS in pH buffers (pH 1.4-8.0) with
5% dimethyl sulfoxide (DMSO) as a co-solvent at 37°C. Samples were removed
periodically and BPS concentration was quantified using an HPLC method. The HPLC
system consisted of a Milton Roy CM 4000 pump, an LDC Analytical 3200 absorbance
detector, a Hewlett Packard 3395 integrator, and a Spectra Physics SP 8780 autosampler.
A 3.9*75 mm C8 column (Nova-Pak) along with a mobile phase consisting of 60%
acetonitrile and 40% 10 mM pH 2.8 NaH2P04 solution was used to separate and
determine intact BPS from their degradation products at 210 nm (Buyuktimkin et al.,
1993). The flow rate was set at 1.0 ml/min. The chemical degradation rate constant was
obtained after plotting the peak height of the intact BPS over time and used to create BPS
pH hydrolysis rate profile.
To determine the hydrolysis rate of BPS in biological systems, a porcine esterase
was used. Varying units of esterase were incubated at 37°C with constant amount of BPS
in a 5% DMSO pH 7.0 buffer solution. Aliquot was removed at a periodic interval and
peak height of the intact BPS was measured with the HPLC method described above.
The biological rate constants of three BPS were calculated to compare their
biodegradability.

56
Cellular Toxicity Test of Biodegradable pH-Sensitive Surfactants
The cellular toxicity of biodegradable pH-sensitive surfactants (BPS) was
monitored in an SKnSH human neuroblastoma cell line and in a CV-1 monkey kidney
fibroblast cell line with a calcein-AM assay (Lichtenfels et al., 1994). To confirm the
toxic effect of DIP and MIL as a result of their metabolites, imidazole, 1-imidazole
methanol, dodecanol, and lauric acid were used to test their individual cytotoxicity in a
CV-1 cell line.
Briefly, for the SKnSH cell line, subconfluent monolayered cultures were
incubated in a 96-well plate (105 cells/well) with 200 pi of RPMI 1640 growth medium
(100 U/ml penicillin, 100 pg/ml streptomycin, and 10% fetal bovine serum) at 37°C, 5%
C02, and 100% humidity environment for 24 h. For the CV-1 cell line, MEM growth
medium (100 U/ml penicillin, 100 pg/ml streptomycin, 1 mM MEM sodium pyruvate
solution, IX MEM amino acids solution, and 10% heated fetal bovine serum) was used
instead of RPMI 1640. The growth medium was then removed and BPS added from 1
pM to 1 M in 200 pi of fresh growth medium. The cells were maintained for an
additional 48 h. After the incubation, cells were washed three times with phosphate
buffered saline and incubated with 100 pi of calcein-AM (1 pM) for 30 min at room
temperature.
Calcein fluorescence intensity was then measured at an excitation wavelength of
496 nm and observed at 517 nm on a Perkin Elmer LS 50B Spectrophotometer. The
percentage of live cells was calibrated as in the following equation:

57
{Sample - Min)
Live{%) = —— 77—— * 100% , where Max is the fluorescence signal from cells
{Max - Min)
without any treatment, Min is the fluorescence signal without cells, and Sample is
fluorescence signal from each sample. To compare the difference among different
treatments, a parameter ID50 was used. ID50 was defined as the drug concentration
required to reduce the absorbance of calcein by 50%, thus indicating 50% cell death.
Statistical Analysis
Statistical differences between the treatments were determined using analysis of
variance where appropriate (StatView 4.53, Abacus Concepts, Inc., Berkeley, CA) with
p<0.05 considered statistically significant and Fisher’s (PLSD) post hoc t-test was
applied.
Results
Critical Micelle Concentration Determination
An important parameter in characterizing surfactants is the concentration at which
micelles form. All experiments were conducted at pH 3.0 to ensure that all biodegradable
pH-sensitive surfactants (BPS) were in an ionized state (>99.9%). The critical micelle
concentrations (CMCs) of the ionized BPS were determined by using surface tension
measurements in different counter ion solution. As BPS concentration increased, the
surface tension of the solution sharply decreased until the formation of micelles occurred
(Figure 4-1). In general, N-dodecyl imidazole (DI) had the highest CMCs while methyl

58
Figure 4-1: Critical micelle concentration (CMC) measurement of three ionized
biodegradable pH-sensitive surfactants (BPS). The CMC (meanlstandard deviation
(SD)) was calculated in a pH 3.0 hydrofluoric acid solution at room temperature with an
interfacial tensiometer (n=3). The concentration region at which surface tension
stabilized was recorded as the CMC. The CMC was determined to be 1.0-1.2 mM for
ionized dodecyl 2-(l’-imidazolyl) propionate (DIP) (■), 0.6-0.8 mM for ionized methyl
1-imidazolyl laureate (MIL) (♦), and 1.0-1.2 mM for N-dodecyl imidazole (DI) (A).

59
1-imidazolyl laureate (MIL) had the lowest CMCs (Table 4-1) in all four counter ion
solutions. For all three BPS, HI, HBr, HC1, and HF caused the CMC to decrease in
descending order.
Table 4-1: The critical micelle concentration of dodecyl 2-(l’-imidazolyl) propionate
(DIP), methyl 1-imidazolyl laureate (MIL), and N-dodecyl imidazole (DI) in four
different counter ion solutions (n=3).
Solution
BPS (mM)
DIP
MIL
DI
HF
1.0-1.2
0.6-0.8
1.0-1.2
HC1
0.9-1.1
0.6-0.7
0.9-1.1
HBr
1.7-1.9
1.0-1.2
1.8-2.2
HI
1.6-1.7
0.8-1.0
2.5-2.5
Effective Release Ratio Determination of Biodegradable pH-Sensitive Surfactants
Effective release ratio (Re) describes the molar ratio of a surfactant to the total
amount of lipid required to release 50% liposomal contents. Re was determined by fitting
a sigmoidal curve of calcein release from liposomes at increasing molar ratios of
biodegradable pH-sensitive surfactants (BPS) to lipid using the Scientist computer
program (Micromath; Salt Lake City, Utah). Re was determined to be 3.0, 6.0, and 13.2
for dodecyl 2-(l’-imidazolyl) propionate (DIP), methyl 1-imidazolyl laureate (MIL), and
N-dodecyl imidazole (DI), respectively (Figure 4-2).

60
Figure 4-2: Ability of various biodegradable pH-sensitive surfactants (BPS) to induce
calcein release (mean±SD) at increasing molar ratios of ionized BPS from an external
environment to liposomes when incubated in a pH 4.2 buffer solution for 30 min (n=3).
The effective release ratio was then determined to be 3.0, 6.0 and 13.2 for dodecyl 2-(l
imidazolyl) propionate (DIP) (•), methyl 1-imidazolyl laureate (MIL) (■), and N-
dodecyl imidazole (DI) (A), respectively.

61
pH Sensitivity of Biodegradable pH-Sensitive Surfactants on Liposomal Calcein
Release
To determine whether biodegradable pH-sensitive surfactants (BPS) become
effective in acidic environments but have limited effect at extracellular biological pH,
calcein containing liposomes were incubated with increasing amounts of BPS at four pHs
(4.2, 6.0, 6.5, and 8.0). An increase in fluorescence intensity indicated calcein release
that correlated to membrane lysis.
Calcein was released sigmoidally at pH 4.2 as the concentration of dodecyl 2-(l
imidazolyl) propionate (DIP) increased (Figure 4-3a). When the pH of the incubation
environment was increased, the percentage of calcein release was decreased at the same
molar ratio of DIP to lipid. With the decreased surface active properties of DIP at pH 8.0,
calcein release was only slightly increased at higher DIP concentration. This release was
most likely due to saturation of the space between the lipid bilayers with increasing
amount of DIP. Since distribution between the aqueous environment and lipid bilayer
must occur for DIP to elicit membrane lysis, no significant differences between calcein
release and pH were observed until the molar ratio reached 2 (p<0.01).
Similar profiles were seen with methyl 1-imidazolyl laureate (MIL) induced
calcein release (Figure 4-3b). Significant differences (p<0.01) on calcein release were
observed at different pHs when the molar ratio of MIL to lipid equaled to 2 or above.
However, compared to the calcein release caused by DIP at the same molar ratio, MIL
showed less pH sensitivity and less calcein release at pH 6.0 and 6.5.

62
Figure 4-3: Effect of different biodegradable pH-sensitive surfactants (BPS) on calcein
release from liposomes when added from an external environment (n=3). The
percentages (meaniSD) of release were calculated after 30 min of incubation in four
buffer solutions (pH 4.2 (♦), 6.0 (X), 6.5 (A), and 8.0 (■)). a) Dodecyl 2-(l’-
imidazolyl) propionate (DIP); b) Methyl 1-imidazolyl laureate (MIL); c) N-dodecyl
imidazole (DI).

Release
63
Figure 4-3—continued
(b)

64
Figure 4-3—continued
(c)

65
When DIP was replaced by N-dodecyl imidazole (DI), similar profiles were seen
on calcein release at low pH (Figure 4-3 c). However, no significant difference was seen
on the calcein release among the four tested pHs at all observed molar ratios.
Membrane Lysis Profile of Biodegradable pH-Sensitive Surfactants when
Incorporated into Liposomes
Since the ultimate goal was to incorporate biodegradable pH-sensitive surfactants
(BPS) into liposomes, we determined the ability of unionized BPS incorporated into
liposomes to be protonated at lower pHs, thereby facilitating the release of entrapped
materials. Liposomes containing calcein were prepared with increasing molar ratios
(R=0, R=0.2, and R=0.4) of BPS and incubated at decreasing pHs.
Minimal calcein release was observed when no dodecyl 2-(l’-imidazolyl)
propionate (DIP) was incorporated into the liposomes (Figure 4-4a). As the pH
decreased, calcein release increased gradually in all groups. At each ratio group, calcein
release increased significantly (p<0.05) as pH dropped from 6.0 to 5.0. Significant
differences (p<0.05) of the calcein release were also observed among all molar ratio
groups (R=0, R=0.2, and R=0.4) at all observed pHs. Like DIP, both methyl 1-
imidazolyl laureate (MIL) (Figure 4-4b) and N-dodecyl imidazole (DI) (Figure 4-4c)
caused calcein release in a similar pH-dependent manner. Compared to other groups, the
system at the R=0.4 group was relatively unstable at physiological pH probably due to the
alternations in lipid packing of the liposomes.
When comparing the calcein release caused by three BPS, significant differences
(p<0.05) were observed at the R=0.4 group as pH was 6.0 or 5.0. However, no

66
4 5 6 7 8
pH
(a)
Figure 4-4: Membrane lysis profile of various biodegradable pH-sensitive surfactants
(BPS) when incorporated into liposomes (n=3). The effect of pH and BPS/liposome
molar ratio (R=0 (♦), R=0.2 (■), and R=0.4 (A)) on BPS-induced calcein release from
liposomes were plotted after 30 min (mean±SD). a) Dodecyl 2-(l ’-imidazolyl)
propionate (DIP); b) Methyl 1-imidazolyl laureate (MIL); c) N-dodecyl imidazole (DI).

67
a>
m
ea
o
0
6
pH
8
Figure 4-4—continued
(b)

68
4 5 6 7 8
pH
(c)
Figure 4-4—continued

69
significant difference of the calcein release was seen at the other molar ratio groups
(R=0.1 and R=0.2) among these three BPS.
Chemical and Biological Stabilities of Biodegradable pH-Sensitive Surfactants
After releasing oligonucleotides to cytoplasm, an ideal pH-sensitive surfactants
must be degraded in the intercellular milieu, thus limiting its potential toxicity.
Biodegradable pH-sensitive surfactants (BPS) should be able to be degraded by ester
hydrolysis either chemically or enzymatically. The hydrolytic stability of BPS was
assessed by incubating the compound in pH buffers and monitoring the amount of the
starting material remaining intact (Figure 4-5). Using the Scientist program to fit the
degradation curve, the pH-dependent pseudo-first order degradation rate constants (k)
was calculated.
Dodecyl 2-(l ’-imidazolyl) propionate (DIP) was at its most stable state (k=0.055
day'1) at pH 2.8. The degradation rate constant reached a plateau after pH 6.0 (k=0.70
day'1) (Figure 4-6). Similar to DIP, methyl 1-imidazolyl laureate (MIL) and N-dodecyl
imidazole (DI) showed the greatest stability at pH 2.8 (k=0.050 day'1 and k=0.0055 day"1,
respectively). DI exhibited the lowest rate constant among the three BPS from pH 1.3 to
pH 7.0 solutions. No difference in the degradation rate constant of BPS was seen when
the pH was adjusted to 8.0. However, since each pH buffer solution was composed of
different compounds, it might have its individual catalyst effect on the chemical rate
constant.
To assess the enzymatic stability of BPS, we used various amounts of porcine
esterase as a model enzyme. Similar to the determination of chemical degradation rate

70
Time (day)
Figure 4-5: Chemical degradation profile of dodecyl 2-(l ’-imidazolyl) propionate (DIP)
at pH 1.4 and 37°C over time (n=3). The peak heights of intact DIP were obtained at
various time points (mean±SD). Using the Scientist program to fit the degradation curve,
the pseudo-first order degradation rate constant (k) was determined to be 0.36 (/day).

71
Figure 4-6: Chemical degradation pH-rate profiles of three biodegradable pH-sensitive
surfactants (BPS). The rate constant (mean±SD) measured at 37°C of dodecyl 2-(l
imidazolyl) propionate (DIP) (■), methyl 1-imidazolyl laureate (MIL) (♦), and N-
dodecyl imidazole (DI) (A) was plotted against pH (n=3).

72
stability of BPS was assessed by monitoring the amount of the starting material
remaining intact in a pH 7.0 buffer solution (Figure 4-7). Figure 4-8 showed the
biological degradation rate profile of BPS at various ratios of esterase/BPS. MIL was
more biodegradable than DIP through out the tested ratios. Furthermore, DI was almost
insensitive to the addition of the esterase.
Cellular Toxicity Test of Biodegradable pH-Sensitive Surfactants
A more important parameter than biodegradability of biodegradable pH-sensitive
surfactants (BPS) is the cytotoxicity. Biodegradability of BPS relates to levels of cellular
toxicity. A commonly used calcein-AM assay was used to measure the cellular toxicity
of BPS. Calcein-AM is a lipophilic ester compound without fluorescent activity. After
diffusion into cells, calcein-AM is degraded into calcein, a fluorescence chemical, by
endogenous enzymes. The percentage of live cells is then calculated indirectly from the
fluorescence signal of calcein. The higher the signal, the more cells surviving.
In SKnSH cells, the IDS0 (Figure 4-9a) was determined to be 1 mM, 8 mM, and
0.07 mM for dodecyl 2-(l’-imidazolyl) propionate (DIP), methyl 1-imidazolyl laureate
(MIL) and N-dodecyl imidazole (DI), respectively. In CV-1 cells, the ID50 (Figure 4-9b)
was measured at 20 mM, 300 mM, and 0.3 mM for DIP, MIL, and DI, respectively. For
the possible metabolites from DIP and MIL, the ID50 was 70 mM for imidazole, 7 mM for
dodecanol, 800 mM for 1-imidazole methanol, and 9 mM for lauric acid in CV-1 cells
(Figure 4-10).
DI showed the highest cytotoxic effect and was one and two orders of the
magnitude more toxic than DIP in SKnSH and CV-1 cells, respectively. On the contrary,

73
Figure 4-7: Biological degradation profile of 0.65 pmole of dodecyl 2-(l ’-imidazolyl)
propionate (DIP) when incubated with 0.9 U of porcine esterase at pH 7.0 and 37°C over
time. The peak heights of the intact DIP were obtained at various time points. Using the
Scientist program to fit the degradation curve, the pseudo-first order degradation rate
constant (k) was determined to be 0.99 (/h).

74
5
y = 1.7453x + 0.1221
Figure 4-8: Biological degradation rate profile of biodegradable pH-sensitive surfactants
(BPS). Various ratios (U/pmole) of porcine esterase to three BPS (dodecyl 2-(l
imidazolyl) propionate (DIP) (■), methyl 1-imidazolyl laureate (MIL) (♦), and N-
dodecyl imidazole (DI) (A)) were plotted against degradation rate constants (mean±SD)
in a pH 7.0 buffer solution at 37°C (n=4). The linear relationship between the
degradation rate constant of each BPS and the ratio of esterase to BPS was also shown.

75
Cone. (mM)
(a)
Figure 4-9: Cytotoxic effect of biodegradable pH-sensitive surfactants (BPS) on two cell
lines measured by a calcein-AM assay after 48 h of incubation. Data were expressed as
mean±SD. a) In SKnSH cells, the ID5o (50% live cells) of methyl 1-imidazolyl laureate
(MIL) (A), dodecyl 2-(l’-imidazolyl) propionate (DIP) (■), andN-dodecyl imidazole
(DI) (♦) was visually determined to be 8 mM, 1 mM, and 0.07 mM, respectively (n=4).
b) In CV-1 cells, the ID50 of MIL (A), DIP (■), and DI (♦) was visually determined to
be 300 mM, 20 mM, and 0.3 mM, respectively (n=4).

% Live Cells
76
Figure 4-9—continued
++â–º

77
Cone. (mM)
Figure 4-10: Cytotoxic effect of possible metabolites from biodegradable pH-sensitive
surfactants (BPS) on CV-1 cells measured by a calcein-AM assay after 48 h of
incubation. The ID50 (50% live cells) was determined to be 70 mM for imidazole (♦), 7
mM for dodecanol (•), 800 mM for 1-imidazole methanol (■), and 9 mM for lauric acid
(X), respectively (n=4). Data are expressed as mean±SD.

78
MIL exhibited the least cytotoxicity compared to the other two BPS and was one order of
the magnitude less toxic than DIP in both cell lines. The possible metabolites, imidazole
and dodecanol, from DIP were more toxic than those metabolites, 1-imidazole methanol
and lauric acid, from MIL in CV-1 cells.
Discussion
Imidazolyl based lipids have been used successfully for in vitro delivery of
nucleic acids (Solodin et al., 1995) establishing a rational for the use of imidazole. Most
non-viral delivery systems enter cells through endocytosis. The most significant
characteristic of endosomes is the pH gradient from inside the endosóme to the
intracellular space.
Dodecyl 2-(l’-imidazolyl) propionate (DIP), methyl 1-imidazolyl laureate (MIL),
and N-dodecyl imidazole (DI) are imidazolyl based surfactants in the biodegradable pH-
sensitive surfactants (BPS) family which have been proposed to facilitate the transport of
nucleic acid through the endosomal pathway. BPS take advantage of the acidic
environment within endosomes to protonate a lysosomotropic amine thus increasing their
surface active properties. After BPS become ionized, they can assist the destabilization
of the endosomal membrane. To lessen adverse effects of the ionized BPS, an ester bond
was introduced into DIP and MIL’s structure making it biodegradable. In a preliminary
study (Hughes et al., 1996), DIP has been shown to reduce the concentration of
oligonucleotides required to produce a biological effect using a tissue culture system. In
this report, the physicochemical properties of three BPS were systematically
characterized.

79
The number of possible analogs of the BPS family can be immense. In order to
determine what physicochemical parameters influence the biological activity of BPS, a
series of evaluative tests associated with surfactants were established. Presently, it is not
clear which measured parameter would be useful in the characterization of BPS for
nucleic acid delivery. The current established methods have been optimized for studying
small molecule transport instead of macromolecules such as oligonucleotides and plasmid
DNA.
The first parameter determined for BPS was the critical micelle concentration
(CMC). The more hydrophilic a surfactant, the higher the CMC (Rosen, 1989). From the
chemical structures, DI, DIP, and MIL had decreasing orders of hydrophilicity. As a
result of this difference, DI, DIP, and MIL had decreasing orders of CMC in general. As
the size of the counter ion increased, the surfactant became more hydrophilic thus
obtaining the higher CMC.
CMC is an essential parameter in describing surfactants (i.e., hydrophilicity,
surface excess); however, it may not be the best parameter to measure the ability of a
surfactant to cause membrane lysis (Lichtenberg, 1985; Ruiz et al., 1988). Therefore, the
effective release ratio (Re) was utilized to describe the ability of BPS to lyse membranes.
The lower the Re, the less surfactant required to lyse membranes.
For basic amines such as BPS, the intrinsic ionization constant (pKa 5-7) and
local pH environment determine the percent ionized. The relationship can be expressed
\BH+1
in Henderson-Hasselbach equation: pKa = pH + log , where [BH+] and [B]
[B]
represents the ionized and un-ionized bases, respectively. From the perspective of their

80
chemical structures, DI, DIP, and MIL were expected to have a decreasing order of pKa.
This difference between the pKa reflects the different amount of ionized BPS that can be
converted at the same pH environment. While pKa is an important factor that determines
the amount of ionized BPS and further influence the liposomal content release, the ability
of different BPS partition into the liposomal membranes at different pHs should be also
considered.
Comparing these three agents, with the highest pKa and no chemical hindrance
(i.e., straight hydrocarbon chain) that facilitated partition into liposomes more easily, DI
was almost indiscriminate to various pH environments resulting in a similar calcein
release profile. For the MIL treated liposome group, on the other hand, a limited amount
of calcein was detected at pH 6.0, 6.5, and 8.0 because of its lowest pKa and chemical
hindrance effect (i.e., ester linker) that might decrease the partition of MIL into
liposomes.
However, with the median pKa value and chemical hindrance (i.e., ester linker
and branched methyl group) of DIP, more calcein was released than MIL as pH was
decreased from 8.0 to 6.5 or 6.0. With these two factors (i.e., pKa and partition
coefficient), the calcein release profiles were therefore established in various pH
environments.
The ability of BPS to release liposome-entrapped molecules was tested as a proof
of the compound’s pH sensitivity based on the principle experiment. As the
BPS/liposome molar ratio was increased and/or pH decreased, more calcein was released
from the liposome model system. Calcein release induced by BPS when incorporated
into the liposome system was slightly higher than that caused by BPS when added to a

81
solution containing calcein liposomes. The greater release could be due to the dilution of
BPS or may be related to partitioning baniers.
An interesting finding was observed between the release of liposome entrapped
calcein when BPS were added externally as compared to direct incorporation into the
liposome matrix. All three BPS demonstrated similar release profiles when the uninoized
BPS agent was incorporated into the liposome illustrating they most likely have similar
mobility within the bilayer. When the three agents were added from the external phase to
preformed liposomes there was a drastic difference between the release profiles of BPS
with ester linkages (e.g., DIP and MIL) as compared to the alkyl chain analog (DI) which
demonstrated little pH dependency.
This effect is also possibly due to the change of pKa at the interface between the
monolayer of the liposome and the solution. However, it is unlikely that this lack of pH
dependency is solely due to changes in the pKa of BPS. The different profiles may
reflect the ease of BPS to partition into the bilayer. BPS with a more polar head group
(e.g., ester containing) would be expected to have added resistance in membrane
partitioning.
After releasing membrane entrapped molecules in a pH-sensitive manner, the
biodegradability of BPS was confirmed. Due to its possible increased stable metabolites
and lack of chemical hindrance, MIL was more biodegradable than DIP (Figure 4-8). On
the other hand, without any biodegradable bond (e.g., ester), DI showed no difference of
the degradation rate constants when incubated in biological media. The degradation rate
of BPS in vivo would be expected to be greater due to the higher number of esterase (e.g.,

82
lipase) molecules. After the release of BPS from the liposomal membrane, these
molecules would be available for BPS metabolism.
A major concern with the use of agents to enhance oligonucleotide delivery is that
any compound added to a delivery vector might contribute to the toxicity of the system.
The toxicity of a given compound is often related to its stability; therefore, the
biodegradability of BPS may decrease its cellular toxicity. In the calcein-AM assay, the
addition of an ester group to the surfactant resulted in a less toxic effect as compared to
N-dodecyl imidazole, a first generation lysosomotropic detergent (De Duve et al., 1974),
by one to two orders of magnitude in two different cell lines. It was also shown that the
less toxic effect of MIL compared to DIP was the possible result of the less toxic
metabolites from MIL than those from DIP. While this study implies that ester
containing imidazole based surfactants are less toxic than their straight chain analogs, it is
unclear how the toxicity of BPS alone will compare to cationic liposome mediated
delivery.
The rate and efficiency of oligonucleotide release from lipid compartments
depend on the characteristics of the surfactant. Biodegradable pH-sensitive surfactants
(BPS) must be stable enough in the biological milieu to induce a therapeutic effect.
However, after facilitating oligonucleotides to their sites of action, BPS must be
metabolized into less toxic lysosomotropic amines and hydrocarbon chain components to
prevent cellular toxicity. BPS are similar to the first generation lysosomotropic
detergents with their long lasting surface active property in a pH-dependent manner.
However, they have much higher biodegradability because of the bridge connector which
can be hydrolyzed into less toxic compounds.

83
Conclusion
The ultimate goal of this project was to adjust chemical characteristics of BPS
components, head and tail groups, in order to optimize BPS-induced release of
oligonucleotides from membrane compartments. Furthermore, BPS would have
minimum cellular toxicity with the introduction of a biodegradable linker. At present, the
critical micelle concentration (CMC), effective release ratio (Re), biodegradability, and
cytotoxicity of these three BPS have little biological relevance. As more diverse BPS are
established and characterized with these mentioned parameters, it is hoped that particular
physicochemical properties will be predictive of promoting oligonucleotide biological
activity.

CHAPTER 5
DELIVERY SYSTEM EVALUATION OF BIODEGRADABLE pH-SENSITIVE
SURFACTANTS
Introduction
Oligonucleotides must reach their sites of action (cytoplasm or nucleus) to
hybridize with their targets to exert their effect (Agrawal & Iyer, 1997). However, the
cellular delivery of free oligonucleotides is very poor (Akhtar et ah, 19916; Stein &
Cheng, 1993). One strategy to improve the delivery of the oligonucleotide is to use
liposomes which can carry oligonucleotides with the vehicles and increase the
intracellular accumulation via endocytosis. Liposomes can deliver oligonucleotides to
the cells but the obstacle of escaping from endosomes still remains. A series of
biodegradable pH-sensitive surfactants (BPS) were developed to conquer the potential
pitfall that the liposome delivery system might have and to further enhance cellular
delivery of the oligonucleotide.
In previous studies, the physicochemical properties of BPS were characterized.
They were shown to be able to induce membrane lysis in a pH- and molar ratio-
dependent manner (Chapter 4). Nevertheless, the BPS-liposome system has not yet been
tested using a biological system. Therefore, we further evaluated the BPS-liposome
delivery system in vitro.
84

85
To evaluate oligonucleotide cellular uptake with the BPS-liposome system, flow
cytometry was employed. Another screening method that addressed oligonucleotide
cellular delivery was the use of laser scanning confocal microscopy (Fisher et al., 1993).
We used this technique to investigate oligonucleotide cellular uptake and distribution that
the BPS-liposome delivery system affected. Additionally, we quantitatively monitored
the inhibition effect of oligonucleotides in BPS-liposomes on luciferase enzyme activity
(Brasier et ah, 1989).
Materials
Chemical
Dihydrogen potassium phosphate, EDTA, formaldehyde solution, and Triton X-
100 were bought from Fisher Scientific (Pittsburgh, PA). Adenosine triphosphate,
dithiothreitol (DTT), magnesium sulfate, and tricine were purchased from Sigma (St.
Louis, MO). BCA Protein Assay Kit was obtained from Pierce (Rockford, IL) and Label
IT fluorescein Nucleic Acid Labeling Kit from Mirus (Madison, WI). The pGL3 plasmid
DNA was obtained from Mr. Fuxing Tang in the Department of Pharmaceutics,
University of Florida. D-luciferin was purchased from Molecular Probes (Eugene, OR).
L-a-lecithin and N-[l-(l-2,3-dioleoloxy)propyl]-N,N,N-trimethylammonium
methylsulfate (DOTAP) was purchased from Avanti Polar Lipids (Alabaster, AL). Three
biodegradable pH-sensitive surfactants (BPS), dodecyl 2-(l’-imidazolyl) propionate
(DIP), methyl 1-imidazolyl laureate (MIL), and dodecyl imidazole (DI) were synthesized

86
as previously reported (Chapter 3). All purchased or obtained chemicals were used
directly without additional purification.
Cell
The CV-1 luciferase expressing cell line was a generous gift from Dr. M. C. Cho
of the University of North Carolina. The RAW 264.7 (TIB-71) cell line was purchased
from the American Type Culture Collection (Rockville, MD).
Methods
Oligonucleotide and Plasmid DNA Cellular Uptake Evaluation Using Flow
Cytometry
Oligonucleotide synthesis and liposome preparation
Phosphorothioate oligonucleotides (15 bases; 5’-TGG CGT CTT CCA TTT-3’)
labeled with fluorescein isothiocyanate (FITC) at the 5’-end were synthesized in the DNA
Core Synthesis Lab at the University of Florida. Oligonucleotides were used directly
without additional purification. pGL3 plasmid DNA with a size of 5256 bp and purity of
1.9 (A260/A280) was labeled with fluorescein using a Mirus Label IT Nucleic Acid
labeling Kit. Simply, 100 pg of the plasmid DNA (2.25 mg/ml) reacted with 100 pi of
fluorescein reagent at 1:1 (w/v) ratio was dilute to a final concentration of 0.1 mg/ml in
IX labeling buffer and incubated at 37°C for 1 h. The unreacted labeling reagent was
removed from labeled nucleic acid by sephadex G50 spin columns at a force of720*g for
2 min and then the purified sample collected.

87
Cationic N-[l-(l-2,3-dioleoloxy)propyl]-N,N,N-trimethylammonium
methylsulfate (DOTAP) liposomes were made as a control vector for nucleic acid
delivery. Biodegradable pH-sensitive surfactants (BPS) at various molar ratios were
combined with DOTAP to create liposomes. After liposomes were rehydrated, a Sonic
Dismembrator 60 probe was used to form small unilamellar vesicles by applying 5 Watts
of power for 10 s to the liposome suspension and keeping on ice for 30 s. The cycle was
repeated until a clear solution was seen. The size of the liposomes (volume-weight
Gaussian distribution) was determined to be 55.2+26.1 nm (standard deviation) by a
dynamic light scattering method using a NICOMP 380 ZLS Zeta Potential/Particle Sizer
(Santa Barbara, CA).
Cell preparation
Monolayered CV-1 (monkey kidney fibroblast) cells were incubated in 24-well
plates (5*105 cells/well) with 1 ml of MEM growth medium in each well at 37°C, 5%
C02, and a 100% humidity environment for 24 h. The medium included 100 U/ml
penicillin, 100 pg/ml streptomycin, 1 mM MEM sodium pyruvate solution, IX MEM
amino acids solution, and 10% heated fetal bovine serum.
Experimental procedures
Either 37.5 nmole of the liposome (DOTAP or DOTAP-dodecyl 2-(l’-imidazolyl)
propionate (DIP) at molar ratio 0.3 (R=0.3)) was complexed with 0.25 nmole of the
FITC-oligonucleotide or 20 pg of the liposome with 1 pg of the plasmid DNA in each
well for 30 min. The charge ratio (+/-) of DOTAP to oligonucleotide was set to be 10
which was proposed as the optimal ratio in other similar study (Zelphati & Szoka,

88
1996a). The charge ratio (+/-) of DOTAP to plasmid DNA was also set to be 10 to
exclude the possible charge interference when these two types of nucleic acid were
compared. The liposome-nucleic acid complex was added into 500 pi of serum free
MEM growth medium after the MEM growth medium in each well was removed. After 4
h of incubation, the serum free MEM growth medium including the liposome-nucleic
acid complex was discarded and replaced with 1 ml of fresh growth medium. The cells
were harvested at various time points and analyzed by flow cytometry.
In the second part of the experiments, all three BPS (DIP, methyl 1-imidazolyl
laureate (MIL), and N-dodecyl imidazole (DI)) were incorporated into cationic DOTAP
liposomes at four molar ratios (R=0, R=0.1, R=0.2, and R=0.3). After a 30-min period of
complexation, either 37.5 nmole of the liposome with 0.25 nmole of the fluorescein-
labeled oligonucleotide or 20 pg of the liposome with 1 pg of the fluorescein-labeled
plasmid DNA with at a charge ratio of 10 (+/-) was incubated with CV-1 cells in 500 pi
of serum free growth medium. After 4 h, the cells were harvested and then analyzed by
flow cytometry.
Flow cytometry
In order to minimize the nucleic acids adsorbed onto cells, the cells were washed
twice with phosphate buffered saline (PBS) and lifted from the wells before analyzing the
samples. The cells were then transferred to tubes and centrifuged at 1,200 rpm for 5 min.
The supernatant (800 pi) was decanted and resuspended with 800 pi of PBS. The above
procedure was repeated twice and the samples kept on ice until analysis. To address

89
nucleic acid cellular uptake in some studies, rather than using PBS in the final step, 2%
formaldehyde was added into the tubes for 30 min to fix the cells.
The signals emitted from the FITC-oligonucleotide (or fluorescein-plasmid DNA)
were performed by a Becton Dickinson FACSort flow cytometer (San Jose, CA). Green
fluorescence was monitored with a 530/30 nm bandpass filter, and photomultiplier tube
pulses were amplified logarithmically. Ten thousand cells were counted at a flow rate
between 100 and 200 cells per second. Cells were gated with their morphological
properties, forward scatter and side scatter, set on logarithmic mode. The mean
fluorescence intensity of the related populations of cells was calculated using histograms
and expressed in arbitrary units corresponding to an intensity channel number ranging
from 0 to 1,023 using a LYSYS II program (Becton Dickinson; San Jose, CA).
Oligonucleotide Cellular Uptake and Distribution Evaluation with Confocal
Microscopy
Oligonucleotide synthesis and liposome preparation
Fifteen bases of poly-A phosphorothioate oligonucleotides labeled with
fluorescein isothiocyanate (FITC) at the 5’-end were synthesized in the DNA Core
Synthesis Lab at the University of Florida. They were used directly without additional
purification.
Two different lipid formulations were used: L-a-lecithin and L-a-lecithin with
dodecyl 2-(l ’-imidazolyl) propionate (DIP) at molar ratio 0.3 (R=0.3). The lipid
rehydration method was used to form neutral liposomes as FITC-labeled oligonucleotides
dissolved in the aqueous solvent. To increase the encapsulation efficiency of

90
oligonucleotides into the liposomes, five freeze-and-thaw cycles were employed after a
30-min period of hand shaking.
The liposomes were then passed three times through 600-nm polycarbonate
membranes (Poretics; Livermore, CA) using a high pressure extruder (Lipex
Biomembrane Inc.; Vancouver, Canada). The concentration of the phospholipid was
calculated as previously reported (Chapter 4). The volume-weight Gaussian distribution
of the liposome size was determined to be 630+265 nm (standard deviation) by a dynamic
light scattering method using a NICOMP 380 ZLS Zeta Potential/Particle Sizer (Santa
Barbara, CA).
Cell preparation
Before plating RAW 264.7 (mouse monocyte-macrophage) cells, a cover slip was
placed in each well of 12-well plates for later observation on a confocal microscope.
Each well containing a cover slip was treated with 300 pi of collagen in a 0.02 M acetic
acid solution (50 pg/ml) and incubated at room temperature. After 1 h, the solution was
removed by rinsing with phosphate buffered saline (PBS) three times. The plates were
then ready for use.
Subconfluent monolayered RAW 264.7 cells were cultured in the 12-well plates
(2*105 cells/well) with 1 ml of DMEM growth medium in each well at 37°C, 5% C02,
and a 100% humidity environment for 24 h. The medium included 100 U/ml penicillin,
100 pg/ml streptomycin, and 10% fetal bovine serum.

91
Experimental procedures
After the incubation, the growth medium in each well was replaced with 500 pi of
serum free medium containing same amount of starting FITC-oligonucleotides as the
following preparations.
• FITC-oligonucleotide
• FITC-oligonucleotide+liposome (100 nmole)
• FITC-oligonucleotide+DIP-liposome (R=0.3) (120 nmole)
Confocal microscopy
After 4 h of incubation in serum free medium, 10% fetal bovine serum was added.
At the end of each sampling time (4 h, 8 h, and 24 h), the wells were washed with PBS
and cells fixed in 2% formaldehyde for 30 min. The cover slips were transferred to the
slides with Gel/Mount. Cellular uptakes and distributions of the oligonucleotides were
then viewed and compared.
Cells incubated with FITC-labeled oligonucleotides were imaged using a Biorad
MRC-600 laser scanning confocal microscope equipped with a krypton/argon laser at the
Center of Structural Biology, University of Florida. Images were collected on an
Olympus IMT-2 inverted microscope using the 488/568 nm line at which the excitation
light was attenuated with a 1 % neutral density filter to minimize photobleaching and
photodamage.

92
Quantitative Effect Evaluation of Luciferase Activity
Oligonucleotide synthesis and liposome preparation
Phosphorothioate oligonucleotides with an antisense sequence (15 bases; 5’-TGG
CGT CTT CCA TTT-3’) and a sense sequence (15 bases; 5’-TTT ACC TTC TGC Gel¬
s’) were synthesized in the DNA Core Synthesis Lab at the University of Florida. They
were used directly without further purification.
Since molar ratio did not play a significant role in characterizing oligonucleotide
cellular uptake induced by BPS-liposomes, to minimize cytotoxicity associated with DIP,
a molar ratio 0.1 of DIP to DOTAP (R=0.1) was used in our subsequent biological
evaluation study. A cationic lipid, N-[l-(l-2,3-dioleoloxy)propyl]-N,N,N-
trimethylammonium methylsulfate (DOTAP) with or without dodecyl 2-(l ’-imidazolyl)
propionate (DIP) were mixed to create liposomes. After liposomes were rehydrated, as in
the previous set of experiment, a Sonic Dismembrator 60 probe was used to form small
unilamellar vesicles (30-70 nm) and size distribution of the liposomes confirmed by a
NICOMP 380 ZLS Zeta Potential/Particle Sizer (Santa Barbara, CA).
Cell preparation
Subconfluent CV-1 luciferase expressing cells were grown in 24-well plates
(2*105 cells/well) with 1 ml of MEM growth medium in each well at 37°C, 5% C02, and
a 100% humidity environment for 24 h. The medium included 100 U/ml penicillin, 100
Lig/'ml streptomycin, 1 mM MEM sodium pyruvate solution, IX MEM amino acid
solution, and 10% heated fetal bovine serum.

93
Experimental procedures
For each lipid formulation (DOTAP or DIP-DOTAP (R=0.1)), there were four
treatments to investigate the effect which DIP might have on inhibiting enzyme
expression.
• Antisense oligonucleotide+liposome
• Sense oligonucleotide+liposome
• Antisense oligonuleotide
• Liposome
Before adding oligonucleotides into wells, various concentrations of the liposome
were complexed with the oligonucleotide at a charge ratio of 10 (+/-) for 30 min. The
growth medium in each well was removed and the liposome-oligonucleotide complex
added into 500 pi of serum free growth medium. After 4 h of incubation, the growth
medium including the liposome-oligonucleotide complex was discarded and replaced
with new growth medium containing 10% fetal bovine serum. The system was incubated
for another 20 h before the final analysis of luciferase activity.
Luciferase assay
The growth medium was removed and the cells washed with 1 ml of pH 7.4
phosphate buffered saline (PBS). After removing PBS, cells were lysed with 100 pi of
pH 7.8 luciferase lysis buffer (0.1M dihydrogen potassium phosphate, 2 mM EDTA, 1%
Triton X-100, and 1 mM dithiothreitol (DTT)). The plate was then shaken gently for 15
min. The lysates were transferred into 1.5-ml tubes and centrifuged at 14,000 rpm for 5
min. A mixture of 20 pi of the supernatant and 100 pi of the pH 7.8 luciferase assay

94
buffer (30 xnM tricine, 3 mM adenosine triphosphate (ATP), 15 mM magnesium sulfate,
and 10 mM DTT) were added to a 100-pl injection of 1 mM D-luciferin (pH 6.1-6.5).
Luciferase activity of the samples was measured with a Monolight 2010 Luminometer.
The luminometer read light production for 10 s at room temperature.
Protein assay
To standardize cell numbers in each well of the cluster plate, the total amount of
protein was quantified using a BCA Protein Assay Kit. Briefly, 100 pi of PBS was
placed in each well of a 96-well plate. A standard calibration curve was constructed for
each assay using bovine serum albumin. The supernatant of the lysate (10 pi) from each
well was placed in the appropriate well of the 96-well plate and mixed gently. The BCA
reagent (100 pi) was then added to each well. The plate was incubated at 37°C for 30 min
and read at 630 nm on an EL 340 Bio Kinetics Reader. The amount of protein in each
well was then calculated by substituting the absorbent to the calibration curve created in
the same plate.
Statistical Analysis
Statistical differences between the treatments were determined using analysis of
variance where appropriate (StatView 4.53, Abacus Concepts, Inc., Berkeley, CA) with
p<0.05 considered statistically significant and Fisher’s (PLSD) post hoc t-test was
applied.

95
Results
Oligonucleotide and Plasmid DNA Cellular Uptake Evaluation Using Flow
Cytometry
To examine the effect of biodegradable pH-sensitive surfactants (BPS) in the
cationic N-[l-(l-2,3-dioleoloxy)propyl]-N,N,N-trimethylammonium methylsulfate
(DOTAP) liposomes on oligonucleotide (or plasmid DNA) cellular uptake, a flow
cytometry technique was used (Figure 5-1). Fluorescein isothiocyanate (FITC)-labeled
oligonucleotides (or fluorescein-labeled plasmid DNA) were complexed with cationic
DOTAP liposomes plus or minus dodecyl 2-(l ’-imidazolyl) propionate (DIP) at molar
ratio 0.3 (R=0.3) for 30 min. The complex was then incubated with CV-1 cells, and the
fluorescence intensity emitted from the oligonucleotides was recorded at various time
points with or without cell fixation.
The fluorescence intensities in both types of cells (fixed or live) from the free
oligonucleotide were significantly lower than those from the oligonucleotide with
delivery systems at all observed time points (Figure 5-2). Also, the fluorescence signals
from DIP treated liposomes in live cells at early time points (4 h and 5 h) were
significantly (p<0.001) higher than those from liposomes without DIP (Figure 5-2a).
When the cells were fixed to equalize the intracellular compartments with regard to pH,
no difference in the fluorescence signal was seen between these two groups (Figure 5-2b).
Oligonucleotide cellular uptake caused by three BPS (DIP, methyl 1-imidazolyl
laureate (MIL), and N-dodecyl imidazole (DI)) at different molar ratios (R=0, R=0.1,
R=0.2, and R=0.3) was further investigated. After 4 h of incubation in the live CV-1

96
OI
'ai
I
s
O
m
m
o
(O
o
io
CM i
O
O Ӓ
CM
9 ’
(O
o ^
o
***
UD *
#•
* * *.
if m w
w* ,
*21
' *
i
IM
y
**'* Hjff*.
* #’
Iw
*»
* *
- * .**
* f *5 ,
; rh f * * i-* J M *
v , « ■* •p- i r ##4*
■« * * fca> # * ,**# **B*
Jw * í ¿i*4** lija
r vv >*í£¡í|P
' ' * ■ ,. •** 1
J* tw fW*»’ -
**. !***'â– '
»%*•£?* »
V,V*v«¥ -
â–¼ â– *
i» f
vk-; ^v í
* ¿••Ai*’.*: ,
;wv*//, *
tf* ;íw«
Vf
."5 J* " „ * «
+ #*
fW** 4 J,
" * \ *
■rTfum1»*1!" i nregr^yw imir ,,>*’,fw wi;
10° 101 10
FL1-H\FL1-Height
1
10'
10
Figure 5-1: Distribution of the live CV-1 cells versus the fluorescence intensity measured
by flow cytometry. Before analyzing the fluorescence signals, 0.25 nmole of the
fluorescein-labeled oligonucleotide complexed with N-[l-(l-2,3-dioleoloxy)propyl]-
N,N,N-trimethylammonium methylsulfate (DOTAP) liposomes at a charge ratio of 10
(+/-) were incubated in the well for 4 h. The relative fluorescence intensity of the first
10,000 cells was calculated to be 884.

97
(a)
Figure 5-2: Time course study of cationic liposomes on fluorescein-labeled
oligonucleotide cellular uptake in CV-1 cells (n=3). Oligonucleotide (0.25 nmole)
without (A) and with a delivery system (i.e., N-[l-(l-2,3-dioleoloxy)propyl]-N,N,N-
trimethylammonium methylsulfate (DOTAP) liposomes plus (■) or minus (♦) dodecyl
2-(l’-imidazolyl) propionate (DIP) with molar ratio 0.3 (R=0.3) at a charge ratio of 10
(+/-)) were compared. The fluorescence intensities (meantstandard deviation (SD)) were
recorded by flow cytometry in two cell conditions, a) Live CV-1 cells; b) Fixed CV-1
cells.

98
1800
(b)
Figure 5-2—continued

99
cells, significant differences (p<0.001) in the fluorescence intensity were observed
between the DIP treated liposome groups (R=0.1, R=0.2, and R=0.3) and blank
liposomes (Figure 5-3a). However, no difference in the fluorescence intensity was seen
among the DIP treated liposome groups. When CV-1 cells were fixed, no difference in
the fluorescence intensity was observed among all liposome groups (Figure 5-3a).
Similar fluorescence intensity profiles were also seen in the other two BPS (MIL and DI)
treated liposomes among all molar ratio groups (R=0, R=0.1, R=0.2, and R=0.3) in both
live and fixed cells (Figure 5-3b and Figure 5-3c). In addition, no difference of the
fluorescence intensity was observed among these three BPS at any molar ratio groups.
WTien FITC-oligonucleotides were replaced by fluorescein-plasmid DNA, no
difference in the fluorescence intensity between DOTAP liposomes and DIP treated
DOTAP liposomes (R=0.3) was observed throughout the entire time course in the live
CV-1 cells (Figure 5-4a). When the cells were fixed before analysis, no difference of the
fluorescence signal was seen (Figure 5-4b).
Plasmid DNA cellular uptake caused by three BPS (DIP, MIL, and DI) at
different molar ratios (R=0, R=0.1, R=0.2, and R=0.3) was also investigated. After 4 h of
incubation in both live and fixed CV-1 cells, no difference in the fluorescence signal was
seen among all DIP-liposome groups (R=0, R=0.1, R=0.2, and R=0.3) (Figure 5-5a).
Similar fluorescence intensity profiles were also seen in the other two BPS-liposome
groups (MIL and DI) in both the live and fixed cells (Figure 5-5b and Figure 5-5c).

100
(a)
Figure 5-3: Effect of cationic N-[l-(l-2,3-dioleoloxy)propyl]-N,N,N-trimethylammonium
methylsulfate (DOTAP) liposomes containing three biodegradable pH-sensitive
surfactants (BPS) at various molar ratios (R=0 (open bar), R=0.1 (solid bar), R=0.2
(vertical bar), and R=0.3 (horizontal bar)) on fluorescein-labeled oligonucleotide cellular
uptake in CV-1 cells (n=3). Oligonucleotides (0.25 nmole) were complexed with
liposomes at a charge ratio of 10 (+/-). The fluorescence signals (mean±SD) were
recorded by flow cytometry after 4 h of incubation while the cells were either alive or
fixed, a) Dodecyl 2-(l’-imidazolyl) propionate (DIP); b) Methyl 1-imidazolyl laureate
(MIL); c) N-dodecyl imidazole (DI).

101
Fixed Cells

102
Figure 5-3—continued
(c)

103
(a)
Figure 5-4: Time course study of cationic liposomes on fluorescein-labeled plasmid DNA
cellular uptake in CV-1 cells (n=3). Plasmid DNA (1 pg) without (A) and with a carrier
system (i.e., N-[l-(l-2,3-dioleoloxy)propyl]-N,N,N-trimethylammonium methylsulfate
(DOTAP) liposomes plus (■) or minus (♦) dodecyl 2-(l ’-imidazolyl) propionate (DIP)
with molar ratio 0.3 (R=0.3) at a charge ratio of 10 (+/-)) were compared. The
fluorescence signals (mean+SD) were recorded by the flow cytometry in two cell
conditions, a) Live CV-1 cells; b) Fixed CV-1 cells.

104
(b)
Figure 5-4—continued

105
160
140 -
120 -
100
>
«s-s
'1 80 -
cu
- 60
40
20 -
0
i
Figure 5-5: Effect of cationic N-[l-(l-2,3-dioleoloxy)propyl]-N,N,N-trimethylammonium
methylsulfate (DOTAP) liposomes containing three biodegradable pH-sensitive
surfactants (BPS) at various molar ratios (R=0 (open bar), R=0.1 (solid bar), R=0.2
(vertical bar), and R=0.3 (horizontal bar)) on fluorescein-labeled plasmid DNA cellular
uptake in CV-1 cells (n=3). Plasmid DNA (1 pg) were complexed with liposomes at a
charge ratio of 10 (+/-). The fluorescence signals were recorded by flow cytometry after
4 h of incubation with live or fixed cells, a) Dodecyl 2-(l ’-imidazolyl) propionate (DIP);
b) Methyl 1-imidazolyl laureate (MIL); c) N-dodecyl imidazole (DI).

106
160
140 -
120 -
100 -
1 80 -
(V
+-•
- 60 -
40
20
Figure 5-5—continued

107
Figure 5-5—continued
(c)

108
Oligonucleotide Cellular Uptake and Distribution Evaluation with Confocal
Microscopy
Using confocal microscopy, the effect of neutral L-a-lecithin liposomes
containing dodecyl 2-(l’-imidazolyl) propionate on oligonucleotide cellular delivery was
further investigated. There is a direct correlation between cellular associated
oligonucleotides and the intensity of photograph. Without any delivery carrier (e.g.,
liposomes), there was little oligonucleotide associated with the cells at three observed
time points (Figure 5-6). Liposomes minus (Figure 5-7) or plus (Figure 5-8) DIP at
molar ratio 0.3 (R=0.3) were used, more oligonucleotides were taken into the cells as
compared to those without delivery systems at all observed time points.
The cellular distributions of the oligonucleotides once brought into the cells
through two delivery systems (i.e., liposomes and DIP-liposomes (R=0.3) liposomes)
were subsequently compared. For those oligonucleotides that were taken into cells
through L-a-lecithin liposomes, perinuclear localization of the oligonucleotides was seen
in the observed time points (Figure 5-7). When DIP was added into the neutral liposomes
(R=0.3), much well dispersion of the oligonucleotides in the cells was observed (Figure
5-8).
Quantitative Effect Evaluation of Luciferase Activity
To investigate the impact on oligonucleotide activity of dodecyl 2-(l ’-imidazolyl)
propionate (DIP) in cationic N-[l-(l-2,3-dioleoloxy)propyl]-N,N,N-trimethylammonium
methylsulfate (DOTAP) liposomes, a CV-1 luciferase expressing cell line was used.

109
Figure 5-6: Cellular uptake/distribution of fluorescein-labeled oligonucleotides in RAW
264.7 cells. Cells were observed by confocal microscopy at three time points, a) 4 h; b)
8 h; c) 24 h.

110

Ill
(C)
Figure 5-6—continued

112
Figure 5-7: Cellular uptake/distribution of fluorescein-labeled oligonucleotides when
encapsulated in the lecithin liposomes in RAW 264.7 cells. Cells were observed by
confocal microscopy at three time points, a) 4 h; b) 8 h; c) 24 h.

113
Figure 5-7—continued

114

115
(a)
Figure 5-8: Cellular uptake/distribution of fluorescein-labeled oligonucleotides when
encapsulated in the lecithin liposomes containing dodecyl 2-(l’-imidazolyl) propionate
(DIP) at molar ratio 0.3 (R=0.3) in RAW 264.7 cells. Cells were observed by confocal
microscopy at three time points, a) 4 h; b) 8 h; c) 24 hr.

116

117
Figure 5-8—continued

118
After 24 h of incubation, the percentages of inhibition (luciferase activity) were
determined. The positive control (0% inhibition) referred to no treatment in the cells
while the negative control (100% inhibition) was the background interference. Other
appropriate controls including sense oligonucleotide+liposome, antisense
oligonucleotide, and liposome were also used.
No inhibition of luciferase expression was detected at the observed concentration
of free antisense oligonucleotides (Figure 5-9a). However, when (antisense or sense)
oligonucleotide-DOTAP liposome complex was used, greater inhibitions of luciferase
activity were observed (Figure 5-9a). As the concentration of (antisense or sense)
oligonucleotide reached 0.5 pM (0.25 nmol/0.5 ml), no extra inhibition was seen.
Significant differences (p<0.005) in the percentage of inhibition between antisense and
sense oligonucleotides were obtained when the concentration of oligonucleotide was 0.5
pM (0.25 nmol/0.5 ml) or higher.
When DIP at molar ratio 0.1 (R=0.1) was added to the cationic liposomes, similar
profiles were noticed (Figure 5-9b). Comparing the luciferase inhibition effect
throughout the entire observed concentration range, more inhibition (p<0.005) was seen
when antisense oligonucleotides were complexed with the DIP treated (R=0.1) DOTAP
liposomes than when antisense oligonucleotides were complexed with the DOTAP
liposomes. A same phenomenon was also observed when antisense oligonucleotides
were replaced by sense oligonucleotides between DIP-DOTAP liposomes and DOTAP
liposomes.

119
(a)
Figure 5-9: Effect of oligonucleotide activity when complexed with two cationic
liposomes of different lipid compositions at a charge ratio of 10 (+/-) on CV-1 luciferase
expressing cells (n=4). The percentage of inhibition on luciferase activity (meaniSD) in
each treatment (antisense oligonucleotide+liposome (♦), sense oligonucleotide+liposome
(â– ), antisense oligonucleotide (A), and liposome (X)) was calculated and plotted against
oligonucleotide concentration after 24 h of incubation. It should be noted that for the
liposome subset experiment (X), the data indicated that the inhibition effect was the
same for the amount of liposome used in the antisense oligonucleotide+liposome subset
experiment (♦) but no oligonucleotides were used, a) N-[l-(l-2,3-dioleoloxy)propyl]-
N,N,N-trimethylammonium methylsulfate (DOTAP); b) DOTAP containing dodecyl 2-
(l’-imidazolyl) propionate (DIP) at molar ratio 0.1 (R=0.1).

120
70
Figure 5-9

121
Discussion
After characterizing these three biodegradable pH-sensitive surfactants (BPS),
their biological effects in vitro were further evaluated. Studies were initially conducted to
investigate the impact of BPS in cationic liposomes on oligonucleotide (or plasmid DNA)
cellular uptake in terms of incubation time and the molar ratio. Increased fluorescence
signals were observed during the early time points when the oligonucleotides were
delivered in the live CV-1 (monkey kidney fibroblast) cells by dodecyl 2-(l’-imidazolyl)
propionate (DIP) treated liposomes compared to control liposomes. Since the
oligonucleotide labeling material, fluorescein isothiocyanate (FITC), is a pH-sensitive
fluorophore, it demonstrates higher fluorescence intensity in base than in acid. The
results indicated that with the addition of DIP, the liposomes could induce either higher
oligonucleotide cellular uptake or different oligonucleotide intracellular distribution (i.e.,
from endosóme to cytoplasm).
When the cells were fixed with formaldehyde to equalize pH intracellularly, no
difference in the fluorescence signal was observed over the entire time course between
these two types of liposomes. This set of experiments ruled out that the difference seen
in intensity was a result of total amount of oligonucleotide entering the cells. Instead,
BPS-liposomes could redistribute oligonucleotides once brought into cells. It suggests
similar oligonucleotide cellular uptake but possible transport of oligonucleotides to a
more acidic environment (e.g., lysosome) with liposomes in the absence of BPS and a
more basic surrounding (e.g., endosóme or cytoplasm) with liposomes in the presence of
BPS.

122
When increasing molar ratios of BPS were used in the same study, greater signals
from the oligonucleotides were observed after 4 h of incubation in the live cells with BPS
treated liposomes than with regular liposomes. Moreover, when the cells were fixed, no
difference in the signal from the oligonucleotides was detected. However, no correlation
between either three BPS or molar ratios and the fluorescence intensity could be
established. Further detailed examinations regarding the selection of BPS, sampling
time, cell type, oligonucleotide concentration, and charge ratio need to expand to
distinguish this relationship.
To further investigate the size of membrane pore that BPS could create when they
destabilized endosomal membrane to release the endocytosed materials, a macromolecule
plasmid DNA was used. From our studies, BPS seemed unable to improve the delivery
of plasmid DNA at either the observed time course or at any molar ratio of BPS to
liposomes. One explanation is that the membrane pore promoted by BPS was not large
enough to rupture the endosomal membranes and transfer the plasmid DNA into the
cytoplasm but could only leak out smaller materials such as oligonucleotide. The reason
for this occurrence is possibly due to the relatively big molecular size of plasmid DNA
(5256 bp) compared to oligonucleotide (15 b). The double strand structure of plasmid
DNA may also hinder its delivery. Moreover, the different backbone of the plasmid
DNA (i.e., phosphorodiester) and the oligonucleotide (i.e., phosphorothioate) in our
studies relating to their respective stability can also change their efficiency. However, the
use of BPS to increase the activity of plasmid DNA has been shown in a previous study
(Liang & Hughes, 1998). As a result, some other mechanism may be involved in BPS-

123
induced plasmid DNA delivery. It is also possible that our experiments were not
sensitive enough to notice the furtive difference.
In addition, the effect of BPS in the liposomes on oligonucleotide cellular
uptake/distribution was assessed. To elude the possible cell line dependent factor on
oligonucleotide delivery, RAW 264.7 (mouse monocyte-macrophage) cells were used.
Moreover, instead of cationic liposomes, DIP was added into different type of neutral
liposomes. It was shown that oligonucleotides that had no delivery carrier were
inefficiently internalized into the cells. When the liposomes were used to deliver the
oligonucleotides, more oligonucleotides were brought into the cells. Nevertheless, they
were localized in the punctuate cytoplasmic spots probably corresponding to their
presence in the endocytotic compartments (endosóme or lysosome) and indicating a
possible lysosomal degradation fate (Vlassov et al., 1994).
In contrast, cells exposed to the BPS-liposomes containing FITC-labeled
oligonucleotides demonstrated diffuse fluorescence supporting their presence in the
cytoplasm. This indicates a possible oligonucleotide cellular pathway into the cytoplasm
or nucleus. As a result, the intra-delivery of the oligonucleotide was improved.
In the final set of experiments, the ability of BPS-liposomes to aid
oligonucleotides in inhibiting the luciferase activity was evaluated quantitatively. To
accurately calculate the amount of oligonucleotide in each treatment, cationic liposomes
were chosen over neutral counterparts. As cationic N-[l-(l-2,3-dioleoloxy)propyl]-
N,N,N-trimethylammonium methylsulfate (DOTAP) liposomes were used as an
oligonucleotide delivery system, additional luciferase activity was restrained in the
observed concentration range than oligonucleotides without the delivery system. When

124
dodecyl 2-(l’-imidazolyl) propionate (DIP) was added as a part of the liposome
composition to deliver the oligonucleotides, an even greater amount of enzyme
expression was inhibited than liposomes without DIP.
Moreover, not only could DIP enhance the efficacy of oligonucleotides in
hampering the synthesis of luciferase, but it could also increase oligonucleotide potency
(i.e., more inhibition at a fixed concentration or same inhibition with a lower amount of
oligonucleotide). This fact implies the usefulness of BPS in enhancing the inhibition of
luciferase expression. However, when only the delivery systems were used, the
inhibitions were also elevated as more liposomes were employed, suggesting a possible
cytotoxic interference of luciferase expression from the cationic DOTAP liposomes and
the DIP-DOTAP liposomes.
In summary, we have showed that with help from a carrier (cationic liposomes),
greater enzyme activities were inhibited by the oligonucleotides. This observation was in
accordance with the other comparable studies (Capaccioli et al., 1993; Takle et al., 1997).
Similar effects were also seen when other cationic liposomes were used (Bennett et al.,
1992; Konopka et al., 1996; Lappalainen et al., 1997; Ollikainen et al., 1996). With the
addition of biodegradable pH-sensitive surfactants (BPS) into the cationic liposomes,
more dramatic increase of the inhibition on luciferase activity by the oligonucleotides was
observed than the cationic liposomes without BPS.
We have also identified that the mechanism of BPS to increase the delivery of
oligonucleotide was not by increasing oligonucleotide cellular uptake in CV-1 cells.
Once oligonucleotides were brought into the intracellular compartment, they were
redistributed by promoting the formation of a small membrane pore. To better

125
characterize the oligonucleotide delivery under the effect of BPS in different RAW 264.7
cells, confocal microscopy was used. The results corroborated that liposomes induced
more cellular uptake of oligonucleotides than that of free oligonucleotides as displayed in
the flow cytometry experiment set. The results confirmed that the oligonucleotide
cellular distribution was ameliorated when BPS were a part of the liposome composition.
Conclusion
The use of liposomes in the delivery of oligonucleotides is still greatly limited by
poor cell membrane permeability ability (Zelphati & Szoka, 1996a). Biodegradable pH-
sensitive surfactants (BPS) were developed and used to enhance the cellular delivery of
nucleic acids. Previous reports (Hughes et al.s 1996; Liang & Hughes, 1998) and current
studies have been shown their potential use. Nevertheless, there is plenty of space to
refine the entire BPS delivery system.
To further optimize the use of BPS, a number of factors need to be considered.
Oligonucleotide cellular delivery by BPS-cationic liposomes relies on a number of
variables including the liposome composition (Bennett et al., 1995; Zhou & Huang,
1994), charge ratio (Arima et ah, 1997; Jaakselainen et ah, 1994; Zelphati & Szoka,
1996a), incubation period (Zelphati & Szoka, 1996a), and presence of serum (Feigner et
ah, 1987). By modulating any one of the above variables, the cellular delivery of the
oligonucleotides by BPS-liposomes can be strongly affected and eventually be enhanced.

CHAPTER 6
MECHANISM OF ACTION INVESTIGATION OF BIODEGRADABLE pH-
SENSITIVE SURFACTANTS
Introduction
Dodecyl 2-(l’-imidazolyl) propionate (DIP), a member of biodegradable pH-
sensitive surfactants (BPS), has been previously demonstrated to increase cytoplasm
delivery of oligonucleotide (Hughes et al., 1996) and plasmid DNA (Liang & Hughes,
1998). It has already been proved that the elevated effect from BPS-liposomes was not to
increase the cellular uptake of oligonucleotides but to redistribution once
oligonucleotides were brought into cells (Chapter 5). However, it remained unclear how
BPS destabilized the endosomal membrane and released the content out of endosomes.
To further utilize and modify BPS in intracellular macromolecule delivery, it is essential
to understand the mechanisms responsible for membrane destabilization. In addition to
DIP, two other BPS, methyl 1-imidazolyl laureate (MIL) and N-dodecyl imidazole (DI),
were used to investigate possible mechanisms.
Instead of serving as a nucleic acid delivery system, liposomes were used as the
experimental membrane model to study the possible membrane destabilization
mechanisms. In order to maintain simplicity, only neutral lipids (e.g., lecithin) were used
in the liposomal membrane system. While the liposomes may not fully represent events
occurring in biological situations they still served as excellent models in addressing
126

127
potential mechanisms of lipid membrane disruption. An aqueous space fusion assay
(Smolarsky et al., 1977) and a lipid mixing resonance energy transfer assay (Struck et al.,
1981) were used to observe membrane destabilization through fusion. A liposome
leakage assay of a marker compound, calcein, was used to observe membrane
destabilization through rupture.
Materials
Chemical
Calcein was purchased from Aldrich (Milwaukee, WI). N-(lissamine rhodamine
B sulfonyl)-phosphatidylethanolamine (Rh-PE), N-(7-nitro-2,l,3-benzoxadiazol-4-yl)-
phosphatidylethanolamine (NBD-PE), L-a-lecithin, l,2-dimyristoyl-sn-glycero-3-
phosphocholine (DMPC), dioleoylphosphatidylethanolamine (DOPE), and cholesterol
were purchased from Avanti Polar Lipids (Alabaster, AL). N, N’-p-xylylenebis-
(pyridinium bromide) (DPX) and l-aminonaphthalene-3,6,8-trisulfonic acid (ANTS)
were purchased from Molecular Probes (Eugene, OR). Three biodegradable pH-sensitive
surfactants (BPS) including dodecyl 2-(l’-imidazolyl) propionate (DIP), methyl 1-
imidazolyl laureate (MIL), and N-dodecyl imidazole (DI) were synthesized as previously
reported (Chapter 3). All purchased or obtained chemicals were used directly without
further purification.

128
Buffer Preparation
All buffers were adjusted with NaCl to an equal ionic strength. The pH of the
buffers and their chemical compositions were as follows: pH 4.2 (150 mM sodium acetate
and 350 mM glacial acetic acid), pH 5.0 (300 mM KH2P04 and 50 mM Na,HP04), pH 6.0
(150 mM KH2P04 and 100 mM Na2HP04), pH 6.5 (120 mM KH2P04 and 100 mM
NajHPOJ, pH 7.0 (80 mM KH2P04 and 120 mM N^HPOJ, and pH 8.0 (200 mM
KH2P04 and 188 mM NaOH).
Liposome Preparation
Liposomes (L-a-lecithin: l,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC):
cholesterol; molar ratio 6:1:8) were prepared and used in most experiments to evaluate
fusion and rupture events. The lipid rehydration method was used to produce vesicles
(Hughes et al., 1994). The liposomes were passed through a high pressure extruder
(Lipex Biomembrane Inc.; Vancouver, BC) with 600 nm polycarbonate membranes three
times. The size of the liposomes (volume-weight Gaussian distribution) was determined
to be 578+113 nm (standard deviation) with a NICOMP 380 ZLS Zeta Potential/Particle
Sizer (Santa Barbara, CA). The concentration of total phospholipids was determined by a
spectrophotometric technique as previously described (Chapter 4).

129
Methods
Time Course Fusion Assay
Increasing molar ratios (R=0, R=0.2, and R=0.4) of biodegradable pH-sensitive
surfactants (BPS) to 75 nmol/ml of liposome (L-a-lecithin: l,2-dimyristoyl-sn-glycero-3-
phosphocholine (DMPC): cholesterol; molar ratio 6:1:8) were used to monitor fusion
mechanism over time in a pH 5.0 buffer solution. Liposome-liposome fusion was
characterized by measuring fluorescence resonance energy transfer between two lipid
head groups (Struck et al., 1981). N-(7-nitro-2,l,3-benzoxadiazol-4-yl)-
phosphatidylethanolamine (NBD-PE) and N-(lissamine rhodamine B sulfonyl)-
phosphatidylethanolamine (Rh-PE) were incorporated into two separate populations of
vesicles at 1% (mol) each. As the two liposome groups interacted, the fluorescence
energy emitted from NBD-PE labeled liposomes was transferred to the Rh-PE labeled
liposomes resulting in a decreased fluorescence signal.
The NBD-PE liposomes were initially added into various pH buffers with the Rh-
PE liposomes and fluorescence intensity measured over a 30-min incubation period at
room temperature. The liposomal suspensions were excited at a wavelength of 470 nm
and observed at 530 nm with a Perkin-Elmer Luminescence Spectrophotometer LS-50B.
The percentage of fusion was defined by the following relationship:
F ...
Fusion{%) = (1 ) * 100, where F and F0 are the fluorescence intensities in the
Fo
presence and absence of the Rh-PE group, respectively (Struck et al., 1981).

130
To corroborate the membrane fusion study, an aqueous content mixing method
was used (Smolarsky et ah, 1977). In this assay, 25 mM of l-aminonaphthalene-3,6,8-
trisulfonic acid (ANTS) and 90 mM of N, N’-p-xylylenebis-(pyridinium bromide) (DPX)
were encapsulated into two separate liposomes (L-a-lecithin: DMPC: cholesterol; molar
ratio 6:1:8). Unencapsulated ANTS and DPX were later removed through centrifugation
(10,000 rpm, 5 min) five times and washed with a pH 7.4 phosphate buffered saline.
These two liposome populations (75 nmol/ml) were mixed and the fluorescence intensity
recorded over time at an excitation wavelength of 353 nm and an emission wavelength of
525 nm.
Mixing of the aqueous contents of ANTS and DPX containing liposomes resulted
in a decrease in fluorescence due to the quenching of ANTS by DPX. The percentage of
fusion was calibrated similarly with the above equation, where F is the fluorescence
intensity in the presence of DPX group and F0 is the fluorescence intensity in the absence
of DPX group in different pH buffer solutions.
Biodegradable pH-Sensitive Surfactants-Induced Membrane Fusion
Both aqueous and lipid mixing techniques were further applied to characterize the
liposome fusion induced by different molar ratios of biodegradable pH-sensitive
surfactants (BPS) after 30 min. Increasing molar ratios (R=0, R=0.2, and R=0.4) of BPS
were incorporated into 75 nmol/ml of liposomes (L-a-lecithin: 1,2-dimyristoyl-sn-
glycero-3-phosphocholine (DMPC): cholesterol; molar ratio 6:1:8) to monitor fusion

131
events with changing pHs (5.0-8.0). The fluorescence intensities were then quantified
after 30 min in both assays.
Biodegradable pH-Sensitive Surfactants-Induced Membrane Rupture
Liposomes (L-a-lecithin: 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC):
cholesterol; molar ratio 6:1:8, 75 nmol/ml) containing 100 mM calcein (> self-quenching
concentration) were prepared using increasing molar ratios of biodegradable pH-sensitive
surfactants (BPS) (R=0, R=0.1, R=0.2, and R=0.4). The liposomes were used to observe
whether membrane rupture occured. The BPS-liposome preparations were incubated
with phosphate buffers (pH 5.0-8.0) for 30 min and calcein release quantified. The
released calcein was excited at 496 nm and observed at 517 nm. The percentage of
released calcein was calculated by the equation /(%) =
7^*100% (Liu & Regen,
vLc *b)
1993). Ix is the 100% fluorescence intensity value when adding excess Triton X-100 (10
mM); Ia and Ib are the fluorescence intensities after incubation with and without BPS,
respectively.
Effects of Liposome Concentration on Membrane Fusion and Rupture
Increasing concentrations of liposomes (L-a-lecithin: 1,2-dimyristoyl-sn-glycero-
3-phosphocholine (DMPC): cholesterol; molar ratio 6:1:8) with biodegradable pH-
sensitive surfactants (BPS) at two molar ratio groups (R=0.2 and R=0.4) were used to
determine their dependency on membrane rupture (3 nmol/ml, 15 nmol/ml, and 75

132
nmol/ml) and fusion (37.5 nmol/ml, 75 nmol/ml, and 150 nmol/ml) in a pH 5.0 buffer
solution. Similar lipid mixing assay protocols were used as described above.
Impact of Cholesterol and Dioleoylphosphatidylethanolamine on Membrane Fusion
Liposomes (135 nmol/ml) containing L-a-lecithin, with or without cholesterol
(weight ratio 3:2; molar ratio 4:5) at two molar ratios (R=0.2 and R=0.4) of dodecyl 2-
(1 ’-imidazolyl) propionate (DIP) were used to determine the effect of the addition of
cholesterol on fusion. A similar study was conducted with cholesterol replaced by
dioleoylphosphatidylethanolamine (DOPE) (weight ratio 3:2; molar ratio 3:2). The
percentage of fusion using the lipid mixing assay was determined in different pH buffers
after 30 min.
To further characterize the difference of fusion events caused by cholesterol and
DOPE, liposomes (135 nmol/ml) with an equal molar ratio of cholesterol or DOPE to L-
a-lecithin were incorporated with DIP at molar ratio 0.4 (R=0.4). The percentage of
fusion using the lipid mixing assay was determined in different pH buffer solutions after
30 min.
Statistical Analysis
Statistical differences between the treatments were determined using analysis of
variance where appropriate (StatView 4.53, Abacus Concepts, Inc., Berkeley, CA) with
p<0.05 considered statistically significant and Fisher’s (PLSD) post hoc t-test was
applied.

133
Results
Time Course Fusion Assay
To identify possible events in the membrane destabilization sequence, fusion
assays were quantified over time with all three biodegradable pH-sensitive surfactants
(BPS)-liposome system using a pH 5.0 buffer. The percentage of membrane fusion
determined by the lipid mixing assay demonstrated a rapid initial event followed by a
plateau (Figure 6-1). On the other hand, for the aqueous mixing assay, the percentage of
fusion increased slightly in the early period and continued over the entire test time course
(Figure 6-1). For both assays, membrane fusion was significantly different (pO.OOl)
among all the molar ratio groups in each BPS over time. No significant difference in
fusion among the three agents was observed.
Biodegradable pH-Sensitive Surfactants-Induced Membrane Fusion
To investigate the fusogenic properties elicited by inclusion of biodegradable pH-
sensitive surfactants (BPS) on membranes, increasing amounts of three BPS were
incorporated into liposomes followed by incubation at different pHs (Figure 6-2). When
fusion was determined with the lipid mixing assay, throughout all pHs tested, the
percentage of fusion with the control liposomes (R=0) increased slightly as the pH
decreased. After incorporating BPS into the liposomes, the percentages of fusion were
statistically higher at all observed pHs. As the molar ratio of BPS in the liposomes was
raised, fusion was significantly increased (pO.OOl) from pH 6.0 to pH 5.0 (Figure 6-2).

134
Lipid Mixing Assay
Time (min)
Aqueous Mixing Assay
40 T
35 .
30
(a)
Figure 6-1: Time course fusion study of liposome (75 nmol/m]) containing different
biodegradable pH-sensitive surfactants (BPS) at three molar ratios (R=0 (♦), R=0.2 (■),
and R=0.4 (A)) in a pH 5.0 buffer solution. For the lipid mixing assay, NBD-PE labeled
liposomes were incubated with Rh-PE labeled liposomes while for the aqueous mixing
assay, liposomes containing 25 mM ANTS were incubated with liposomes containing 90
mM DPX (n=3). Data were expressed as mean+standard deviation (SD). a) Dodecyl 2-
(l’-imidazolyl) propionate (DIP); b) Methyl 1-imidazolyl laureate (MIL); c) N-dodecyl
imidazole (DI).

135
Lipid Mixing Assay
Aqueous Mixing Assay
40 T
35 -
30
(b)
Figure 6-1—continued

% Fusion % Fusion
136
Lipid Mixing Assay
Aqueous Mixing Assay
(c)
Figure 6-1—continued

137
Lipid Mixing Assay
4 5 6 7 8
pH
Aqueous Mixing Assay
Figure 6-2: Fusion study of liposomes (75 nmol/ml) with various biodegradable pH-
sensitive surfactants (BPS) at three molar ratios (R=0 (♦), R=0.2 (■), and R=0.4 (A)) in
different pHs. For the lipid mixing assay, NBD-PE labeled liposomes were incubated
with Rh-PE labeled ones while for the aqueous mixing assay, liposomes containing 25
mM ANTS were incubated with those containing 90 mM DPX (n=3). The percentages of
fusion (mean±SD) were determined after 30 min. a) Dodecyl 2-(l’-imidazolyl)
propionate (DIP); b) Methyl 1-imidazolyl laureate (MIL); c) N-dodecyl imidazole (DI).

138
Lipid Mixing Assay
4 5 6 7 8
pH
Aqueous Mixing Assay
4 5 6 7 8
pH
(b)
Figure 6-2—continued

139
Lipid Mixing Assay
4 5 6 7 8
pH
Aqueous Mixing Assay
Figure 6-2—continued

140
All three BPS exhibited a similar fusion profile but there was no significant difference
between the fusion events among the three agents.
For the fluorescence aqueous mixing method, the percentages of fusion by control
liposomes (R=0) demonstrated no significant difference at all tested pHs. After the
addition of BPS, the percentages of fusion increased at all pHs. As the molar ratio of
BPS was raised, fusion increased significantly (p<0.001) below pH 6.0 (Figure 6-2).
Comparable to the lipid mixing assay, these three BPS exhibited similar fusion profiles
with no significant difference among the evaluated BPS.
Biodegradable pH-Sensitive Surfactants-Induced Membrane Rupture
We determined the ability of liposome incorporated un-ionized biodegradable pH-
sensitive surfactants (BPS) to facilitate the release of entrapped solutes at acidic pHs.
Liposomes containing calcein were prepared with increasing amounts of BPS and
incubated at decreasing pHs. Minimal calcein release was observed at the lower
BPS/liposome molar ratio group (R=0.1), but at the R=0.4 group, calcein release
increased significantly (p<0.001) at all pHs (Figure 6-3). At pH 5.0 and 6.0, significant
differences (p<0.05) in the percentage of released calcein was observed among all molar
ratio groups. Comparing these three BPS, the percentage of release was significant
different (p<0.01) at the R=0.4 group in all observed pHs. However, no significant
difference was observed at the other two ratio groups (R=0.1 and R=0.2) among the three
BPS-liposomes.

141
4 5 6 7 8
pH
(a)
Figure 6-3: Membrane rupture profile with the incorporation of different biodegradable
pH-sensitive surfactants (BPS) at four molar ratios (R=0 (♦), R=0.1 (X), R=0.2 (■), and
R=0.4 (A)) in liposomes (75 nmol/ml). The membrane lysis effects in different pHs
were determined by calcein release (meaniSD) after 30 min (n=3). a) Dodecyl 2-(l
imidazolyl) propionate (DIP); b) Methyl 1-imidazolyl laureate (MIL); c) N-dodecyl
imidazole (DI).

142
4 5 6 7 8
pH
(b)
Figure 6-3—continued

143
4 5 6 7 8
PH
(C)
Figure 6-3—continued

144
Effects of Liposome Concentration on Membrane Fusion and Rupture
To quantify the liposome concentration dependency on membrane fusion and
rupture, the fusion and rupture events of liposomes at three concentrations was calculated.
Measurements were made with liposomes containing biodegradable pH-sensitive
surfactants (BPS) at two molar ratios (R=0.2 and R=0.4) in a pH 5.0 buffer solution. Due
to the sensitivity of the assays, different liposome concentrations were used in the studies
presented. Significant differences of membrane fusion (p<0.05) were observed among
the three liposome concentrations with the lipid mixing assay in both ratio groups (Figure
6-4a and Figure 6-5a). The liposome-liposome fusion was concentration dependent on
each individual BPS. However, no significant differences in released calcein were
observed among the three liposome concentrations on the rupture behavior in both ratio
groups (Figure 6-4b and Figure 6-5b) indicating the concentration independency of BPS-
liposome lysis profiles.
Impact of Cholesterol and Dioleoylphosphatidylethanolamine on Membrane Fusion
For L-a-lecithin liposomes without any additive, the percentages of fusion
increased significantly (p<0.05) as the pH decreased to 5.0 in both ratio groups (R=0.2
and R=0.4). Greater fusion was observed at the higher ratio group (R=0.4) than in lower
one (R=0.2) (Figure 6-6). The incorporation of 40% (weight) cholesterol into the
liposome system resulted in enhanced fusion in both ratio groups. However, when

145
(a)
Figure 6-4: Effect of liposome concentration on membrane fusion and lysis events in a
pH 5.0 buffer solution with biodegradable pH-sensitive surfactants (BPS), dodecyl 2-(l
imidazolyl) propionate (DIP), methyl 1-imidazolyl laureate (MIL), and N-dodecyl
imidazole (DI), into liposomes at a molar ratio of 0.2 (R=0.2). The control group referred
to liposomes without BPS. Data were expressed as mean±SD (n=4). a) A lipid mixing
assay was used to quantify fusion at various liposome concentrations (37.5 nmol/ml
(horizontal line bar), 75 nmol/ml (vertical line bar), and 150 nmol/ml (solid bar)), b) A
calcein release measurement was used to determine the liposome lysis behavior at
increasing liposome concentrations (3 nmol/ml (open bar), 25 nmol/ml (dotted bar), and
75 nmol/ml (vertical line bar).

Release
146
(b)
Figure 6-4—continued

147
(a)
Figure 6-5: Effect of liposome concentration on membrane fusion and lysis events in a
pH 5.0 buffer solution with three biodegradable pH-sensitive surfactants (BPS), dodecyl
2-(l’-imidazolyl) propionate (DIP), methyl 1-imidazolyl laureate (MIL), and N-dodecyl
imidazole (DI), into liposomes at a molar ratio of 0.4 (R=0.4). The control group referred
to liposomes without BPS. Data were expressed as meanlSD (n=4). a) A lipid mixing
assay was used to quantify fusion at various liposome concentrations (37.5 nmol/ml
(horizontal line bar), 75 nmol/ml (vertical line bar), and 150 nmol/ml (solid bar)), b) A
calcein release measurement was used to determine the liposome lysis behavior at
increasing liposome concentrations (3 nmol/ml (open bar), 25 nmol/ml (dotted bar), and
75 nmol/ml (vertical line bar)).

148
Figure 6-5—continued

149
Figure 6-6: Effect of cholesterol and dioleoylphosphatidylethanolamine (DOPE) on
membrane fusion using the NBD-Rh lipid mixing assay (n=4). Liposomes (135 nmol/ml)
containing dodecyl 2-(l’-imidazolyl) propionate (DIP) at various molar ratios (R) without
any co-lipid (♦), with 40% (weight) cholesterol (■), or with 40% (weight) DOPE (A)
were compared in different pH environments. Data are expressed as mean±SD. a)
R=0.2; b) R=0.4.

35
30
25 -
c 20
o
10
5
0
Figure 6-6—continued

151
cholesterol was replaced by DOPE, fusion events decreased significantly in both groups
(pO.OOl). Significant differences of fusion (p<0.001) were observed among the three
separate formulations in both ratio groups when the pH was dropped to 5.0.
With the same molar ratio to a neutral lipid, dioleoylphosphatidylethanolamine
(DOPE) and cholesterol contributed distinctively in the dodecyl 2-(l’-imidazolyl)
propionate (DlP)-liposome fusion behavior over the observed pH (Figure 6-7). The
incorporation of cholesterol into the liposomes resulted in additional fusion (p<0.01) than
the liposomes with L-a-lecithin only as pH dropped to 7.0 or lower. When cholesterol
was replaced by DOPE, fusion decreased significantly (p<0.001) as pH decreased to 5.0.
Fusion affected by cholesterol was significantly higher than that affected by DOPE
(p<0.001) at pH 5.0. When the pH was raised to 6.0 or above, fusion influenced by
DOPE was significantly higher than that by cholesterol (p<0.001).
Discussion
Nucleic acid (e.g., plasmid DNA and oligonucleotide) therapy is a promising
approach for the treatment of a variety of disorders (Sokol & Gewirtz, 1996). Two
delivery methods, viral and non-viral, are being studied for cellular delivery of these
nucleic acid agents. The non-viral systems (e.g., cationic liposomes) are attractive due to
the ease of production, the ability to transfect a variety of cell types, and the lower chance
of immune reactions (Lee & Huang, 1997). While non-viral systems are currently
somewhat inefficient, they will undoubtedly improve with the production of new systems
and determination of rate-limiting mechanisms that govern non-viral nucleic delivery.

152
Figure 6-7: Effect of equal molar ratio of cholesterol and
dioleoylphosphatidylethanolamine (DOPE) to L-a-lecithin on membrane fusion using the
NBD-Rh lipid mixing assay (n=4). Liposomes (135 nmol/ml) containing dodecyl 2-(l
imidazolyl) propionate (DIP) at molar ratio 0.4 (R=0.4) without any co-lipid (♦), with
cholesterol (â– ), and with DOPE (A) were compared in different pH environments. Data
are expressed as mean±SD.

153
Dodecyl 2-(l ’-imidazolyl) propionate (DIP), the prototypical member of the
biodegradable pH-sensitive surfactants (BPS) family, has been shown to increase the
effectiveness of nucleic acid delivery (Hughes et al, 1996; Liang & Hughes, 1998).
However, it is unclear how BPS destabilize the endosomal membrane and elicit the
transfer of the oligonucleotide and plasmid DNA or if this is its main mechanism of
action.
Both membrane fusion and rupture elicited by BPS were pH- and molar ratio-
dependent. As the pH became acidic or BPS intra-liposomal amount increased (e.g,
increasing the amount of ionized BPS), the fusion and rupture events increased. The
extent of membrane rupture and fusion were also elevated when the non-charged BPS
was equal to a molar ratio of 0.4. This effect is possibly due to the alternations in lipid
packing (New, 1990). We were unable to form BPS-liposomes as the molar ratio of BPS
to total lipids equaled to 0.5 (R=0.5) or above.
When comparing two membrane fusion assays, it was found fusion determined by
the lipid mixing assay was consistently (i.e., 5-10%) higher than those by the aqueous
content mixing assay. This discrepancy was attributed to the sensitivity of lipid
aggregation for the lipid mixing assay. When membranes aggregate, the fluorescence
energy from one liposome group can also transfer to the other group (Duzgunes et al.,
1985; Yeagle, 1993). The difference in these two assays could be also due to the
hemifusion stage in liposome-liposome interaction. At this stage, lipids from the external
leaflets will mix together which proceeds liposomal content mixing. Both events might
then lead to a perceivable percentage of fusion. However, with the membrane
aggregation or at the hemifusion stage, no aqueous mixing events would be observed.

154
The time course study using both fusion assays supports the above findings.
Liposome fusion reached a plateau in the early period with the lipid mixing assay. With
the aqueous mixing assay, however, fusion gradually increased during the entire time
period. The results indicate the liposome fusion process caused by BPS is at least
composed of three components, initial liposome aggregation followed by lipid mixing
and aqueous mixing.
Membrane fusion is not only dependent on the pH and liposome formulation, but
also directly proportional to the probability of the liposomes to associate with each other
(Ellens, 1984). It is therefore an inter-membrane interaction and liposome concentration
dependent. If the calcein release is the result of leakage during the observed period
caused by fusion, it should be concentration dependent (Ellens, 1984). On the other hand,
if membrane rupture, an intra-membrane effect, is responsible for the majority of the
calcein release, it should be independent from the liposome concentration. The extent of
calcein release caused by BPS depended only on the pH and liposome formulation. The
above results indicated that both rupture and fusion are responsible for the BPS-induced
membrane defect.
To understand BPS-induced membrane destabilization, it is necessary to
comprehend the interactions between the various lipid molecules. A simple means to
explain the lipid molecule interaction (e.g. hexagonal II (Hu) phase, lipid bilayer phase,
and micellar phase) is to describe the packing of lipids into a bilayer by considering the
A
shape of the molecule. This can be represented more quantitatively by Pr - , where Pr
A.
is the packing ratio, Ah is the effective cross-sectional area of the head group, and Ac is

155
the effective cross-sectional area of the hydrocarbon chain region (Yeagle, 1993). When
P <1 (e.g. phosphatidylethanolamine), the inverse cone shape would lead to an inverse
packing (Hu phase). When P«T (e.g. phosphatidylcholine), the cross-sectional area of the
head group is similar to the cross-sectional area of the hydrocarbon chains. The resulting
cylindrical shape would lead to a lipid bilayer. When Pr>l (e.g. Triton X-100), cone
shapes would lead to micelle formation.
To support the above conclusion that BPS membrane fusion and rupture are parts
of the same membrane destabilization continuum, additional studies were conducted with
alternative liposome formulations. In these studies, the impact of cholesterol and
dioleoylphosphatidylethanolamine (DOPE) on liposome fusion was assayed. Cholesterol
is known to increase the rigidity of the liposome structure (New, 1990) while DOPE can
destabilize the liposome structure with its tendency to form the Hn phase (Siegel, 1986;
Yeagle, 1994; Yeagle, 1993). When cholesterol was present in a lecithin liposome, the
addition of BPS resulted in increased fusion at the acidic pH. Cholesterol has also been
reported to promote fusion of lipid bilayers in other studies (Nussbaum et al, 1992; Vogel
et al., 1992). However, when cholesterol was replaced by DOPE, the liposome fusion
events were significantly decreased at acidic pHs.
It is thought that this alteration in membrane fusion is simply due to the chemical
structure configurations among the liposome formulations. Cholesterol can fill in the
gaps between the hydrocarbon chains of adjacent lipid molecules, which will result in
similar ratios of head group to tail group and solidify the liposome. The ensuing rigidity
of the cholesterol-liposome with reduced ability to accommodate other lipophilic

156
compounds within the bilayer can then facilitate membrane fusion after the conversion of
the protonated BPS.
When DOPE was present in the liposome formula, the packing ratio of BPS
(Pr fusogenic than liposomes without DOPE at an alkaline pH. This effect was more evident
in the presence of extra 10% (mol) DOPE in the liposomes. As the pH decreased, the
protonated BPS (Pr>l) became complementary to DOPE (Pr fusion, the liposomes would then tend to remain as an intact bilayer structure as the pH
decreased. When the amount of BPS was 20% (mol) more than that of DOPE at which
BPS outweighed DOPE, the fusion was relatively stable in the pH range of 6-8 and
increased at pH 5.0 (data not shown). However, as expected, the increased fusion was
less than the liposomes without DOPE.
Based upon the generated data and existing literature regarding membrane fusion
and rupture mechanism, we proposed a BPS-induced membrane defect mechanism
(Figure 6-8). It is suggested that at an alkaline pH, un-ionized BPS having a relatively
small head group as compared to the ionized species will reside within the lipid bilayer or
inner leaflet. With the loose lipid packing from the incorporation of un-ionized BPS, the
swollen mixed bilayers tend to be less stable, leading to easier fusion and leakage (Figure
6-2 and Figure 6-3), corresponding to the increasing amounts of BPS packed within the
bilayer. As the amount of BPS in the membrane increases, the swollen mixed bilayer
may be transformed into a humpbacked mixed bilayer at which BPS will reside within
regions of high negative curvature. The imidazole head group which is more
hydrophobic than lecithin’s head group would require less dehydration to form the stalk,

157
Rupture
Protonated BPS
¥ «
Fusion
Figure 6-8: Proposed mechanism of liposome destabilization elicited by biodegradable
pH-sensitive surfactants (BPS). At an alkaline pH, BPS will be neutral and reside within
the lipid bilayer. When additional BPS are incorporated into liposomes, a humpbacked
mixed bilayer will be built from the swollen mixed bilayer. With less dehydration
required to form the stalk, the humpbacked mixed bilayer membrane may interact with
each other more easily. As a consequence of the decreased pH, a portion of BPS will be
protonated and facilitate the formation of a stalk pore at which the fusion process will be
finished. Without interaction with other mixed bilayers, the humpbacked mixed bilayer
will be transformed into a mixed bilayer sheet leading to rupture.

158
the first step in the membrane fusion process, thus favoring this transition and resulting in
the lipid mixing effects (Figure 6-1 and Figure 6-2). When a portion of BPS is
protonated (P >1) as a consequence of the acidic pH present in the system, a stalk pore
will be created to complete the fusion process resulting the content mixing effects (Figure
6-1 and Figure 6-2). Without any subsequent interaction between other humpbacked
mixed bilayers (Figure 6-4 and Figure 6-5), the protonated BPS may transform the
humpbacked mixed bilayer to a mixed bilayer sheet (Lasch, 1995) in which only a
rupture event will be observed (Figure 6-3). The above hypothetical membrane fusion
description is only one of the reported mechanisms that have been proposed for
membrane fusion (Yeagle, 1997). Other models of fusion such as inverted micelle
intermediates may also show BPS dependency but were not addressed in the current
study.
BPS may induce membrane defects leading to fusion and rupture by alternative
routes in comparison of other fusogenic compounds. The difference in events is partially
attributed to BPS’ unique chemical features. When BPS are un-ionized (pH 7.4), it can
be incorporated into the liposome hydrophobic regions but once ionization occurs, it will
behave as a surfactant. Traditionally, commonly studied surfactants (e.g. sodium dodecyl
sulfate) have always interacted with the first membrane encountered; in most cases the
outer lipid leaflet of a bilayer. When surfactants are added to the external aqueous
environment of biological membranes, they will interact with the membranes leading to
mixed micelles.
A recent report (Melikyan et ah, 1997) indicated the inner membrane leaflet
controlled membrane fusion when a hemagglutinin-peptide was the triggering agent. If

159
the results from this study can be applied to our model, agents which alter the inner
leaflet may then increase membrane fusion. Due to the uncharged lipophilic nature of
BPS at pH 7.4, it was hypothesized that BPS would distribute to the inner leaflet similar
to other compounds with relative small head groups (Huang et ah, 1974; Michaelson et
ah, 1973) and demonstrate a portion of its activity at this location. In the current
discussion the influence of the transmembrane pH gradient formed by the incubating the
liposomes at acidic pH was not addressed. Ongoing studies will study this phenomenon
which may result in redistribution of BPS within the lipid bilayer.
In the preceding discussion we have considered three BPS (dodecyl 2-(l
imidazolyl propionate (DIP), methyl 1-imidazolyl laureate (MIL), and N-dodecyl
imidazole (DI)) as one category. At the onset of the experiments a greater difference was
expected between the individual BPS members with regard to fusion and rupture. In
previous studies, by an interfacial tensiometer the critical micelle concentration (CMC) of
three BPS in an ionized state were determined to be 1.0 mM, 0.7 mM, and 1.0 mM for
DIP, MIL, and DI, respectively, in a pH 3.0 hydrochloric acid solution (1 pM). It was
speculated that there would be a correlation between these values and membrane
destabilization.
Furthermore, with the various chemistries used in the attachment of the imidazole
head group to the aliphatic hydrocarbon tail, the pKa values for the three head groups
would be altered. The difference in pKa was expected to affect the pH dependency of
BPS membrane destabilization. This effect was not observed when incorporated into the
liposome bilayer and an unexpected result was obtained when BPS were added to the

160
external environment where DI lost its pH dependency. These findings indicate that the
various BPS will likely have different partitioning into lipid bilayers but may demonstrate
similar intra-bilayer movement. The only statistical difference among BPS was observed
in the release of calcein. In these experiments DIP was noted to have the greatest effect
on membrane leakage. It is unclear why DIP increases leakage over the other two agents
but this may be related to the branched methyl group near the head group whose steric
effect may facilitate membrane destabilization.
Conclusion
To enhance membrane fusion, negative curvature (packing ratio (Pr) compounds can facilitate the formation of the stalk and positive curvature (P >1)
compounds are preferred for the formation of the stalk pore to complete the membrane
fusion process (Chemomordik et al., 1995). The chemical structure of biodegradable pH-
sensitive surfactants (BPS) allows coexistence of both un-ionized (Pr (P >1) species during the fusion process. Instead of causing membrane fusion with the
hexagonal II (Hn) phase similar to dioleoylphosphatidylethanolamine (DOPE), BPS
seemed to be able to participate in membrane fusion at different stages (stalk and stalk
pore). The above results imply examples of how BPS can induce both membrane fusion
and rupture in pH and concentration dependent manner. These findings suggested important
implications in the development of BPS mediated oligonucleotide delivery system. It should be
noted, however, that there was little structure activity relationship between BPS and their
membrane destabilization effects in the study. To clarify the structure activity

161
relationship and more fully understand the membrane destabilization effects of BPS,
more work in respect to diverse head groups, linkers, and aliphatic tails is required.

CHAPTER 7
CONCLUSION AND FUTURE PROSPECT
Conclusion
An ideal oligonucleotide should be stable in serum before exerting its therapeutic
effect. In vitro/in vivo stability of oligonucleotides must therefore be considered.
Chemical modification of oligonucleotides can be employed to increase their stability.
However, modified oligonucleotides should still be able to form a stable complex with
their target sequence. An alternative to enhance the stability of oligonucleotides is to use
a delivery system that can resist oligonucleotides from enzymatic degradation.
After stabilizing oligonucleotides in serum, the second barrier, cellular uptake,
must be overcome. Free oligonucleotides that can be internalized into cells are highly
inefficient. Only a small portion of free unmodified oligonucleotides are able to enter
cells. To increase cellular uptake, oligonucleotides and/or their delivery system can be
further modified in a number of ways (see Chapter 2 for more details).
Most oligonucleotides either with or without a delivery system are taken into cells
through endocytosis and eliminated by lysosomes, the later endocytotic stage. Hence, the
endosomal membrane represents the third rate limiting step. To increase oligonucleotide
162

163
transfer to the cytoplasm, oligonucleotides can be modified to bypass endocytosis and/or
the delivery system can be designed to deal with membrane destabilization.
By increasing oligonucleotide transfer at each step during the delivery process, the
amount of oligonucleotides that reach the cytoplasm can be maximized. Therefore,
optimization would decrease toxic effects associated with a large amount of the system
from oligonucleotides and/or the delivery system.
Biodegradable pH-sensitive surfactants (BPS) were proposed and designed to
focus on disrupting the third delivery barrier by destabilizing the endosomal membrane.
Incorporating BPS into liposomes as an oligonucleotide delivery system would protect
the oligonucleotides from degradation (first delivery barrier) and increase oligonucleotide
cellular uptake (second delivery barrier). As the pH decreased during endocytosis, the
hydrogen ion would protonate BPS and activate the membrane destabilization process so
that the endosomal contents, including oligonucleotides, would be released (third delivery
barrier). After releasing oligonucleotides to their sites of action, BPS would be digested
into less toxic metabolites by the existing digestive enzymes in the cytoplasm. The
additional cytotoxic effects from the delivery system would thus be minimized.
To fulfill these requirements, three agents, dodecyl 2-(l’-imidazolyl) propionate
(DIP), methyl l’-imidazolyl laureate (MIL), andN-dodecyl imidazole (DI), loosely
grouped as BPS were synthesized. Critical micelle concentration and effective release
ratio of the protonated BPS were calculated as a proof of surfactant characteristics. The
question of whether BPS have surface activities when they are protonated and able to
induce liposomal content release was identified at different pHs. When incorporated into
liposomes, BPS released calcein in a pH-dependent manner. As pH decreased, more

164
calcein, index of liposomal content, was freed at the same molar ratio of BPS to
liposomes.
Systems to evaluate the chemical and biological stabilities of BPS were
established and used for testing the biodegradability of BPS. Without a cleavable linker
between its head and tail group, DI exhibited no sensitivity to the esterase. With an ester
connector, stable metabolites, and no chemical hindrance, MIL showed the most
biodegradability among these three BPS. Cellular toxicity of BPS was determined and
correlated to their biodegradability. As reflected from the biodegradability, DI, DIP, and
MIL had a descending order of cytotoxicity.
In vitro biological effects of BPS when incorporated into liposomes were studied
to investigate their impact on oligonucleotide cellular uptake. Compared to liposomes
without BPS, those containing BPS did not increase the cellular uptake of
oligonucleotides (second delivery barrier). Both liposome groups exhibited a similar
cellular uptake profile. However, BPS-liposomes promoted subcellular redistribution of
oligonucleotides after they were endocytosed into cells (third delivery barrier). This
suggests the disruption of endosomal membranes caused by BPS and followed by the
release of oligonucleotides to cytoplasms. To gain insight into oligonucleotide activity,
the degree of the luciferase enzyme expressed by CV-1 cells was monitored in BPS-
liposomes. More enzyme activities were inhibited when BPS were a part of liposomes
compared liposomes without BPS.
The mechanisms involved in disturbing endosomal membranes by BPS were
finally investigated and pictured using a liposome-liposome interaction model. It is
known that fusogenic compounds having a packing ratio (area occupied by the head

165
group to that by the tail group) less than 1 can form the hexagonal II (Hn) phase to start
membrane fusion at the stalk stage. In contrast, surfactants having a packing ratio larger
than 1 can form the mixed micelle phase and contribute to membrane fusion at the other
stalk pore stage. The following BPS-induced membrane defect mechanisms were
proposed based upon the existing literature evidence and our data,. At an alkaline
environment, a BPS is predominated by its hydrocarbon tail group over its
lysosomotropic amine head group, and thus has a packing ratio less than 1. When it is
protonated at an acidic environment, the hydrophilicity of the agent from its head group
increases significantly, thereby reversing the packing ratio to be larger than 1. BPS are
therefore suggested to be able to join the membrane fusion process at both stages (stalk
and stalk pore). It was proposed that when BPS-liposomes along with oligonucleotides
were internalized into cells through endocytosis, BPS would behave like fusogenic
compounds and initiate membrane fusion at pH 7.4. As pH decreased during the
endocytotic process, some BPS would be protonated and facilitate the fusion process like
surfactants. Oligonucleotides would therefore be released from endosomes to cytoplasms
through the pore that BPS induced, bind to their targets, and eventually exert their
biological effect.
Future Aims
Establishment of a Better Biological Evaluation System
Antisense oligonucleotide activity caused by the biodegradable pH-sensitive
surfactants (BPS)-liposomes resulted in better enzyme inhibition than liposomes without

166
BPS. However, both the sense strand of the oligonucleotide and liposomes alone
restrained a certain degree of luciferase activity. The issue of specificity must therefore
be solved before BPS are fully evaluated. Several interfering factors need to be
considered and excluded. When analyzing luciferase activity, the amount of protein was
used to correct the total number of cells in each treatment. However, this may not be a
good index of the total number of cells. Protein debris from the dead cells due to the
toxic effect from oligonucleotides and/or liposomes can mask the participated live cells.
To circumvent this drawback, the endogenous enzymes (e.g., lactate
dehydrogenase) that are not inhibited by specific oligonucleotides represent a good
candidate to rectify the total number of live cells. Since synthesized oligonucleotides
were used directly without purification, this might also decrease their specificity.
Organic simple extraction can be used to purify oligonucleotides and possibly increase
their specificity.
As the possible interferences are cleared, endocytosis inhibitors such as
cytochalasin B, chloroquine, NH4C1, and low temperature (4°C) can be used as additional
controls to strengthen BPS mediated oligonucleotide activity.
Creation of a Structure Activity Relationship
None of the properties that were determined in the previous experiments could
correlate with the structures of the three biodegradable pH-sensitive surfactants (BPS).
These three agents were used to corroborate the findings in our current studies. In order
to draw any conclusions about the structure activity relationship, more BPS in terms of
lysosomotropic amine head groups, hydrocarbon tail groups, and connecting bridges must

167
be synthesized. The parameters including critical micelle concentration (CMC), effective
release ratio (Re), pH sensitivity, chemical rate constant, biodegradability, cytotoxicity,
oligonucleotide cellular uptake, oligonucleotide biological effect, and mechanism of
action will be assessed. Newly synthesized BPS will then be compared to address their
individual impact on BPS mediated oligonucleotide cellular delivery. Thereafter, the
biological effect of oligonucleotides caused by BPS-liposomes can be predicted from the
perspective of the established parameters.

REFERENCES
Agrawal, S., and Iyer, R. P. (1997) Perspectives in antisense therapeutics. Pharmacol.
Ther. 76 11-31: 151-160
Agrawal, S., Goodchild, J., Civeira, M. P., Thornton, A. H., Sarin, P. S., and Zamecnik,
P. C. (1988) Oligodeoxynucleoside phosphoramidates and phosphorothioates as
inhibitiors of human immunodeficiency virus. Proc. Natl. Acad. Sci. USA 85 (19): 7079-
7083.
Akhtar, S., Basu, S., Wickstrom, E., and Juliano, R. L. (1991a) Interactions of antisense
DNA oligonucleotide analogs with phospholipid membranes (liposomes). Nucleic Acids
Res. 19 1201: 5551-5559.
Akhtar, S., Kole, R., and Juliano, R. L. (19916) Stability of antisense DNA
oligodeoxynucleotide analogs in cellular extracts and sera. Life Sci. 49 (24): 1793-1801.
Akhtar, S., and Juliano, R. L. (1992) Cellular uptake and intracellular fate of antisense
oligonucleotides. Trends Cell Biol. 2: 139-144.
Alahari, S. K., Dean, N. M., Fisher, M. H., Delong, R., Manoharan, M., Tivel, K. L., and
Juliano, R. L. (1996) Inhibition of expression of the multidrug resistance-associated P-
glycoprotein of by phosphorothioate and 5' cholesterol-conjugated phosphorothioate
antisense oligonucleotides. Mol. Pharmacol. 50 (4): 808-819.
Anazodo, M. I., Wainberg, M. A., Friesen, A. D., and Wright, J. A. (1995) Sequence-
specific inhibition of gene expression by a novel antisense oligodeoxynucleotide
phosphorothioate directed against a nonregulatory region of the human
immunodeficiency virus type 1 genome. J. Virol. 69 131: 1794-1801.
Aoki, M., Morishita, R., Higaki, J., Moriguchi, A., Kida, I., Hayashi, S., Matsushita, H.,
Kaneda, Y., and Ogihara, T. (1997) In vivo transfer efficiency of antisense
oligonucleotides into the myocardium using HVJ-liposome method. Biochem. Biophvs.
Res. Commun. 231 (3): 540-545.
Arima, H., Aramaki, Y., and Tsuchiya, S. (1997) Effect of oligodeoxynucleotides on the
physicochemical characteristics and cellular uptake of liposomes. J. Pharm. Sci. 86 (4):
438-442.
168

169
Aronsohn, A. L, and Hughes, J. A. (1997) Nuclear localization signal peptides enhance
cationic liosome-mediated gene therapy. J. Drug Targeting 5 (3): 163-169.
Barry, E. L., Gesek, F. A., and Friedman, P. A. (1993) Introduction of antisense
oligonucleotides into cells by permeabilization with streptolysin O. Biotechniques 15
(6): 1016-1018, 1020.
Belousov, E. S., Afonina, I. A., Podyminogin, M. A., Gamper, H. B., Reed, M. W.,
Wydro, R. M., and Meyer, R. B. (1997) Sequence-specific targeting and covalent
modification of human genomic DNA. Nucleic Acids Res. 25 (17): 3440-3444.
Bennett, C. F., Chiang, M. Y., Chan, H., Shoemaker, J. E., and Mirabelli, C. K. (1992)
Cationic lipids enhance cellular uptake and activity of phosphorothioate antisense
oligonucleotides. Mol. Pharmacol. 41 (6): 1023-1033.
Bennett, M. J., Nantz, M. H., Balasubramaniam, R. P., Gruenert, D. C., and Malone, R.
W. (1995) Cholesterol enhances cationic liposome-mediated DNA transfection of human
respiratory epithelial cells. Biosci. Rep. 15 (1): 47-53.
Bentz, J., Ellens, H., Lai, M. Z., and Szoka, F. C., Jr. (1985) On the correlation between
HII phase and the contact-induced destabilization of phosphatidylethanolamine-
containing membranes. Proc. Natl. Acad. Sci. USA 82 (17): 5742-5745.
Bergan, R., Hakim, F., Schwartz, G. N., Kyle, E., Cepada, R., Szabo, J. M., Fowler, D.,
Gress, R., and Neckers, L. (1996) Electroporation of synthetic oligodeoxynucleotides: a
novel technique for ex vivo bone marrow purging. Blood 88 (2): 731-741.
Bielinska, A., Kukowska Latallo, J. F., Johnson, J., Tomaba, D. A., and Baker, J. R., Jr.
(1996) Regulation of in vitro gene expression using antisense oligonucleotides or
antisense expression plasmids transfected using starburst PAMAM dendrimers. Nucleic
Acids Res. 24 (11): 2176-2784.
Blake, K., Murakami, A., Spitz, S. A., Glave, S. A., Reddy, M. P., Ts'o, P. O., and Miller,
P. S. (1985) Hybridization arrest of globin synthesis in rabbit reticulocyte lysates and
cells by oligodeoxyribonucleoside methylphosphonates. Biochemistry 24 (22): 6139-
6145.
Blondel, D., harmison, G. G., and Schubert, M. (1990) Role of matrix protein in
cytopathogenesis of vesicular stomatitis vims. J. Virol. 64 141: 1716-1725.
Bock, L. C., Griffin, L. C., Latham, J. A., Vermass, E. H., and Toole, J. J. (1992)
Selection of single-stranded DNA molecules that bind and inhibit human thrombin.
Nature 355 (6360): 564-566.

170
Bodor, N., Kaminski, J. J., and Selk, S. (1980) Soft drugs. 1. Labile quaternary
ammonium salts as soft antimicrobials. J. Med. Chem. 23 (5): 469-474.
Bonfils, E., Depierreux, C., Midoux, P., Thuong, N. T., Monsigny, M., and Roche, A. C.
(1992) Drug targeting: synthesis and endocytosis of oligonucleotide-neoglycoprotein
conjugates. Nucleic Acids Res. 20 (171: 4621 -4629.
Bongartz, J. P., Aubertin, A. M., Milhaud, P. G., and Lebleu, B. (1994) Improved
biological activity of antisense oligonucleotides conjugated to a fusogenic peptide.
Nucleic Acids Res. 22 (22): 4681-4688.
Boutorin, A. S., Gus’kova, L. V., Ivanova, E. M., Kobetz, N. D., Zarytova, V. F., Ryte,
A. S., Yurchenko, L. V., and Vlassov, V. V. (1989) Synthesis of alkylating
oligonucleotide derivatives containing cholesterol or phenazinium residues at their 3’-
terminus and their interaction with DNA within mammalian cells. FEBS Lett. 254 (1-2):
129-132.
Brasier, A. R., Tate, J. E., and Habener, J. F. (1989) Optimized use of the firefly
luciferase assay as a reporter gene in mammalian cell lines. Biotechniques 7 (10): 1116-
1122.
Bunnell, B. A., Askari, F. K., and Wilson, J. M. (1992) Targeted delivery of antisense
oligonucleotides by molecular conjugates. Somat. Cell Mol. Genet. 18 (6): 559-569.
Buyuktimkin, S., Buyuktimkin, N., and Rytting, J. H. (1993) Synthesis and enhancing
effect of dodecyl 2-(N,N-dimethylamino) propionate on the transepidermal delivery of
indomethacin, clonidine, and hydrocortisone. Pharm. Res. 10 (11): 1632-1637.
Capaccioli, S., Di Pasquale, G., Mini, E., Mazzei, T., and Quattrone, A. (1993) Cationic
lipids improve antisense oligonucleotide uptake and prevent degradation in cultured cells
and in human serum. Biochem. Biophvs. Res. Commun. 197 (2): 818-825.
Carrasco, L., (1994) Entry of animal viruses and macromolecules into cells. FEBS Lett.
350 (2-3): 151-154.
Chaudhuri, G. (1997) Scavenger receptor-mediated delivery of antisense mini-exon
phosphorothioate oligonucleotide to Leishmania-infected macrophages. Selective and
efficient elimination of the parasite. Biochem. Pharmacol. 53 (3): 385-391.
Chavany, C., Le Doan, T., Couvreur, P., Puisieux, F., and Helene, C. (1992)
Polyalkylcyanoacrylate nanoparticles as polymeric carriers for antisense oligonucleotides.
Pharm. Res. 9 (4): 441-449.
Chavany, C., Saison-Behmoaras, T., Le Doan, T., Puisieux, F., Couvreur, P., and Helene,
C. (1994) Adsorption of oligonucleotides onto polyisohexylcyanoacrylate nanoparticles

171
protects them against nucleases and increases their cellular uptake. Pharm. Res. 11 (9):
1370-1378.
Chen, J. K., Weith, H. L., Grewal, R. S., Wang, G., and Cushman, M. (1995) Synthesis of
novel phosphoramidite reagents for the attachment of antisense oligonucleotides to
various regions of the benzophenanthridine ring system. Bioconjug. Chem. 6 (4): 472-
483.
Chemomordik, L., Kozlov, M. M., and Zimmerberg, J. (1995) Lipids in biological
membrane fusion. J. Membr. Biol. 146111: 1-14.
Chin, D. J., Green, G. A., Zon, G., Szoka, F. C., Jr., and Straubinger, R. M. (1990) Rapid
nuclear accumulation of injected oligodeoxyribonucleotides. New Biol. 2 (12): 1091-
1100.
Chu, C. J., Dijkstra, J., Lai, M. Z., Hong, K., and Szoka, F. C. (1990) Efficicency of
cytoplasmic delivery by pH-sensitive liposomes to cells in culture. Pharm. Res. 7 (8):
824-834.
Citro, G., Perrotti, D., Cucco, C., D. Agnano, I., Sacchi, A., Zupi, G., and Calabretta, B.
(1992) Inhibition of leukemia cell proliferation by receptor-mediated untake of c-myb
antisense oligodeoxynucleotides. Proc. Natl. Acad. Sci. USA 89 (15): 7031-7035.
Citro, G., Szczylik, P., Ginobbi, P., Zupi, G., and Calabretta, B. (1994) Inhibition of
leukaemia cell proliferation by folic acid-polylysine-mediated introduction of c-myb
antisense oligodeoxynucleotides into HL-60 cells. Br. J. Cancer 69 (3): 463-467.
Connolly, B. A., Potter, B. V., Eckstein, F., Pingoud, A., and Grotjahn, L. (1984)
Synthesis and characterization of an octanucleotide containing the EcoRI recognition
sequence with a phosphorothioate group at the cleavage site. Biochemistry 23 (15):
3443-3453.
Connor, J., Yatvin, M. B., and Huang, L. (1984) pH-sensitive liposomes: acid-induced
liposome fusion. Proc. Natl, Acad. Sci. USA 81 Í6L 1715-1718.
Cooney, M., Czemuszewicz, G., Postel, E. H., Flint, S. J., and Hogan, M. E. (1988) Site-
specific oligonucleotide binding represses transcription of the human c-myc gene in vitro.
Science 241 (4864): 456-459.
Cowsert, L. M., Fox, M. C., Zon, G., and Mirabelli, C. K. (1993) In vitro evaluation of
phosphorothioate oligonucleotides targeted to the E2 mRNA of papillomavirus: potential
treatment for genital warts. Antimicrob. Agents Chemother. 37 (2): 171-177.

172
Cristina De Oliveira, M, Fattal., E., Ropert, C., Malvy, C., and Couvreur, P. (1997)
Delivery of antisense oligonucleotides by means of pH-sensitive liposomes. J. Contr.
Rel. 48: 179-184.
Crooke, S. T. (1993) Progress toward oligonucleotide therapeutics: pharmacodynamic
properties. FASEB J. 7 (6): 533-539.
De Duve, C. (1983) Lysosomes revisited. Eur. J. Biochem. 137 (3): 391-397.
De Duve, C., De Barsy, T., Poole, B., Trouet, A., Tulkens, P., and Van Hoof, F. (1974)
Lysosomotropic agents. Biochem. Pharmacol. 23 (18): 2495-2531.
Dean, R. T., Jessup, W., and Roberts, C. R. (1984) Effects of exogenous amines on
mammalian Cells, with particular reference to membrane flow. Biochem. J. 217 (1): 27-
40.
Degols, G., Leonetti, J. P., Gagnor, C., Lemaitre, M., and Lebleu, B. (1989) Antiviral
activity and possible mechanisms of action of oligonucleotides-poly(L-lysine) conjugates
targeted to vesicular stomatitis virus mRNA and genomic RNA. Nucleic Acids Res. 17
(22): 9341-9350.
Degols, G., Leonetti, J. P., Mechti, N., and Lebleu, B. (1991) Antiproliferative effects of
antisense oligonucleotides directed to the RNA of c-myc oncogene. Nucleic Acids Res.
19 (4): 945-948.
Delong, R., Stephenson, K., Loftus, T., Fisher, M., Alahari, S., Nolting, A., and Juliano,
R. L. (1997) Characterization of complexes of oligonucleotides with polyamidoamine
starburst dendrimers and effects on intracellular delivery. J. Pharm. Sci. 86 (6): 762-764.
Deshpande, D., Toledo Velasquez, D., Thakkar, D., Liang, W., and Rojanasakul, Y.
(1996) Enhanced cellular uptake of oligonucleotides by EGF receptor-mediated
endocytosis in A549 cells. Pharm. Res. 13 (1): 57-61.
Duzgunes, N., Straubinger, R. M., Baldwin, P. A., Friend, D. S., and Papahadjopoulos, D.
(1985) Proton-induced fusion of oleic acid-phosphatidylethanolamine liposomes.
Biochemistry 24 (13): 3019-3098.
Dzau, V. J., Mann, M. J., Morishita, R., and Kaneda, Y. (1996) Fusigenic viral liposome
for gene therapy in cardiovascular diseases. Proc. Natl. Acad. Sci. USA 93 (21): 11421-
11425.
Ellens, H., Bentz, J., and Szoka, F. C. (1984) pH-induced destabilization of
phosphatidylethanolamine-containing liposomes: role of bilayer contact. Biochemistry
23 (7): 1532-1538.

173
Ellington, A. D. (1994) RNA selection. Aptamers achieve the desired recognition. Curr.
Biol. 4 (5): 427-429.
Fattal, E., Nir, S., Párente, R. A., and Szoka, F. C., Jr. (1994) Pore-forming peptides
induce rapid phospholipid flip-flop in membranes. Biochemistry 33 (21): 6721-6731.
Feigner, P. L., Gadek, T. R., Holm, M., Roman, R., Chan, H. W., Wenz, J. P., Ringold,
G. M., and Danielsen, M. (1987) Lipofection: a highly efficient, lipid-mediated DNA-
transfection procedure. Proc. Natl, Acad. Sci. USA 84 121): 7413-7417.
Fenster, S. D., Wagner, R. W., and Froehler, B. C., and Chin, D. J. (1994) Inhibition of
human immunodeficiency virus type-1 env expression by C-5 propyne oligonucleotides
specific for Rev-respnse element stem-loop V. Biochemistry 33 (28): 8391-8398.
Firestone, R. A., Pisano, J. M., Bailey, P. J., Sturm, A., Bonney, R. J., Wightman, P.,
Devlin, R., Fin, C. S., Keller, D. L., and Tway, P. C. (1982a) Fysosomotropic agents. 4.
carbobenzoxyglycylphenylalanyl, a new protease-sensitive masking group for
introduction into cells. J. Med. Chem. 25 (5): 539-544.
Firestone, R. A., Pisano, J. M., and Bonney, R. J. (1982h) Lysosomotropic agents. 2.
synthesis and cytotoxic action of lysosomotropic detergents. In International Symposium
on Solution Behavior of Surfactants: Theoretical and Applied Aspects. (Mittal, K. L., and
Fendler, E. J., editors) pp. 1455-1464. Plenum Press, New York.
Firestone, R. A., Pisano, J. M., and Bonney, R. J. (1979) Lysosomotropic agents. 1.
synthesis and cytotoxic action of lysosomotropic detergents. J. Med. Chem. 22 (9): 1130-
1133.
Fisher, T.L., Terhorst, T., Cao, X., and Wagner, R. W. (1993) Intracellular disposition
and metabolism of fluorescently-labeled unmodified and modified oligonucleotides
microinjected into mammalian cells. Nucleic Acids Res. 21 (16): 3857-3865.
Flanagan, W. M., and Wagner, R. W. (1997) Potent and selective gene inhibition using
antisense oligodeoxynucleotides. Mol. Cell Biochem. 172 (1-2): 213-225.
Forster, S., Scarlett, L., and Lloyd, J. B. (1987) The effect of lysosomotropic detergents
on the permeability properties of the lysosome membrane. Biochim. Biophys, Acta 924
(3): 452-457.
Fukui, K., and Tanaka, K. (1996) The acridine ring selectively intercalated into a DNA
helix at various types of abasic sites: double strand formation and photophysical
properties. Nucleic Acids Res. 24 GO): 3962-3967.

174
Galbraith, W. M., Hobson, W. C., Giclas, P. C., Schechter, P. J., and Agrawal, S. (1994)
Complement activation and hemodynamic changes following intravenous administration
of phosphorothioate oligonucleotides in the monkey. Antisense Res. Dev. 4 (3): 201-206.
Camper, H. B., Reed, M. W., Cox, T., Virosco, J. S., Adams, A. D., Gall, A. A., Scholler,
J. K., and Meyer, R. B. (1993) Facile preparation of nuclease resistant 3' modified
oligodeoxynucleotides. Nucleic Acids Res. 21 G): 145-150.
Gao, W. Y., Stein, C. A., Cohen, J. S., Dutschman, G. E., and Cheng, Y. C.Gao, W. Y.
(1989) Effect of phosphorothioate homo-oligodeoxynucleotides on herpes simplex virus
type 2-induced DNA polymerase. J. Biol. Chem. 264 1191: 11521-11516.
Gershon, H., Chirlando, R., Guttman, S. B., and Minsky, A. (1993) Mode of formation
and structural features of DNA-cationic liposome complexes used for transfection.
Biochemistry 32 (28): 7143-7153.
Ghosh, M. K., Ghosh, K., Dahl, O., and Cohen, J. S. (1993) Evaluation of some
properties of a phosphorodithioate oligodeoxyribonucleotide for antisense application.
Nucleic Acids Res. 21 (24): 5761-5766.
Ginobbi, P., Geiser, T. A., Ombres, D., and Citro, G. (1997) Folic acid-polylysine carrier
improves efficacy of c-myc antisense oligodeoxynucleotides on human melanoma (Ml4)
cells. Anticancer Res. 17 (la): 29-35.
Giovannangeli, C., Perrouault, L., Escude, C., Nguyen, T., and Helene, C. (1996) Specific
inhibition of in vitro transcription elongation by triplex-forming oligonucleotide-
intercalator conjugates targeted to HIV proviral DNA. Biochemistry 35 (32): 10539-
10548.
Godard, G., Boutorine, A. S., Saison-Behmoaras, E., Helene, C. (1995) Antisense effects
of cholesterol-oligodeoxynucleotide conjugates associated with poly(alkylcyanoacrylate)
nanoparticles. Eur. J. Biochem. 232 (2): 404-410.
Griffey, R. H., Monia, B. P., Cummins, L. L., Freier, S., Greig, M. J., Guinosso, C. J.,
Lesnik, E., Manalili, S. M., Mohan, V., Owens, S., Ross, B. R., Sasmor, H., Wancewicz,
E., Weiler, K., Wheeler, P. D., and Cook, P. D. (1996) 2-O-aminopropyl ribonucleotides:
a zwitterionic modification that enhances the exonuclease resistance and biological
activity of antisense oligonucleotides. J. Med. Chem. 39 (26): 5100-5109.
Grigoriev, M., Praseuth, D., Robin, P., Hemar, A., Saison-Behmoaras, T., Dautry Varsat,
A., Thuong, N. T., Helene, C., and Harel Bellan, A. (1992) A triple helix-forming
oligonucleotide-intercalator conjugate acts as a transcriptional repressor via inhibition of
NF kappa B binding to interleukin-2 receptor alpha-regulatory sequence. J. Biol. Chem,
267 (5): 2289-3395.

175
Guvakova, M. A., Yakubov, L. A., Vlodavsky, I., Tonkinson, J. L., and Stein, C. A.
(1995) Phosphorothioate oligodeoxynucleotides bind to basic fibroblast growth factor,
inhibit its binding to cell surface receptors, and remove it from low affinity binding sites
on extracellular matrix. J. Biol. Chem. 270 (6): 2620-2627.
Guy Caffey, J. K., Bodepudi, V., Bishop, J. S., Jayaraman, K., and Chaudhary, N. (1995)
Novel polyaminolipids enhance the cellular uptake of oligonucleotides. J. Biol. Chem.
270 1521: 39391-39396.
Habus, I., Zhao, Q., and Agrawal, S. (1995) Synthesis, hybridization properties, nuclease
stability, and cellular uptake of the oligonucleotide-amino-beta-cyclodextrins and
adamantane conjugates. Bioconjug. Chem. 6 (4): 327-331.
Hassner, A., and Alwxanian, V. (1978) Direct room temperature esterification of
carboxylic acids. Tetrahvdron Lett. 46: 4475-4478.
Hatta, T., Nakagawa, Y., Takai, K., Nakada, S., Yokota, T., and Takaku, H. (1996)
Inhibition of influenza virus RNA polymerase and nucleoprotein genes expression by
unmodified, phosphorothioated, and liposomally encapsulated oligonucleotides.
Biochem. Biophvs. Res. Commun. 223 (2): 341-346.
Hatta, T., Takai, K., Nakada, S., Yokota, T., and Takaku, H. (1997) Specific inhibition of
influenza virus RNA polymerase and nucleoprotein genes expression by liposomally
endocapsulated antisense phosphorothioate oligonucleotides: penetration and localization
of oligonucleotides in clone 76 cells. Biochem. Biophvs. Res. Commun. 232 (2): 545-
549.
Helene, C., and Toulme, J. J. (1990) Specific regulation of gene expression by antisense,
sense and antisense nucleic acids. Biochim. Biophvs. Acta 1049 (2): 99-125.
Huang, C. H., Sipe, J. P., Chow, S. T., and Martin, R. B. (1974) Differential interaction of
cholesterol with phosphatidylcholine on the inner and outer surfaces of lipid bilayer
vesicles. Proc. Nath Acad. Sci. USA 71 (2): 359-362.
Hug, P., and Sleight, R. G. (1994) Fusogenic virosomes prepared by partitioning of
vesicular stomatitis virus G protein into preformed vesicles. J. Biol. Chem. 269 (6):
4050-4056.
Hughes, J. A., Aronsohn, A. I., Avrutskaya, A. V., and Juliano, R. L. (1996) Evaluation
of adjuvants that enhance the effectiveness of antisense oligodeoxynucleotides. Pharm.
Res. 13 (3): 404-410.
Hughes, J. A., Bennett, C. F., Cook, P. D., Guinosso, C. J., Mirabelli, C. K., and Juliano,
R. L. (1994) Lipid membrane permeability of 2'-modified derivatives of phosphorothioate
oligonucleotides. J. Pharm. Sci. 83 (4): 597-600.

176
Jaaskelainen, I., Monkkonen, J., and Urtti, A. (1994) Oligonucleotide-cationic liposome
interactions. A physicochemical study. Biochim. Biophys. Acta 1195 (1): 115-123.
Juliano, R. L., and Akhtar, S. (1992) Liposomes as a drug delivery system for antisense
oligonucleotides. Antisense Res. Dev. 2 12): 165-176.
Kabanov, A. V., Vinogradov, S. V., Ovcharenko, A., V., Krivonos, A. V., Melik-
Nubarov, N. S., Kiselev, V. I., and Severin, E. S. (1990) A new class of antivirals:
antisense oligonucleotides combined with a hydrophobic substituent effectively inhibit
influenza virus reproduction and synthesis of virus-specific proteins in MDCK cells.
FEBS Lett. 259 (2): 327-330.
Khaled, Z., Benimetskaya, L., Zeltser, R., Khan, T., Sharma, H. W., Narayanan, R., and
Stein, C. A. (1996) Multiple mechanisms may contribute to the cellular anti-adhesive
effects of phosphorothioate oligodeoxynucleotides. Nucleic Acids Res. 24 (4): 737-745.
Klysik, J., Kinsey, B. M., Hua, P., Glass, G. A., and Orson, F. M. (1997) A 15-base
acridine-conjugated oligodeoxynucleotide forms triplex DNA with its IL-2R alpha
promoter target with greatly improved avidity. Bioconjug. Chem. 8 (3): 318-326.
Konopka, K., Pretzer, E., Feigner, P. L., and Duzgunes, N. (1996) Human
immunodeficiency virus type-1 (HIV-1) infection increases the sensitivity of
macrophages and THP-1 cells to cytotoxicity by cationic liposomes. Biochim. Biophys.
Acta 1312 (3): 186-196.
Krieg, A. M., Yi, A. K., Matson, S., Waldschmidt, T. J., Bishop, G. A., Teasdale, R.,
Koretzky, G. A., and Klinman, D. M. (1995) CpG motifs in bacterial DNA trigger direct
B-cell activation. Nature 374 165221: 546-549.
Kukowska-Latallo, J. F., Bielinska, A. U., Johnson, J., Spindler, R., Tomaba, D. A., and
Baker, J. R., Jr. (1996) Efficient transfer of genetic material into mammalian cells using
starbust polyamidoamine dendrimers. Proc. Natl. Acad. Sci. USA 93 (10): 4897-4902.
Kumar, M., Hassan, M. Q., Tyagi, S. K., and Sarkar, D. P. (1997) A 45,000-M(r)
glycoprotein in the Sendai virus envelope triggers virus-cell fusion. J. Virol. 71 (9):
6398-6406.
Lacoste, J., Francois, J. C., and Helene, C. (1997) Triple helix formation with purine-rich
phosphorothioate-containing oligonucleotides covalently linked to an acridine derivative.
Nucleic Acids Res. 25 G0L 1991-1998.
Lamprecht, R., Hazvi, S., and Dudai, Y. (1997) cAMP response element-binding protein
in the amygdala is required for long-but not short-term conditioned taste aversion
memory. J. Neurosci. 17 1211: 8443-8450.

177
Lappalainen, K., Miettinen, R., Kellokoski, J., Jaaskelainen, I., and Syrjanen, S. (1997)
Intracellular distribution of oligonucleotides delivered by cationic liposomes: light and
electron microscopic study. J. Histochem. Cytochem. 45 (2): 265-274.
Lappalainen, K., Pirila, L., Jaaskelainen, I., Syrjanen, K., and Syijanen, S. (1996) Effects
of liposomal antisense oligonucleotides on mRNA and protein levels of the HPV 16 E7
oncogene. Anticancer Res. 16 (5a): 2485-2492.
Lappalainen, K., Urtti, A., Soderling, E., Jaaskelainen, I., Syrjanen, K., and Syrjanen, S.
(1994) Cationic liposomes improve stability and intracellular delivery of antisense
oligonucleotides into CaSki cells. Biochim. Biophvs. Acta 1196 (2): 201-208.
Lasch, J. (1995) Interaction of detergents with lipid vesicles. Biochim. Biophvs. Acta
1241 (2): 269-292.
Lee, R. J., and Huang. L. (1997) Lipidie vector systems for gene transfer. Crit. Rev.
Ther. Drug. Carrier. Svstm. 14 (2): 173-206.
Lefebvre d'Hellencourt, C., Diaw, L., and Guenounou, M. (1995) Immunomodulation by
cytokine antisense oligonucleotides. Eur. Cytokine Netw. 6 (1): 7-19.
Lemaitre, M., Bayard, B., and Lebleu, B. (1987) Specific antiviral activity of a poly (L-
lysine)-conjugated oligodeoxyribonucleotide sequence complementary to vesicular
stomatitis virus N protein mRNA initiation site. Proc. Natl. Acad. Sci. USA 84 (3): 648-
652.
Leonetti, J. P., Machy, P., Degols, G., Lebleu, B., and Leserman, L. (1990) Antibody-
targeted liposomes containing oligodeoxyribonucleotides complementary to viral RNA
selectively inhibit viral replication. Proc. Natl Acad. Sci. USA 87 (7): 2448-2451.
Leonetti, J. P., Mechti, N., Degols, G., Gagnor, C., and Lebleu, B. (1991) Intracellular
distribution of microinjected antisense oligonucleotides. Proc. Natl. Acad. Sci. USA 88
(7): 2702-2706.
Leonetti, J. P., Rayner, B., Lemaitre, M., Gagnor, C., Milhaud, P. G., Imbach, J. L., and
Lebleu, B. (1988) Antiviral activity of conjugates between poly(L-lysine) and synthetic
oligodeoxyribonucleotides. Gene 72 (1-2): 323-332.
Letsinger, R. L., Zhang, G. R., Sun, D. K., Ikeuchi, T., and Sarin, P. S. (1989)
Cholesteryl-conjugated oligonucleotides: synthesis, properties, and activity as inhibitors
of replication of human immunodeficiency virus in cell culture. Proc. Natl. Acad. Sci.
USA 86 1171: 6553-6556.

178
Levina, A. S., Tabatadse, D. R., Khalimskaya, L. M., Prichodko, T. A., Shishkin, G. V.,
Alexandrova, L. A., and Zarytova, V. P. (1993) Oligonucleotide derivatives bearing
reactive and stabilizing groups attached to C5 of deoxyuridine. Bioconjug. Chem. 4 (5):
319-325.
Liang, E., and Hughes, J. A. (1998) Characterization of a pH-sensitive surfactant,
dodecyl-2-(r-imidazolyl) propionate (DIP), and preliminary studies in liposome
mediated gene transfer. Biochim. Biophvs. Acta 1369 (1): 39-50.
Liang, W. W., Shi, X., Deshpande, D., Malanga, C. J., and Rojanasakul, Y. (1996)
Oligonucleotide targeting to alveolar macrophages by mannose receptor-mediated
endocytosis. Biochim. Biophvs. Acta 1279 (2): 227-234.
Lichtenberg, D. (1985) Characterization of the solubilization of lipid bilayers by
surfactants. Biochim. Biophvs. Acta 821. (3): 470-478.
Lichtenberg, D., Zilberman, Y., Greenzaid, P., and Zamir, S. (1979) Structural and
kinetic studies on the solubilization of lecithin by sodium deoxycholate. Biochemistry 18
(16): 3517-3525.
Lichtenfels, R., Biddison, W. E., Schulz, H., Vogt, A. B., and Martin, R. (1994) CARE-
LASS (calcein-release-assay), An improved fluorescence-based test system to measure
cytotoxic T lymphocyte activity. J. Immunol. Methods 172 (2): 227-239.
Litzinger, D. C., Brown, J. M., Wala, I., Kaufman, S. A., Van, G. Y., Farrell, C. L., and
Collins, D. (1996) Fate of cationic liposomes and their complex with oligonucleotide in
vivo. Biochim. Biophvs. Acta 1281 (2): 139-149.
Liu, Y., and Regen, S. L. (1993) Control over vesicle rupture and leakage by membrane
packing and by the aggregation state of an attacking surfactant. J. Am. Chem. Soc. 115
(2): 708-713.
Loke, S. L., Stein, C., Zhang, X., Avigan, M., Cohen, J., and Neckers, L. M. (1988)
Delivery of c-myc antisense phosphorothioate oligodeoxynucleotides to hematopoietic
cells in culture by liposome fusion: specific reduction in c-myc protein expression
correlates with inhibition of cell growth and DNA synthesis. Curr. Top. Microbiol.
Immunol. 141: 282-289.
Loke, S. L., Stein, C. A., Zhang, X. H., Mori, K., Nakanishi, M., Subasinghe, C., Cohen,
J. S., and Neckers, L. M. (1989) Characterization of oligonucleotide transport into living
cells. Proc. Natl. Acad. Sci. USA 86 (10): 3474-3478.
Ma, D. D., and Wei, A. Q. (1996) Enhanced delivery of synthetic oligonucleotides to
human leukaemic cells by liposomes and immunoliposomes. Leuk. Res. 20 (11-12): 925-
930.

179
Mann, M. J., Morishita, R., Gibbons, G. H., von der Leyen, H. E., and Dzau, V. J. (1997)
DNA transfer into vascular smooth muscle using fusigenic Sendai virus (HVJ)-
liposomes. Mol. Cell Biochem. 172 (1-2): 3-12.
Marchand, C., Bailly, C., Nguyen, C. H., Bisagni, E., Garestier, T., Helene, C., and
Waring, M. J. (1996) Stabilization of triple helical DNA by a benzopyridoquinoxaline
intercalator. Biochemistry 35 (10):5022-5032.
Marsh, M. (1984) The entry of enveloped viruses into cells by endocytosis. Biochem. .1.
210(1): 1-10.
Martin, A. (1993) Physical Pharmacy. Lea & Febiger, Philadelphia.
Marzo, A. L., Fitzpatrick, D. R., Robinson, B. W., and Scott, B. (1997) Antisense
oligonucleotides specific for transforming growth factor beta2 inhibit the growth of
malignant mesothelioma both in vitro and in vivo. Cancer Res. 57 (15): 3200-3207.
Maxñeld, F. R. (1985) Acidification of endocytic vesicles and lysosomes. In
Endocytosis. (Pastan, L, and Willingham, M. C., editors) pp. 235-257. Plenum Press,
New York.
McConnaughie, A. W., and Jenkins, T. C. (1995) Novel acridine-triazenes as prototype
combilexins: synthesis, DNA binding, and biological activity. J. Med. Chem. 38 (18):
3488-3501.
McGraw, T. E., and Maxfield, F. R. (1991) Intemalizaiton and sorting of
macromolecules: endocytosis. In Targeted Drug Delivery. (Juliano, R. L., editor) pp. 11-
42. Springer-Verlag, New York.
Melikyan, G. ., Brener, S. A., Ok, D. C., and Cohen, F. S. (1997) Inner but not outer
membrane leaflets control the transition from glycosylphosphatidylinositol-anchored
influenza hemagglutinin-induced hemifusion to full fusion. J. Cell Biol. 136 (5): 995-
1005.
Michaelson, D. M., Horwitz, A. F., and Klein, M. P. (1973) Transbilayer asymmetry and
surface homogeneity of mixed phospholipids in cosonicated vesicles. Biochemistry 12
(14): 2637-2645.
Miller D. K., Griffiths, E., Lenard, J., and Firestone, R. A. (1983) Cell killing by
lysosomotropic detergents. J. Cell Biol. 97 161: 1841-1851.
Morishita, R., Gibbons, G. H., Ellison, K. E., Nakajima, M., Zhang, L., Kaneda, Y.,
Ogihara, T., and Dzau, V. J. (1993) Single intraluminal delivery of antisense cdc2 kinase

180
and proliferating-cell nuclear antigen oligonucleotides results in chronic inhibition of
neointimal hyperplasia. Proc. Natl. Acad. Sci. USA 90 (181: 8474-8478.
Moser, H. E., and Dervan, P. B. (1987) Sequence-specific cleavage of double helical
DNA by triple helix formation. Science 238 (4827): 645-650.
Murakami, A., Blake, K. R., and Miller, P. S. (1985) Characterization of sequence-
specific oligodeoxyribonucleoside methylphosphonates and their interaction with rabbit
globin mRNA. Biochemistry 24 (15): 4041-4046.
New, R. R. C. (1990) Liposomes: a practical approach. Oxford University Press, New
York.
Nussbaum, O., Rott, R., and Loyter, A. (1992) Fusion of influenza virus particles with
liposomes: requirement for cholesterol and virus receptors to allow fusion with and lysis
of neutral but not of negatively charged liposomes. .1. Gen. Virol. 73 (Pt 11): 2831-2837.
Ogo, H., Hirai, Y., Miki, S., Nishio, H., Akiyama, M., and Nakata, Y. (1994) Modulation
of substance P/neurokinin-1 receptor in human astrocytoma cells by antisense
oligodeoxynucleotides. Gen. Pharmacol. 25 161: 1131-1135.
Okada, Y., Koseke, I., Kim, J., Hashimoto, T., Kanno, Y., and Matsui, Y. (1975)
Modification of cell membranes with viral envelopes during fusion of cells with HVJ
(Sendai virus). Exp. Cell Res. 93: 368-378.
O’Keefe, R. T., Mayeda, A., Sadowski, C. L., Krainer, A. R., and Spector, D. L. (1994)
Disruption of pre-mRNA splicing in vivo results in reorganization of splicing factors. L
Cell Biol. 124 (3): 249-260.
Ollikainen, H., Lappalainen, K., Jaaskelainen, I., Syijanen, S., and Pulkki, K. (1996)
Liposomal targeting of bcl-2 antisense oligonucleotides with enhanced stability into
human myeloma cell lines. Leuk. Lymphoma 24 (1-2): 165-174.
Pardridge, W. M., and Boado, R. J. (1991) Enhanced cellular uptake of biotinylated
antisense oligonucleotide or peptide mediated by avidin, a cationic protein. FEBS Lett.
288 (1-2): 30-32.
Párente, R. A., Nir, S., and Szoka, F. C., Jr. (1990) Mechanism of leakage of
phospholipid vesicle contents induced by the peptide GALA. Biochemistry 29 (37):
8720-8728.
Perez, J. R., Li, Y., Stein, C. A., Majumder, S., Van Oorschot, A., and Narayanan, R.
(1994) Sequence-independent induction of Spl transcription factor activity by
phosphorothioate oligodeoxynucleotides. Proc. Natl. Acad. Sci. USA 91 (13): 5957-
5961.

181
Perlaky, L., Saijo, Y., Busch, R. K., Bennett, C. F., Mirabelli, C. K., Crooke, S. T., and
Busch, H. (1993) Growth inhibition of human tumor cell lines by antisense
oligonucleotides designed to inhibit pl20 expression. Anticancer Drug Des. 8 (1): 3-14.
Phillips, N. C., and Emili, A. (1991) Immunogenicity of immunoliposomes. Immunol.
Lett. 30131: 291-296.
Pinnaduwage, P., Schmitt, L., and Huang, L. (1989) Use of a quaternary ammonium
detergent in liposome mediated DNA transfection of mouse L-cells. Biochim. Biophys.
Acta 985 ill: 33-37.
Poxon, S. W., Mitchell, P. M., Liang, E., and Hughes, J. A. (1996) Dendrimer delivery of
Oligonucleotides. Drug Del. 3 (4): 255-261.
Quattrone, A., Papucci, L., Morganti, M., Coronnello, M., Mini, E., Mazzei, T., Colonna,
F. P., Garbesi, A., and Capaccioli, S. (1994) Inhibition of MDR1 gene expression by
antimessenger oligonucleotides lowers multiple drug resistance. Oncol. Res. 6 (7): 311-
320.
Renneisen, K., Leserman, L., Matthes, E., Schroder, H. C., and Muller, W. E. (1990)
Inhibition of expression of human immunodeficiency virus-1 in vitro by antibody-
targeted liposomes containing antisense RNA to the env region. J. Biol. Chem. 265 (27):
16337-16342.
Ropert, C., Lavignon, M., Dubemet, C., Couvreur, P., and Malvy, C. (1992)
Oligonucleotides encapsulated in pH sensitive liposomes are efficient toward Friend
retrovirus. Biochem. Biophys. Res. Commun. 183 (21: 879-885.
Ropert, C., Malvy, C., and Couvreur, P. (1993) Inhibition of the Friend retrovirus by
antisense oligonucleotides encapsulated in liposomes: mechanism of action. Pharm. Res.
10(10): 1427-1433.
Ropert, C., Mishal, Z., Jr., Rodrigues, J. M., Malvy, C., and Couvreur, P. (1996)
Retrovirus budding may constitute a port of entry for drug carriers. Biochim. Biophys.
Acta 1310 tU: 53-59.
Rose, J. K., Buonocore, L., and Whitt, M. A. (1991) A new cationic liposome reagent
mediating nearly quantitative transfection of animal cells. Biotechniques 10 (4): 520-
525.
Rosen, M. J. (1989) Surfactants and Interfacial Phenomena. John Wiley & Sons, New
York.

182
Ruiz, J., Goni, F. M., and Alonso, A. (1988) Surfactant-induced release of liposomal
contents. A survey of methods and results. Biochim. Biophys. Acta 937 11): 127-134.
Sagata, N., Oskarsson, M., Copeland, T., Brumbaugh, J., and Vande-Woude, G. F. (1988)
Function of c-mos proto-oncogene product in meiotic maturation in Xenopus oocytes.
Nature 335 (6190): 519-525.
Saijo, Y., Perlaky, L., Valdez, B. C., Wang, H., Henning, D., and Busch, H. (1993)
Cellular pharmacology of pi 20 antisense oligodeoxynucleotide phosphorothioate ISIS
3466. Oncol. Res. 5 181: 283-291.
Saison-Behmoaras, T., Tocque, B., Rey, I., Chassignol, M., Thuong, N. T., and Helene,
C. (1991) Short modified antisense oligonucleotides directed against Ha-ras point
mutation induce selective cleavage of the mRNA and inhibit T24 cells proliferation.
EMBO.T. 10151 1111-1118.
Sarmiento, U. M., Perez, J. R., Becker, J. M., and Narayanan, R. (1994) In vivo
toxicological effects of rel A antisense phosphorothioates in CD-I mice. Antisense Res.
Dev. 4 (2): 99-107.
Scanlon, K. J., Ohta, Y, Ishida, H., Kijima, H., Ohkawa, T., Kaminski, A., Tsai, J.,
Homg, G., and Kashani-Sabet, M. (1995) Oligonucleotide-mediated modulation of
mammalian gene expression. FASEB J. 9 (13): 1288-1296.
Schaal, H., Klein, M., Gehrmann, P., Adams, O., and Scheid, A. (1995) Requirement of
N-terminal amino acid residues of gp41 for human immunodeficiency virus type 1-
mediated cell fusion. J. Virol. 69 (6): 3308-3314.
Schwab, G., Chavany, C., Duroux, I., Goubin, G., Lebeau, J., Helene, C., and Saison-
Behmoaras, T. (1994) Antisense oligonucleotides adsorbed to polyalkylcyanoacrylate
nanoparticles specifically inhibit mutated Ha-ras-mediated cell proliferation and
tumorigenicity in nude mice. Proc. Natl. Acad. Sci. USA 91 (22): 10460-10464.
Selvam, M. P., Buck, S. M., Blay, R. A., Mayner, R. E., Mied, P. A., and Epstein, J. S.
(1996) Inhibition of HIV replication by immunoliposomal antisense oligonucleotide.
Antiviral. Res. 33 Ill: 11-20.
Sharma, H. W., and Narayanan, R. (1995) The therapeutic potential of antisense
oligonucleotides. Bioessavs 17 (12): 1055-1063.
Sharon, N., and Lis, H. (1989) Lectins as cell recognition molecules. Science 246 (4927):
227-234.

183
Shaw, J. P., Kent, K., Bird, J., Fishback, J., and Froehler, B. (1991) Modified
deoxyoligonucleotides stable to exonuclease degradation in serum. Nucleic Acids Res.
19 (4); 747-750.
Shea, R. G., Marsters, J. C., and Bischofberger, N. (1990) Synthesis, hybridization
properties and antiviral activity of lipid-oligodeoxynucleotide conjugates. Nucleic Acids
Res. IB (13): 3777-3783.
Siegel, D. P. (1986) Inverted micellar intermediates and the transitions between lamellar,
cubic, and inverted hexagonal lipid phases. II. Implications for membrane-membrane
interactions and membrane fusion. Biophvs. 1. 49 (6): 1171-1183.
Silver, G. C., Nguyen, C. FL, Boutorine, A. S., Bisagni, E., Garestier, T., and Helene, C.
(1997) Conjugates of oligonucleotides with triplex-specific intercalating agents.
Stabilization of triple-helical DNA in the promoter region of the gene for the alpha-
subunit of interleukin 2 (IL-2R alpha). Bioconiug. Chem. 8 (1): 15-22.
Slepushkin, V. A., Simoes, S., Dazin, P., Newman, M. S., Guo, L. S., Pedroso De Lima,
M. C., and Duzgunes, N. (1997) Sterically stabilized pH-sensitive liposomes.
Intracellular delivery of aqueous contents and prolonged circulation in vivo. J. Biol.
Chem. 272 (4): 2382-2388.
Smith, C. C., Aurelian, L., Reddy, M. P., Miller, P. S., and Ts'o, P. 0. (1986) Antiviral
effect of an oligo(nucleoside methylphosphonate) complementary to the splice junction of
herpes simplex virus type 1 immediate early pre-mRNAs 4 and 5. Proc. Natl. Acad. Sci.
USA 83 (9): 2787-2791.
Smolarsky, M., Teitelbaum, D., Sela, M., and Gitler, C. (1977) A simple fluorescent
method to determine complement-mediated liposome immune lysis. J. Immunol.
Methods 15 (3): 255-265.
Sokol, D. L., and Gewirtz, A. M. 1996. Gene therapy: basic concepts and recent
advances. Crit. Rev. Eukarvot. Gene Expr. 6 (1): 29-57.
Solodin, L, Brown, C. S., Bruno, M. S., Chow, C., Jang, E., Debs, R. J., and Health, T. D.
(1995) A novel series of amphiphilic imidazolinium compounds for in vitro and in vivo
gene delivery. Biochemistry 34 1411: 13537-13544.
Stein, C. A., and Cheng, Y. C. (1993) Antisense oligonucleotides as therapeutic agents- is
the bullet really magical? Science 261 (51241: 1004-1012.
Stein, C. A., and Cohen, J. S. (1988) Oligodeoxynucleotides as inhibitors of gene
expression: a review. Cancer Res. 48 (10): 2659-2668.

184
Stein, C. A., Mori, K., Loke, S. L., Subasinghe, C., Shinozuka, K., cohén, J. S., and
Neckers, L. M. (1988) Phosphorothioate and normal oligodeoxyribonucleotides with 5’-
linked acridine: characterization and preliminary kinetics of cellular uptake. Gene 72 (1-
2): 333-341.
Stevenson, M., and Iversen, P. L. (1989) Inhibition of human immunodeficiency virus
type 1-mediated cytopathic effects by poly(L-lysine)-conjugated synthetic antisense
oligodeoxyribonucleotides. J. Gen. Virol. 70 (Pt 10): 2673-2682.
Stewart, A. J., Pichón, C., Meunier, L., Midoux, P., Monsigny, M., and Roche, A. C.
(1996) Enhanced biological activity of antisense oligonucleotides complexed with
glycosylated poly-L-lysine. Mol. Pharmacol. 50 161: 1487-1494.
Stewart, J. C. (1980) Colorimetric determination of phospholipids with ammonium
ferrothiocyanate. Anal. Biochem. 104 til: 10-14.
Still, W. C., Kahn, M. and Mitra, A. (1978) Rapid chromatographic technique for
preparative separations with moderate resolution. J. Org. Chem. 43 (14): 2923-2926.
Struck, D. K., Ploekstra, D., and Pagano, R. E. (1981) Use of resonance energy transfer to
monitor membrane fusion. Biochemistry 20 (14): 4093-4099.
Szoka, F., and Papahadjopoulos, D. (1978) Procedure for preparation of liposomes with
large internal aqueous space and high capture by reverse-phase evaporation. Proc. Natl.
Acad. Sci. USA 75 (9): 4194-4198.
Takle, G. B., Thierry, A. R., Flynn, S. M., Peng, B., White, L., Devonish, W., Galbraith,
R. A., Goldberg, A. R., and George, S. T. (1997) Delivery of oligoribonucleotides to
human hepatoma cells using cationic lipid particles conjugated to ferric protoporphyrin
IX (heme). Antisense Nucleic Acid Drug Dev. 7 131: 177-185.
Thierry, A. R., and Dritschilo, A. (1992) Intracellular availability of unmodified,
phosphorothioated and liposomally encapsulated oligodeoxynucleotides for antisense
activity. Nucleic Acids Res. 20 1211: 5691-5698.
Toulme, J. J., Krisch, H. M., Loreau, N., Thuong, N. T., and Helene, C. (1986) Specific
inhibition of mRNA translation by complementary oligonucleotides covalently linked to
intercalating agents. Proc. Natl. Acad. Sci. USA 83 151: 1227-1231.
Trouet, A., Deprez-De Campaneere, D., and De Duve, C. (1972) Chemotherapy through
lysosomes with a DNA-daunorubicin complex. Nat. New Biol. 239 1911: 110-112.
Uhlman, E., and Peyman, A. (1990) Antisense oligonucleotides: a new therapeutic
principle. Chem. Rev. 90: 543-584.

185
Uhlman, E., Peyman, A., and Will, D. W. (1997) Antisense: chemical modification. In
Encyclopedia of Cancer. (Bertino, J. R., editor) pp. 64-81. Academic Press, San Diego.
Verspieren, P., Comelissen, A. W., Thuong, N. T., Helene, C., and Toulme, J. J. (1987)
An acridine-linked oligodeoxynucleotide targeted to the common 5' end of trypanosome
mRNAs kills cultured parasites. Gene 61 (3): 307-315.
Vlassov, V. V., Balakireva, L. A., and Yakubov, L. A. (1994) Transport of
oligonucleotides across natural and model membranes. Biochim. Biophys. Acta 1197
(2): 95-108.
Vogel, S. S., Chemomordik, L. V., Zimmberberg, J. (1992) Calcium-triggered fusion of
exocytotic granules requires proteins in only one membrane. J. Biol. Chem. 267 (36):
25640-25643.
Wagner, R. W., Matteucci, M. D., Lewis, J. G., Gutierrez, A. J., Moulds, C., and
Froehler, B. C. (1993) Antisense gene inhibition by oligonucleotides containing C-5
propyne pyrimidines. Science 260 (51131: 1510-1513.
Walter, N. G. (1995) Modelling viral evolution in vitro using exo- Klenow polymerase:
continuous selection of strand displacement amplified DNA that binds an
oligodeoxynucleotide to form a triple-helix. J. Mol. Biol. 254 (5): 856-868.
Wang, S., Lee, R. J., Cauchon, G., Gorenstein, D. G., and Low, P. S. (1995) Delivery of
antisense oligodeoxyribonucleotides against the human epidermal growth factor receptor
into cultured KB cells with liposomes conjugated to folate via polyethylene glycol. Proc.
Natl. Acad. Sci. USA 92 (8): 3318-3322.
Wielbo, D., Simon, A., Phillips, M. L, and Toffolo, S. (1996) Inhibition of hypertension
by peripheral administration of antisense oligodeoxynucleotides. Hypertension 28 (1):
147-151.
Wilson, P. D., Fireston, R. A., and Lenard, J. (1987) The role of lysosomal enzymes in
killing of mammalian cells by the lysosomotropic detergent N-dodecylimidazole. J. Cell
Biol. 104 (5): 1223-1229.
Wilson, P. D., Hreniuk, D., and Lenard, J. (1989) Reduced cytotoxicity of the
lysosomotropic detergent N-dodecylimidazole after differentiation of HL60
promyelocytes. Cancer Res. 49 (31: 507-510.
Wu, G. Y., and Wu, C. H. (1992) Specific inhibition of hepatitis B viral gene expression
in Vitro by targeted antisense oligonucleotides. J. Biol. Chem. 267 (18): 12436-12439.
Yeagle, P. L. (1997) Membrane fusion intermediates. Curr. Top. Membr. 44: 375-401.

186
Yeagle, P. L. (1994) Lipids and lipid-intermediate structures in the fusion of biological
membranes. Curr. Top. Membr. 40:197-214.
Yeagle, P. L. (1993) The Membranes of Cells. Academic Press, San Diego.
Yeoman, L. C., Dañéis, Y. J., and Lynch, M. J. (1992) Lipofectin enhances cellular
uptake of antisense DNA while inhibiting tumor cell growth. Antisense Res. Dev. 2 (1):
51-59.
Yonemitsu, Y., Kaneda, Y., Muraishi, A., Yoshizumi, T., Sugimachi, K., and Sueishi, K.
(1997) HVJ (Sendai virus)-cationic liposomes: a novel and potentially effective
liposome-mediated technique for gene transfer to the airway epithelium. Gene Ther. 4
(7): 631-638.
Yu, Y. G., King, D. S., and Shin, Y. K. (1994) Insertion of a coiled-coil peptide from
influenza virus hemagglutinin into membranes. Science 266 (5183): 274-276.
Zabner, J., Fasbender, A. J., Moninger, T., Foellinger, K. A., and Welsh, M. J. (1995)
Cellular and molecular barriers to gene transfer by a cationic lipid. J. Biol. Chem. 270
(32): 18977-19007.
Zamecnik, P. C. (1996) History of antisense oligonucleotides. In Antisense Therapeutics.
(Agrawal, S., editor) pp. 1-11. Humana Press, Totowa.
Zamecnik, P. C., and Stephenson, M. L. (1978) Inhibition of Rous sarcoma virus
replication and cell transformation by a specific oligodeoxynecleotide. Proc. Natl. Acad.
Sci. USA 75 (1): 280-284.
Zelphati, O., Imbach, J. L., Signoret, N., Zon, G., Rayner, B., and Leserman, L. (1994)
Antisense oligonucleotides in solution or encapsulated in immunoliposomes inhibit
replication of HIV-1 by several different mechanisms. Nucleic Acids Res. 22 (20): 4307-
4314.
Zelphati, O., and Szoka, F. C., Jr. (1996a) Intracellular distribution and mechanism of
delivery of oligonucleotides mediated by cationic lipids. Pharm. Res. 13 (9): 1367-1372.
Zelphati, O., and Szoka, F. C., Jr. (19966) Mechanism of oligonucleotide release from
cationic liposomes. Proc. Natl. Acad. Sci. USA 93 (21): 11493-11498.
Zelphati, Q., Zon, G., and Leserman, L. (1993) Inhibition of HIV-1 replication in cultured
cells with antisense oligonucleotides encapsulated in immunoliposomes. Antisense Res.
Dev. 3 f41: 323-338.

187
Zhang, R., Lu, Z., Zhao, H., Zhang, X., Diasio, R. B., Habus, L, Jiang, Z., Iyer, R. P., Yu,
D., and Agrawal, S. (1995) In vivo stability, disposition and metabolism of a "hybrid"
oligonucleotide phosphorothioate in rats. Biochem. Pharmacol. 50 (4): 545-556.
Zhao, Q., Temsamani, J., and Agrawal, S. (1995) Use of cyclodextrin and its derivatives
as carriers for oligonucleotide delivery. Antisense Res. Dev. 5 (3): 185-192.
Zhou, X., and Huang, L. (1994) DNA transfection mediated by cationic liposomes
containing lipopolylysine: characterization and mechanism of action. Biochim. Biophvs.
Acta 1189 (2): 195-203.

BIOGRAPHICAL SKETCH
Chih-Wei Earvin Liang was bom on December 21, 1971 in Chang-Hwa, Taiwan.
Earvin entered the Taipei Medical College in October 1989 and obtained his Bachelor of
Science degree in pharmacy in June 1993. He was then admitted to the Department of
Pharmaceutics at the University of Florida in August 1994. He received his Doctor of
Philosophy in pharmaceutical sciences in August 1998 under the supervision of Dr.
Jeffrey A. Hughes. During his free time, Earvin enjoys jogging, working out, listening to
music, and watching all sorts of ball games.
188

I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy. ,f / j /j
Jeffrey ^. 'Hughes, Chair
Assistant Professor of Pharmaceutics
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
bfo 0, Y)áM¿QüuJ
Gayle A. Brazeau
Associate Professor of Pharmaceutics
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
•if
cfo, ^
Hartmut Derendorf
Professor of Pharmaceutics
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
Laszlo Prokai
Associate Professor of Pharmaceutics
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, hi scope and quality,
as a dissertation for the degree of Doctor of Philosophy. f\
ion Schusfer
Professor of Biochemistry and Molecular
Biology

I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
>>
A*>Q
Ian R. Tebbett
Professor of Medicinal Chemistry
This dissertation was submitted to the Graduate Faculty of the College of .
Pharmacy and to the Graduate School and was accepted fas partiahfulfillment pf the
requirements for the degree of Doctor of Philosophy. / /,/ /{/] A /////'}
August, 1998
Dean, Col
harmacy
Dean, Graduate School