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1 NOVEL LIGHT ACTIVATED TUMOR TARGETING DRUG CARRIERS USING SICK L E RED BLOOD CELLS By SE WOON CHOE A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR T HE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2011
2 2011 Se woon Choe
3 To parents, wife and lovely children: Younghoon Choe and Younja Son, Yoonsun Choi, Jean Choe and Andrew Hanwool Choe
4 ACKNOWLEDGMENTS I would lik e to sincerely than k to my advisor, Dr. Brian S. Sorg, for his excellent support and guidance to finish my Ph.D. study. Dr. Sorg always inspired me how to be an independent researcher with more creative and critical pers pective. His voice was always calm b ut powerful. W ithout his advice this dissertation would not be possible. I am indebted to my committee members, Dr. Dietmar W. Siemann Dr. Johannes (Hans) van Oostrom and Dr. Rosalind J. Sadleir for their constructive criticism and assistance. I also wou ld like to express my gratitude to Dr. Angela E Rivers and Dr. Richard Lottenberg for their collaboration I am grateful to Dr. Paul S. Oh for his interest and support, which helped improve the quality of this dissertation. I also thank his lab members Ch ul Han, Yong h wan Kim and Dr. Chul Song, for giving me a warm welcome whenever I visit to ask their help. I have been very fortunate to work with all past and current Ke Sorg le lab members I would like to acknowledge Dr. Mamta Wankhede Raymond Kozikowski Jennifer Lee Casey deDeugd, Nikita Agarwal Phillip Barish, and Dr. Abhinav Acharya for their helpful discussion I also thank my Korean friends, Dr. Soondo Yoon, Dr. Sungho Oh, Choongheon Lee, Kyungpyo Hong, Dr. Sahngmin Han, Hyochul Ahn, Myungsang Kim, Jintaek Ock and Jinseok Hong for their cheerful encouragement and friendship. I greatly appreciate my parents and parents in law Younghoon Choe Younja Son, Sangkyun C h oi and Soohee Yu, for their endless love and support. Especially, my parents always ch eer and strengthen me throughout my life. I also appreciate my sisters and brothers Koheun Choe Soeun Choe Arma Choe Soomin Park, Soonhyoung Joung, and Yoongyu Choi for their love and concern. I am so happy to be a proud father
5 to my kids, Jean Choe an d Andrew Hanwool Choe who are the most precious gifts in my life Last but not least, I am so grateful to my soul mate, lovely wife Yoonsun Choi
6 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 9 LIST OF FIGURES ................................ ................................ ................................ ........ 10 ABSTRACT ................................ ................................ ................................ ................... 12 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 13 Current Drug Delivery Strategies to Tumors ................................ ........................... 13 Enhanced Permeability and Retention Eff ect Based Passive Targeting to Solid Tumors ................................ ................................ ................................ ................ 15 Resealed Erythrocytes as a Drug Carrier ................................ ............................... 18 Sickle Red Blood Cells as a Potential Drug Carrier ................................ ................ 21 Controlled Drug Release by Photoactivation using ................................ ................. 23 Gompertz Function ................................ ................................ ................................ 26 Microdialysis Method for in vivo Delayed Photohemolysis Measurement ............... 27 Motivation and Goal of this Study ................................ ................................ ........... 34 2 MATERIALS AND METHODS ................................ ................................ ................ 35 Window Chamber Installed Mouse M odel ................................ ............................... 35 Imaging System ................................ ................................ ................................ ...... 36 Imaging Acquisition ................................ ................................ ................................ 37 Hyperspectral Imaging of Hemoglobin Saturation map ................................ ........... 37 Delayed Photohemolysis Me asurements ................................ ................................ 39 In vitro Delayed Photohemolysis Measurement Systems ................................ 39 In vivo Delayed Photohemolysis Measurement using Microdialy sis Tubing ..... 40 3 INTRAVITAL MICROSCOPY IMAGING OF MACROPHAGE LOCALIZATION TO IMMUNOGENIC PARTICLES AND CO LOCALIZED TISSUE OXYGEN SATURATION ................................ ................................ ................................ ......... 46 Materials and Methods ................................ ................................ ............................ 48 Microparticles ................................ ................................ ................................ ... 48 Macrophage Cell Line ................................ ................................ ...................... 50 Animal Model ................................ ................................ ................................ .... 50 Intravital Fluorescence and Spectral Imaging ................................ ................... 52 Results ................................ ................................ ................................ .................... 53 Hyperspectral and Fluorescence Imaging ................................ ........................ 53
7 Quantitative A nalysis for Hemoglobin Saturation Map, Microparticles and Macrophages ................................ ................................ ................................ 54 Statistical Analysis for A ccumulated Region of Interests D ata ......................... 55 Discussion ................................ ................................ ................................ .............. 64 4 PREFERENTIAL ACCU MULATION OF SICKLE RED BLOOD CELL IN TUMORIC ENVIRONMENT ................................ ................................ .................... 69 Materials and Methods ................................ ................................ ............................ 71 Tumor Cells ................................ ................................ ................................ ...... 71 Animal M odel ................................ ................................ ................................ .... 72 Window C hamber I nstallation ................................ ................................ ........... 72 Preparation of Blood Cells ................................ ................................ ................ 73 Intravital Spectral and Fluorescence I maging ................................ ................... 74 Histology ................................ ................................ ................................ ........... 76 Results ................................ ................................ ................................ .................... 77 Monitoring Hb Saturation Map ................................ ................................ .......... 77 In vivo Numerical Analysis of Accumulated Sickle Red Blood Cells using a Single Fluorescent Microscopic Imag e ................................ .......................... 77 In vivo Numerical Analysis of Accumulated Sickle Red Blood Cells using Fluorescence Video Recording ................................ ................................ ..... 79 Histological An alysis of Accumulated Sickle Red Blood Cells in Liver, Spleen and Tumor Tissues ................................ ................................ ........... 80 Discussion ................................ ................................ ................................ .............. 88 5 IN VIVO DELAYED PHOTOHEMOLYSI S MEASUREMENT FROM PHOTOSENSITIZED SSRBCs LOADED WITH CALCEIN in 4T1 TUMOR BEARING MOUSE MODEL ................................ ................................ .................... 91 Materials and Methods ................................ ................................ ............................ 93 Mater ials ................................ ................................ ................................ ........... 93 Preparation of Blood Cells ................................ ................................ ................ 93 Preparation of Loaded Blood Cells with Calcein ................................ ............... 94 Preparation of Photosensitized Blood Cells by PpIX ................................ ........ 95 In V itro Delayed Photohemolysis M easurement ................................ ............... 95 Gom pertz F unction ................................ ................................ ........................... 96 Tumor C ells ................................ ................................ ................................ ...... 97 Animal M odels ................................ ................................ ................................ .. 97 In vivo Delaye d Photohemolysis using Microdialysis Tubing ............................ 98 Results ................................ ................................ ................................ .................... 99 Efficacy of Calcein Loading Rate into Sickle Red Blood Cells .......................... 99 In vitro Delayed Photohemolysis Measurement of Photosensitized SSRBCs by PpIX ................................ ................................ ................................ ........ 100 Analysis of in vitro Delayed Photohemolysis Measurem ent by Gompertz Function ................................ ................................ ................................ ...... 101 In vivo Controlled Calcein Release Form Sickle Red Blood Cells .................. 101 Discussion ................................ ................................ ................................ ............ 112
8 6 CONCLUSION S AND FUTURE WORKS ................................ ............................. 115 Summary and Conclusion ................................ ................................ ..................... 115 Future Work s ................................ ................................ ................................ ........ 116 LIST OF REFERENCES ................................ ................................ ............................. 118 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 137
9 LIST OF TABLES Table page 1 1 Drug delivery systems (DDS) in clinical trials and practice (adapted from ) ... 14 1 2 Factors responsible for the EPR effect of macromolecule s in solid tumors (adopted from ) ................................ ................................ ............................. 17 1 3 Comparison of carrier erythrocyte to other DDS in mouse model (adapted from ) ................................ ................................ ................................ ............ 18 1 4 Advantages and disadvantages of erythrocytes in drug delivery (adopted from ) ................................ ................................ ................................ ............ 20 1 5 Comparison of various hypo osmotic lysis methods (adapted from [62, 63]) ..... 21 1 6 Summary of advantages and limitations of microdialysis sampling technique (adopted from ) ................................ ................................ ........................... 29 4 1 Categorized groups for preferential accumulation of SSRBCs to tumoric microvasculature experiment. ................................ ................................ ............. 71 5 1 Categorized groups for in vivo DPH measurement of SSRBCs to tumoric microvasculature experiment. ................................ ................................ ............. 98 5 2 SSRBCs with 25M PpIX irradiated with 0.08W halogen lamp at various irradiation times at fixed irradiation and incubation temperature ....................... 108
10 LIST OF FIGURES Figure page 1 1 Pathophysiology of vaso occlusion by SSRBCs ................................ ................ 30 1 2 DPH measurement from photoactivated RBCs by PpIX with 10 M. ................... 31 1 3 Example plot of age specific mortality rates with random parameters by Gompertz function ................................ ................................ ............................. 32 1 4 Typical microdialysis probe configurations and samplin g process. ..................... 33 2 1 Window chamber installed mouse model ................................ .......................... 41 2 2 Hyperspectral imaging system. ................................ ................................ ........... 42 2 3 Hyperspectral imaging to create HbSat map ................................ ...................... 43 2 4 Experimental setup for in vitro DPH measurements. The sample cuvette in temperature controller at under physiol ogical temperature is exposed by 0.08W halogen lamp. ................................ ................................ .......................... 44 2 5 In vivo DPH measurement setup with 4T1 tumor mass bearing mouse model. 45 3 1. Hyperspectral and fluorescence i mages for a control mouse with saline (carrier) injection without microparticles. ................................ ............................ 57 3 2 Hyperspectral and fluorescence i mages for a LPS( ) mouse with m icroparticles lacking LPS ................................ ................................ ................. 58 3 3 Hyperspectral and fluorescence images for a LPS(+) mouse with LPS ................................ ................. 59 3 4 Hyperspectral and fluroescence images for LPS(++) mouse with LPS ). ................................ .............. 60 3 5 Numerical HbSat data for each group of ROIs at various time points. The ROIs are selected on arterioles and venules separately. ................................ ... 61 3 6 Numerical macrophage signals for each group of ROIs at various time points. The ROIs are selected on arterioles and venules separately. ............................ 62 3 7 The accumulated ROI data overall ti me points are analyzed using ANOVA hoc test. ................................ ................................ ............... 63 4 1 Hyperspectral images of dorsal skinfold window chamber. ................................ 82
11 4 2 Microscopic images of postcapillary venules with transmitted light and fluorescence images were acquired. ................................ ................................ .. 83 4 3 Bar graphs show numerical fluorescence intensities on select ed ROIs in each group (meanSD) ................................ ................................ ..................... 84 4 4 In vivo numerical analysis of accumulated RBCs and SSRBCs using video recording with 20x objective. ................................ ................................ .............. 85 4 5 Fluorescently labeled cells in tissue specimens including liver, spleen and tumor were compared. ................................ ................................ ........................ 87 5 1 Light microscope and fluorescence flow cytometry techniques were employed to analyze the efficiency of hypotonic preswelling method to SSRBCs. ................................ ................................ ................................ .......... 103 5 2 Delayed photohemolysis in normal canine RBCs photosensitized with PpIX were measured at 415nm. ................................ ................................ ................ 104 5 3 Fractional photohemolysis rates of photosensitized SSRBCs by 25 M of PpIX were measured ................................ ................................ ....................... 107 5 4 Parameters for Gompertz function were calculated based on f ractional DPH measurement of SSRBCs carrying calcein with various irradiation time ( t irr ). ... 109 5 5 In vivo fluorescence signal measurement through systemic administration of prepared blood samp le groups ................................ ................................ ........ 111
12 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 NOVEL LIGHT ACTIVATED TUMOR TARG ETING DRUG CARRIERS USING SICKL E RED BLOOD CELLS By Se woon Choe August 201 1 Chair: Brian S. Sorg Major : Biomedical Engineering Conventional drug carriers such as liposomes nanoparticles and red blood ce lls for cancer chemotherapy shield normal tissues from toxic drugs to treat cancer cells in tumors. However, inaccurate tumor targeting, uncontrolled drug release from the carriers and unwanted accumulation in healthy sites can limit treatment efficacy wit h current conventional drug carriers with insufficient concentrations of drugs in the tumors and unwanted side effects (i.e., systemic cytotoxicity) as a result. In this research, we examined the use of sickle red blood cells as a new drug carrier with no vel tumor targeting and controlled release properties. Sickle red blood cells show natural tumor preferential accumulation without any manipulation and controlled drug release is possible using a hemolysis method with photosensitizers. These properties wer e demonstrated in vitro and live animal models. Through this research, we suggest that sickle red blood cells may have the potential to be a new drug carrier with better tumor targeting and controllable drug release for improved chemotherapy in advanced c ancer patients
13 CHAPTER 1 INTRODUCTION Systemic chemotherapy by intravenous administration has been one commonly used and standard method in the treatment of solid tumors due to the ease of administration, rapid reaction,and avoidance of the gastrointest inal tract (GI tract) problems with oral drug administration  Generally, multiple cycle s of chemotherapeutic treatments a re required because the efficacy of treatment is directly proportional to the given drug concentration in target sites  Despite impressive progress in development of chemotherapeutic agents, most challenges remai n such as inadequate therapeutic agent doses to the tumor sites by inaccurate delivery and adverse toxic effects on normal organs (systemic toxicity). For example, highly proliferative normal cells such as bone marrow and GI mucosa a re susceptible to the f atal cytotoxicity of chemotherapeutic agents and reveal the adverse side effects such as a dose response effect by indiscriminate distribution of administered drug. Therefore, new drug delivery strategies that c an improve therapeutic index and adequate co ncentration of drug delivery to tumor sites are needed [1, 2] Current Drug Delivery Strategies to Tumors Numerous drugs have been developed for chemotherapeutics, but the effective drug delivery system (DDS) of th erapeutic agents to tumors has been limited  Among v arious approaches to specific DDS to tumor sites, two targeting methods seem to be most general in clinical trials and practice and summarized in Table 1 1. Active targeting employs molecular interaction in pairs of specific antigen / monoclonal antibody, ligand / receptor and aptamer / counter part between on the surface of pharmaceutical carriers and in targeted cell sites [3, 4]
14 Table 1 1 Drug delivery systems (DDS) in clinical trials and practice (adapted fro m  ) Name Type of DDS Type of t argeting Stage of approval for clinical practice Therapeutic agent Doxil Liposome Passive Approved for clinical use Doxorubicin DaunoXone Liposome Passive Approved for clinical use Daunorubicin Abraxane Albumin based polymer Passive Approved for clinica l use Paclitaxel Bexxar Immunoconjugate Active Approved for clinical use Radioactive iodine SMANCS Nanopolymer Passive Approved for clinical use Neocarzinostatin NK105 Micelle Passive Phase 2 Paclitaxel Xyota Nanopolymer Passive Phase 3 Paclitaxel MBP 426 Liposome Active Phase 1 Oxaliplatin An example of active targeting is the binding of folate to pegylated particles for interaction with the folate receptor. The cells of many cancers overexpress folate receptors on their surface and have a higher bi nding affinity for the nanoparticle folate bound protein than for free folate  However, the rapid clearance of ligand directed immunoliposomes owing to non specific uptake by the cells of reticulo endothelial system (RES) reduced the therapeutic effica cy [7, 8] Antibodies conjugated with liposomes limit targeting to intravascular targets due to the increased size of the liposomes and reduced release rate of entrapped drugs  On the other hand, passive targeting employs the prolonged circulating time of the dr ug carriers in the blood stream with reduced accumulation in the normal organs and sufficient levels of accumulation in tumor sites through the enhanced permeability and retention (EPR) effect [3, 4] Longevity of drug carriers in the circulation increas es the probability for drug loaded carriers to find large fenestrations on the blood vessels with modest loss of drug concentration during circulation in the blood stream.
15 E nhanced P ermeability and R etention Effect B ased Passive Targeting to Solid Tumors The EPR effect is the mechanism by accumulation of macromolecular prodrugs in sites of inflammation or cancer by increased vascular permeability. In 1986, EPR effect was described by Matsumura et al. and was attribute d as the basis for the selective targeting of macromolecular drugs to the site of solid tumors [1 0] In their experiment, Evans blue (which forms a noncovalent, macromolecular complex with albumin) showed decreased clearance rate from tumor tissue for 3 4 weeks compared with healthy tissue less than 1 week  In addition, o ne study reported that EPR is affected by the tumor size, with a greater EPR in smaller tumors, probably because the gre ater vessel density as compared to larger tumors containing an avascular region  Tumor cells tend to aggregate when they proliferat e When the mass of tumor cells reach 2 3 mm in diameter, angiogenesis is induced because of the excessive nutrition and oxygen demands of the growing tumors  As opposed to normal neovasculature, tumor neovasculature differs greatly in an atomical structure  For in stance, the tumor vasculatures are characterized by higher vascular density, unorganized vascular distribution, uneven diameter of blood vessels, and higher permeability with large fenestration  The pores caused by this discontinuity of endothelium are involved in the enhanced permeability or leakiness in tumor microvasculature. Hobbs et al. and Yuan et al. d etermined the pore size of tumor vasculatures using transplanted murine tumors [15 17] It varied from 100 to 780 nm in diameter depending on the anatomic location of the tumor and tumor growth. On the other hand, the fenestration in normal microvasculature with the tight junctions was usually less than 2nm  and larger at up
16 to 6nm in postcapillary venules  Additionally, discontinuous endothelium existing in the kidney glomerulus and the sinusoidal endothelium of the liver and spleen are ranging from 40 to 60nm in width and up to 150nm in width, respectively  Therefore, the major pathway of drug transport across tumor microvascular wall is by extravasation via diffusion and/or convection through the discontinuous endot helial junctions in solid tumors. Also, poor lymphatic drainage in solid tumor decreases the clearance of macromolecular compounds from tumor interstitium [21 23] Consequently, the anatomical and functional defect iveness in solid tumors result in extensive leakage of blood plasma components and enhanced retention in tumor tissues. This phenomenon was recognized as the EPR effect and EPR effect could be observed in almost all human cancers with except for prostate c ancer or pancreatic cancer  Accordingly, EPR effect became most commonly used term and predominant factor to increase specific tumor targeting therapy. The active targeting process cannot be se parated from the passive because it occurs only after passive accumulation in tumors. Additionally, elevated levels of growth factors such as vascular endothelial growth factor (VEGF) [25, 26] basic fibroblast gro wth factor (bFGF)  and other vasoactive factors (bradykinin and nitric oxide) were determined as compared to normal tissues. In Table 1 2, the factors responsible for the EPR effect of macromolecules in solid tumors are summarized. For instance, the overexpressed bradykinin cause vasodilatation and EPR effect in tumors  The principle of the EPR effect has prevailed in numerous strategies including active targeting to overcome systemic delivery limitations 
17 Table 1 2 Factors responsible for the EPR effect of macromolecules in solid tumors (adopted from  ) Anatomical factors Extensive angiogenesis and high vascular density Lack of smooth muscle layer, pericytes, sporadic blood flow passive dilation of vessels in the angiotensin II (AT II) induced hypertensive state more leaka ge Defective vascular architecture extensive leakage Meager lymphatic clearance enhanced retention of macromolecular drugs and lipid particles in the interstitium of tumors Slow venous return accumulation of macromolecules in the interstitium Gene ration of permeability enhancing factors Vascular endothelial growth factor (VEGF) Bradykinin (BK) and/or hydroxypropyl BK Nitric oxide (NO) Peroxynitrite (ONOO ), a reaction product of superoxide radical and NO Prostaglandins (PGs) Matrix metallopro teinases (MMPs) proMMP is activated by ONOO Other proteinases (e.g. kallikrein system) involved in various protease cascades Other cytokines (e.g. tumor necrosis factor, interleukin 2) facilitate EPR effect For example, many drug carriers includ ing nanoparticles  polymer micelles  polymeric conjugate  lipid microemulsion  liposomes  and resealed erythrocytes  have been developed based on EPR effect of solid tumors. In Table 1 3, commonly used DDSs are summarized and compared to other carriers and RBCs Among current drug delivery system (DDS), red blood cell (RBCs) carrier s have longer half life in blood stream than others. RBCs could be potential drug carriers if carrier longevity in the circulation is considered a crucial value in passive targeting because the accumulation of drug carriers is slow, occurring and stabilizi ng over 1 2 days by passive targeting methods [29, 35] However, studies of cytotoxic chemotherapy have revealed that the adverse side effects into normal cells, low concentration level of packaging of the drug int o drug carriers, slow non specific degradation of carriers, and uncontrolled and passive drug release in tumor sites a re the key obstacles in conventional chemotherapeutics 
18 Table 1 3. Comparison of carrier erythro cyte to other DDS in mouse model (adapted from  ) Carriers Size [nm] Shape Half life in blood Diffusion in tissues Murine RBCs 5,000~7,000 Biconcave disc 10~15 days RES openings PEG Lipos omes 50~500 Spheres 3~6 hours Tumors (EPR), endocytosis Polymersomes 50~500 Spheres 10~20 hours Tumors (EPR), endocytosis Filomicelles 4020,000 Filaments 1~3 days Unknown, possibly EPR Polymer micelles 20~300 Spheres 0.1~6 hours Tumors (EPR), endocyt osis Proteins and conjugates 5~5,000 Irregular sphere 10 min~6 hours Diffusion and endocytosis Reddy [2, 8, 36] described that solvents or stabilizers for nanoparticles possibly caused toxicity during nanopartic le production so that consistent and stable substances are needed. Andre son et al.  Chen et al.  and Pakunlu et al.  de monstrated that liposome based formulation s could decrease the systemic cytotoxicity in normal sites, but its rapid clearance owing to non specific uptake by the cells of RES reduced the therapeutic efficacy. Also, s everal limitations including insufficien t accumulation level of drug carrier and drug release into the tumor sites were observed by synthetic drug carriers  Resealed Erythrocytes as a Drug Carrier Among drug carriers, erythrocytes (non nuclear biconc ave discs with a thickness ~2 m, a diameter ~7m and plasma membrane surface area ~160 m 2 ) loaded by therapeutic agents have been exploited extensively for both temporally and spatially contr olled chemotherapeutic carriers. The erythrocytes have been giv en the furthermost interests for their potential applications because of various advantages. The advantages and limitations for erythrocytes as intravenous drug carriers are summarized in table 1 4.
19 Briefly, its relatively long lifespan [39, 40] compared to other drug delivery systems (i.e., <10 hrs for PEG modified liposomes) was considered as a crucial feature for the passive targeting because the accumulation of drug carriers is slowly occurring and stabilized ov er 1 2 days by passive targeting method [29, 35] In addition, restriction of unwanted extravasation except hepatic sinuses and interstitium in the splenic follicles [35, 39 40] high biocompatibility without any undesired immune responses and b iodegradability by macrophages [41, 42] These promising features of analogous RBCs have been attracting more interest as intravenous slow r elease carriers in numerous animals [43 45] and human studies  Gardos tried to encapsulate ATP into human erythrocytes for the first time in 1954  Nineteen years later, Ihler et al. first used the human erythrocyte as the carrier to deli ver enzymes in the human body to heal certain diseases successfully  Consequently, various types of erythrocytes from human beings  and different animal species, s uch as rats  mice  rabbits  and dogs  were applied to deliver anttineoplasms  ,antiparasitics  antiretroviral agents  ,vitamins  ,steroids  antibiotics  and cardiovascular drugs  According to previously documented exam pl es regarding d rug loaded normal RBCs, osmosis based techniques including hypotonic hemolysis, hypotonic dilution hypotonic dialysis, hypotonic preswelling, and osmotic pulse have been deve loped and popularly applied with high entrapment efficacy so far [41, 59 61] Various hypo osmotic lysis methods are reviewed in table 1 5.
20 Table 1 4 Advantages and disadvantages of erythrocytes in drug delivery (adopted from  ) Advantages A remarkable degree of biocompatibility Complete biodegradability and the lack of toxicity resulting fro m the carrier degradation Avoidance of any undesired immune responses against the encapsulated drug Considerable protection of the organism against the toxic effects of the encapsulated drug, e.g., antineoplasms Longer life span of the carrier erythrocyte s in circulation An easily controllable life span within a wide range from minutes to months Considerably uniform size and shape Protection of the loaded compound from inactivation by the endogenous factors Possibility of targeted drug delivery to the R ES organs Relatively inert intracellular environment Wide variety of compounds with the capability of being entrapped within the erythrocytes Possibility of loading a relatively high amount of drug in a small volume of erythrocytes Disadvantages Clearanc e by RES The rapid leakage of certain encapsulated substances from the loaded erythrocytes Several molecules may alter the physiology of the erythrocyte Some inherent variations in their loading and characteristics Possible contaminations Since presw elling method shows relatively higher entrapment efficacy [62, 63] better survival rate in systemic circulation [62, 63] simpler preparation steps among osmosis based loa ding methods  and minimal damage to cells during drug loading procedures  the preswelling method was selected in this experiment. Hypotonic preswelling method is derived from the fact that exposing RBCs in a hypotonic solution cause an enlargement of pores with diameter s of 200 to 500 (20~50 n m) in the me mbrane when it reaches lysis point  At this point, instantly added aqueous solution of the drug can come across through these pores into the interior of the cells. A calcul ated hypertonic buffer is added to restore the tonicity and reseal the openings in membrane. Then, the cell suspension is incubated in 37C for about 30min to reanneal the membrane and washed out twice to remove uncaptured drugs into RBCs  Loss
21 of hemoglobin (Hb) and intracellular components during this loading procedures can be compensated by added aliquo t of a hemolysate. The resealed erythrocytes that retain most of Hb and other cellular contents of normal erythrocytes are re ferred to as  and s uch resealed cells have a circulation half life span comparable to that of normal cells  Table 1 5 Comparison of various hypo osmotic lysis methods (adapted from [62, 63] ) Method Entrapment efficiency [%] Advantages Disad vantages Dilution method 1 8 Fastest and simplest especially for low molecular weight drugs Entrapment efficiency is less Dialysis method 30 50 Better in vivo survival of erythrocytes better structural integrity of membrane Time consuming heterogeneous s ize distribution of resealed erythrocytes Preswelling method 30 90 Good retention of cytoplasm constituents and good survival in vivo Isotonic osmotic lysis method Better in vivo surveillance Impermeable only to large molecules, process is time consum ing Sickle Red Blood Cells as a Potential Drug Carrier Sickle cell disease (SCD) is one of the most prevalently inherited hemolytic anemia in the United States  It affects approximately 70,000 100,000 people of predominantly African descent in the United States. However, Caribbean, East Indian, Arabian, Mediterranean and South and Central American decent al so have high risk genotypes as well  It also affects m illions of individuals worldwide in regions with endemic malaria, such as Africa, the Middle East, and India [70, 71]
22 A single amino acid substitution (Glu Val) globin chain of HbS results in mutated sickle hemoglobin (HbS). This mutation cause s polymerization of HbS, damage to the membrane and cytoskeleton of RBCs, massive cation loss, and increased erythrocyte surface expression of adhesion molecule rec eptors in the presence of decreased pH and hypoxic conditions. These abnormalities result in decreased deformability, increased rigidity and viscosity, and dehydration of sickle RBCs (SSRBCs) [72 76] The mechanica l events lead to hemolysis, reticulocytosis, anemia, vaso occlusive events, reperfusion injury, accumulation of sickle red cells to endothelium, and red cell oxidative stress which play a role to increase oxidative stress and reduce nitric oxide (NO) bioav ailability [77, 78] These pathophysiologies in SCD cause avascular necrosis, abnormal blood cell endothelium interactions, inflammation, infarction and end organ failures [ 78 81] Pathophysiology of vaso occlusion by SSRBCs is shown in Fig. 1 1. It is well known that most tumors are hypoxic and have a lower (more acidic) extracellular pH due to low vascular density, poor vascular organization, irregular vascular geometry, b uild up of products of metabolism (i.e., lactic and carbonic acid) and unbalanced oxygen consumption [82 84] Accordingly, the hypoxic environment in tumor microvasculature could increase HbS polymerization and lea d to form long polymers that distort shape and change flexibility of SSRBCs. In addition, matured SSRBCs express a number of adhesion receptors interacting to endothelial cells such as B CAM/Lu, LW ( ICAM 4 ) and CD47  Z ennadi et al. and Kaul et al. showed that overexpressed LW on SSRBCs was a major receptor for endothelial v 3 integrins contributing to the adhesion in animal models [86 88]
23 Interestingly, v 3 integrin was recog nized as one of the overexpressed counter receptors in t umor microvascular endothelial cells through in vivo screening by Arap et al.  Accordingly, SSRBCs may have a promising combination of molecularly and me chanical ly accumulative factors as a new tumor targeting carrier with preferential accumulation in tumor sites Tumor preferential accumulations by SSRBCs under the hypoxic tumor environment were reported previously. For example, B rown et al. demonstrated that exogen ously administered SSRBCs from human SCD patients loaded with Gd DTPA using hypo osmotic lysis method preferentially aggregated in orthotopic 9L glioma brain tumors in a rat model  On the other hand SSRBCs less than 30% of total circulating RBCs rarely accumulated in normal physiological condition In addition, Milosevic et al. reported case study regarding a 63 year old African American female patient with an advanced squamous cell c arcinoma cervic al cancer  Normally, patients with sickle cell trait do not have anemia and need neither treatment nor occupational restrictions because the c ellular concentration of HbS is too low (less than 50%) for polymerization to occur  However, the polymerization under hypoxic and acidic conditions was confirmed by tu mor punch biopsies  Controlled Drug Release by Photoactivation using It is well known fact that i t is almost imp ossible to achieve sufficien t therapeutic concentration of drug at the tumor site by passive diffusion release without damaging healthy tissues when traditional sy stemic chemotherapy is performed. A possible solution is to encapsulate anticancer drug into specific drug carrier with preferential accumulation in tumor sites and relea se drug from car rier when it reaches at target sites. Preferential accumulations of SSRBCs in tumoric microvasculature were shown above
24 due to the adhesive interaction between molecular receptors on the membrane surface and counter receptor on endothelial cells. In additi on, physical changes of microvascular structure in tumor sites enhanced polymerization of SSRBCs resulting in the accumulation by hypoxic condition. Various methods have been studied for slow efflux of encapsulated drug from RBCs The most important param eter for evaluation of such resealed erythrocy tes is the drug release pattern [42, 65] but release rates vary according to the nature of the encapsulated drug (i.e., polarity and molecular weight) [41, 66] In addition, uncontrolled slow release of drug from carriers with prolonged lifespan does not always show the greatly increased therapeutic efficacy  Therefore, several a ppr oaches to trigger drug release in a controlled manner were proposed by specific stimuli such as magnetic field guided ultrasound  light [95 97] and temperature  over the targeted sites. However, these methods may be restricted to only clinically identified tumor s ites, not for randomly disseminated advanced solid tumors  In addition, only targets within an appropriate penetration depth of the stimulus source may be affected. In response to this challenge, l ight activat ed controlled release method by photosensitizers is employed to control release from resealed SSRBCs. Flynn et al. demonstrated the directly photo dependent release of entrapped thrombolytic agent brinase from photosensitized RBCs by exposure of radiation from a 10mW HeNe laser placed 17cm above the sample  Moreover, they developed the loading erythrocytes with chemotherapeutic agent (methotrexate) for chemotherapy and subsequenty performed photoactivation by hematoporphyrin  Continuous laser stimulus enhanced rapid drug release and then caused decreased Hela cells viability.
25 Photohemolysis react ion of photoactivated RBCs was more precisely analyzed by Al Akhras et al. in 1996  A basic photohemolysis from photosensitized RBCs r equires two distinct steps; photochemical and thermal reactions. Continuous photohemolysis (CPH) measurement is assumed that photohemolysis is attributed to cooperative photochemical and thermal reactions simultaneously. On the other hand, in delayed photo hemolysis measurement (DPH), the photochemical reaction is followed by thermal reaction in dark sequentially. The lysis time, which is the time measured from the start of rupturing the RBCs, was measured from the beginning of irradiation for CPH method and incubation in dark for DPH method  During the incubation step in DPH, the lysis that occurred during the increase in incubation temperature up to physiological temperature in the dark was considered as insignificant offset and ignored. Photohemolysis rate was measured by the absorbance of released Hb in supernatant separated by immediate centrifugation using spectrophotometer and a typical photohemolysis curve has a sigmoidal shape as in Fig. 1 2. Fig. 1 2 showed an example of DPH measurement of bovine RBCs in vitro by Al Akhras et al.  Based on DPH method, photochemical reaction occu rred by irradiation for a defined time interval at lower temperatures to avoid thermal activation, and then thermal reactions were initiated after the solution temperature reached physiological body temperature in the dark The lysis that occurred up to wh en the temperature reached 37C was considered as negligible offset. As shown in Fig. 1 2, DPH was adjustable by only changing radiation time and it demonstrated the controlled Hb release form photoactivated bovine RBCs  Similarly, DPH method may be applicable to perform in vivo experiment using SSRB Cs.
26 For in vivo experiment, photochemically activated SSRBCs loaded with therapeutic drug by hypo osmosis method may be injected in human or animal model. Preferentially accumulated SSRBCs at tumor sites may be observed according to the reasons described in previous sections. Drug release is initiated by thermal reaction due to the body temperature and temporally controllable drug release rate by the adjustment of irradiation time may occur at local tumor sites resulting in a higher peak drug concentration To characterize the released drug concentration rate in a live model, Gompertz function is adapted to examine the characterization in vitro DPH effect by photoactivated SSRBCs in this experiment. The Gompertz function shows a similar fitting curve as mu lti target theory described previously  Using this function, estimated DPH measurements at presumed time points were successfully matc hed with empirical results  Gompertz Functio n elderly mortality rates in 1825  and is determined as follows: ( 1 ) where M(t) is the mortality rate at a give n age t with parameters and The parameter determines the intercept of the curve, also referred to as the basal mortality rate, and is usually set at adolescence. The parameter determin es the rate of increase of the mortality rate over age. This simple model yields a sigmoidal mortality rate curve which shows accelerating growth for younger age and decelerating growth for older age as shown in Fig. 1 3
27 Hemolysis rate of photosensitized RBCs by hypericin and Photofrin showed similar sigmoidal shape as previously described  and Gompertz function was selected as the most applicable function describing photohemolysis process with only two parameters by Al Akhras et al.  A slightly modified Gompertz function was defined as follows: ( 2 ) where H is the percentage of hemolysis during the incubation time t (the time measured from start of rupturing the RBCs at dark incubation), H 0 the initial maximum number of cells, normalized to one, a is a fractional h emolysis ratio and b is the rate of fractional hemolysis change. The theoretical time required for 50% fractional photohemolysis ( t 50 ) could be calculated by equation ( 3 ). ( 3 ) The theoretical values of t 50 from the equat ion was compared with the empirical t 50 This functional parameters a,b, and t 50 were important parameters to estimate the average of empirical DPH rate and this model has been assessed to investigate the hemolysis rate of photosensitized RBC by PpIX. Mi crodialysis Method for in vivo Delayed Photohemolysis Measurement Microdialysis methodology, based on the dialysis principle, has been a powerful sampling technique capable of continuous monitoring during several hours and days the concentration of unbound drugs or biological molecules both for in vitro and in vivo investigation of both endogenous and exogenous substances [103, 104] Although it was originally developed to monitor for neurotransmitter concentrations in animal brain in 1972  it has been applied and broadened for various pharmaceutical researches
28 including the transdermal delivery of drugs  tissue pharmacokinetics  and tissue pharmacodynamics [108, 109] In addition, microdialysis monitoring has been employed in various tissue and organs of human or murine models such as liver  heart [11 1] skin  blood vessels  placenta  stomach  ear  and tumor tissue [116 118] Fig. 1 2 shows the microdialysis sampling process. Briefly, the probe consists of a semip ermeable hollow fiber membrane with a specific molecular weight cutoff connected to an inlet and outlet tubing. The probe is implanted in the region of interest termed th e perfusate, to the extracellular fluid (ECF) surrounding the probe. Lower molecular weight compounds than the membrane cutoff are able to diffuse into or out of the probe lumen in both directions in response to concentration gradients. The collected solut ion that exits the probe, the dialysate, can be analyzed using quantitative techniques like high performance liquid chromatography (HPLC)  Using microdialysis methodology, fluorescence intensities of the coll ected dialysates from tumors were compared to healthy muscle tissues by microplate reader at two different time points. The ratio of the released calcein concentration from resealed SSRBCs in tumors to healthy tissues was determined and compared. The contr ollability of release time for calcein from photoactivated SSRBCs and possible systemic toxicity caused by SSRBCs were evaluated from the numerical comparison.
29 Table 1 6 Summary of advantages and limitations of microdialysis sampling technique (adopted from  ) Advantages Determination of the bioactive concentration of the drug in the target tissue Excellent time and spatial resolution Protein free samples No further enzymatic degradation of the drug On line coupling of analytical determ ination No fluid loss Simultaneous collection of endogenous compounds Monitoring of drug time course in different tissues by multiple microdialysis probes Disadvantages Tissue damage by probe implantation Diluting effect of the microdialysis procedure Nee d of high sensitive analytical methods, specially for drugs with high protein binding Necessity of determination of the in vivo recovery during the experiment Sticking of lipophilic drugs to tubing and probe components Low recovery of large molecules
30 F igure 1 1 Pathophysiology of vaso occlusion by SSRBCs (a) Point mutation in sickle cell disease. (b) Polymerization of HbS under deoxygenation. (c) Red blood cell shape change in response to HbS polymerization. (d) Cells in microvasculature. Abbreviatio n: N::O ,nitric oxide bioavailability. Reticulocyte accumulated to endothelium initiates vaso occlusion by trapping irreversibly sickled cells and forming agg regates with white blood cells (a dapted from  ).
31 Figure 1 2 DPH measurement from photoactivated RBCs by PpIX with 10 M.D ifferent irradiation time was applied with Hg Xe arc lamp at 24 C and incubated at 24 C. The cell were irradiated for open circle 4.5, open square 4.0, open diamond 3.5, open triangle 3 and inverted triangle 2.5 min (adapted from  )
32 Figure 1 3 Example plot of age specific mortality rates with random parameters by Gompertz function
33 Fig ure 1 4 T ypical microdialysis probe configurations and sampling process. (A) flexible CMA probe from CMA product catalogue (B) Sampling process across microdialysis probe membrane from  A perfusion fluid that closely matches the ionic strength and composition of the fluid e xternal to the microdialysis membrane is passed through.
34 Motivation and Goal of this Study Numerous drug carriers such as liposomes, nanoparticles, and polymers for cancer chemotherapy have been investigated to protect normal tissues from toxic drugs to tr eat advanced stage malignant cancer However, currently conventional drug carriers are not free from systemic cytotoxicity, thus therapeutic efficacy is limited by insufficient tumor accumulation of the drug carrier and insufficient drug release into the t umor sites [2, 8] Therefore, alternative drug carrier system is required with effective tumor targeting and controlled drug release rate. SSRBCs may have the potential to be used as a novel drug carrier. According to previous researches, SSRBCs from human SCD patients and animal models caused vaso occlusive events in the deoxygenated microvasculatures [72 76] For example, exogen ously administered SSRBCs from human SCD pati ents demonstrated preferentially aggregated in orthotopic 9L glioma brain tumors in a rat model  In addition, RBCs in a female patient with sickle cell trait were accumulated in the blood vessels near an advan ced squamous cell c arcinoma cervical cancer  Therefore, SSRBCs may have natural tumor preferential accumulation characteristics due to hypoxic circumstances caused by unbalanced oxygen consumption in tumor sites [82 84] Moreover, temporally controlled drug release mechanisms from resealed RBCs were described and performed ex vivo [99 101] To characterize Hb release model, Gompertz function was applied for its numerical analysis to estimate DPH measurement  According to these evidences in this research, it is hypothesized that SSRBCs may have the potential to be a new drug carrier with better tumor target ing and controllable drug release for improved chemotherapy in advanced cancer patients
35 CHAPTER 2 MATERIALS AND METHOD S Window Chamber Installed Mouse M odel Intravital microscopy in window chamber installed animal model allows non invasive, high resoluti on studies of tumor pathophysiology and interaction with therapeutic and diagnostic nanoparticles in solid tumors For these reasons, it is an attractive complementary imaging technique to clinical and preclinical in vivo imaging modalities such as magneti c resonance imaging (MRI), computed tomography (CT), positron emission tomography (PET), and whole body fluorescence imaging  Historically, the window chamber was installed in rabbit ears in 1924 [ 123] and then this technique was employed for study of the tumor vasculature in a hamster cheek pouch window chamber in 1965 [ 124] From that time onward, various chambers such as the mammary imaging window, cranial window chambers, and dorsal skinfold window chambers have been developed for tumor research on hamsters, rats and immunodeficient mice [125 131] Especially, intravital microscopic studies in dorsal skinfold window chamber installed animal models with growing solid tumors provide unique functional and morphological information. For example, microvascular morphology and functio n [132, 133] vascular and physiological response to conventional therapies [134, 135] the extracellular matrix components [136, 1 37] pH and pO 2  growth factor signaling and tumor host interaction [139, 140] and molecular and nanoparticle dynamics [141, 142] were able to be studied. In this exper iment, the tumor bearing immunodeficient mice were surgically implanted with titanium dorsal skinfold window chambers. Transillumination and fluorescence intravital microscopy were employed to monitor the growth rate of tumors,
36 morphological changes of nor mal and tumor microvasculature, Hb saturation, blood flow, and localization / accumulation of fluorescently labeled target cells. The dorsal skinfold window chamber installed on an immunodeficient mouse model with growing tumors is shown in Figure 2 1. Ima ging System A Zeiss microscope (Carl Zeiss, Incorporated, Thornwood, NY) is used as the imaging platform (Fig. 2 2) A 100W tungsten halogen lamp is used for transillumination of the window chamb er while for fluorescence images, a 100W mercury lamp is use d for epiillumination Spectral image datasets are acquired with a monochrome scientific grade CCD camera thermoelectrically cooled to 20C (DVC Company, Austin Tex as) and fluorescence images are acquired with an ANDOR iXon electron multiplying CCD (EMCCD ) camera thermoelectrically cooled to 50C (ANDOR Technology, South Windsor, CT). The long working distance objectives used are 2.5x and 5x Fluars, 10x EC Plan NeoFluar, and a 20x LD Plan NeoFluar (Carl Zeiss, Inc., Thornwood, NY). Hyperspectral image da ta are obtained using a liquid crystal tunable filter ( LCTF, CRI, Cambridge, MA) with a 400 720 nm pass band and 10 nm nominal bandwidth. Fluorescently label ed macrophages and PLGA MPs are imaged using the EMCCD camera with a FITC filter set (Chroma Techno logy Corp., Rockingham, VT; excitation: 480 nm, with 20 nm bandwidth; emission: 550 nm, with 30 nm bandwidth) and a Cy5 filter set (Chroma Technology Corp., Rockingham, VT; excitation: 640 nm, with 20 nm bandwidth; emission: 680 nm, with 30 nm bandwidth), respectively. Additionally, fluorescently labeled RBCs with DiD ( Carbocyanine dye 1,1' dioctadecyl 3,3,3',3' tetramethylindodicarbocyanine 4 chlorobenzenesulfonate salt) are numerically analyzed using a Cy5 filter set (Chroma Technology Corp., Rockingham, Vermont,
37 excitation 640nm with 20nm bandwidth, emission 680nm with 30nm bandwidth) Further i mage processing is performed using Matlab (The Mathworks, Incorporated, Natick, Massachusetts) Imaging Acquisition LabVIEW (National Instruments Corp., Austin, TX) is used to prepare a custom designed virtual instrument for controlling the tuning of the LCTF and operation of the CCD camera. The software enables automated image acquisition using the specifications for camera exposure time and gain for each filter wavelength. Since the LCTF filter transmits less at lower wavelengths and more at higher wavelengths the exposure time for the camera has to be controlled such that the full dynamic range of the camera is utilized. The minimum exposure time used is 400ms w hereas the maximum exposure time used is 1400ms, resulting in a typical acquisition time of approximately 16ms for image acquisition, filter tuning, image transfer, and saving images on external hard drive. One hemoglobin saturation (HbSat) image set compr ised of 16 images are acquired in the wavelength range of 500 575nm with an interval of 5nm. Hyperspectral I maging of Hemoglobin Saturation map Hypoxia caused by the unique characteristics of solid tumor sites such as lowered vascular density, irregular va sculature, longitudinal oxygen gradient, and unbalanced oxygen consumption has decreased therapeutic efficacy in several clinical trials (radiation, chemotherapy and surgery) [143, 144] Hence, t umor oxygenation st udies at microvascular levels are important to provide better understanding of the complexity of microvasculature oxygen transport and exchange with tissue. A known technique, polarographic microelectrodes, was employed to measure pO 2 at the microvasculatu re
38 level, but it is difficult to perform and does not provide significant spatial information of oxygen delivery  On the other hand, hyperspectral imaging is able to provide spatial maps with hemoglobin saturation (HbSat) information for better understanding of the relationship between blood oxygen delivery and hypoxia at microvascular levels during tumor growth  In our experiment, hyperspectral imaging is acquired by stack of spatial images 3 (A). Hypers pectral image data were collected using a LCTF with a 400 720 nm pass band and 10 nm nominal bandwidth. Band limited images were acquired from 500 to 575 nm at 5 nm intervals to create HbSat pseudocolor maps from pure oxy and deoxy Hb reference spectra as described previously  Image processing is performed using Matlab software (The Mathworks, Inc., Natick, MA) to create pseudocolor HbSat maps according to the flow diagram in Fig.2 3 (B) The HbSat values can be calculated from stack of spatial images at a different wavelength based on the followin g model equation using linear least squares regression  (4) where is the absorbance at wavelength I is the pixel value, I 0 is the pixel value of reference light, and are the extinction coefficient for HbO 2 and Hb R at wavelength and are the concentrations of oxy and deoxy Hb, L is the pathlength, and S is a pathlength dependent s cattering term. Using this formulation stack of hyperspectral images can be converted to a single HbSat map.
39 Delay ed Photohemolysis Measurements In vitro Delayed Photohemolysis Measurement Systems In vitro SSRBCs DPH measurement system and techniques are similar to the experimental setup by Al Akhras and Grossweiner and depicted in Fig. 2 3 [99, 100] Photosensitized SSRBCs in 3ml of buffer are transferred in glass cuvette (Starna Cells, Atascadero, CA) and positioned in a temperature controlled cuvette holder (Ocean Optics, Dunedin, FL). The temperature controlled holder for the cuvette can control temporal conditions from 4C to body temperature. The c ell suspension in the cuvette was irradiated by a 0.08W halogen lamp (Fiber lite DC 950, Dolan Jenner, Boxborough, MA) through one face on an orthogonal face of the cuvette for various irradiation times to trigger protoporphyrin IX ( PpIX ) activation at room temperature The SSRBCs solution is gently stirred with a magnetic stir bar to have uniform irradiation and to increase exposing cells to ambient air due to oxygen supply. Aft er the irradiation step, the temporal reaction by i s initiated by increasing the temperature of cuvette holder up to body temperature (37C) in dark. In the DPH method, photohemolysis rates are measured from the beginning of incubation in dark after irradi ation period as discussed in previous chapter When the temperature reached at 37C, 30l blood sample s were c ollected at various time points from 0 min to 48 hrs. The collected blood sample was split into packed SSRBCs and supernatant by immediate centrif ugation and then 20l of supernatant was stored in conical tubes to measure the absorbance for fractional photohemolysis (absorbance) using a spectrophotometer (NanoDrop TM 1000, Nanodrop, Wilmington, DE). T he fractional hemolysis was determined from the am ount of Hb released into the solution normalized to an equivalent solution of fully lysed red blood cells.
40 The empirical results were analyzed by Gompertz function. Unknown parameters such as a and b from equation (2) were calculated to estimate t he theore tical time required for 50% fractional photohemolysis ( t 50 ) by equation ( 3 ). R 2 values were computed for the fit of t he empirical t 50 to the theoretical values of t 50 by the Gompertz function. In vivo Delayed Photohemolysis Measurement using Microdialysis Tubing To investigate the controlled release rates of calcein from photoactivated SSRBCs in ti ssues, microdialysis probes were in serted at the sites for collection of dialysate Microdialysis probes and tubing connectors were nd ethanol for 5min before use. After that, the probes were perfused with to purge air bubbles in tubing for 30min at a flow rate of 2 l min 1 by syringe pump (BASi Syringe Pump, MD 1002, West Lafayette, IN). The probes were then placed 5mm below the surface for tumor tissue through a plastic guide cannula. A probe was placed into the quadriceps muscle as a control site through a small incision made on the skin of implantation site with a small scalpel blade. The probes were inserted in a similar way in tumor tissue and sutured to skin (Fig. 2 4) Dialysate samples were collected by 1 during 4hrs intervals from the tumor tissue and the quadriceps muscle. Each sample was collected a t a variety of time points after the photosensitized SSRBCs cells or free calcein injection via tail vein. Fluorescence signal of calcein collected from tumor t issue and quadriceps muscle was analyzed using a fluorescence plate reader (Synergy HT Multi Mod e Microplate Reader, Biotek, Winooski, VT) and compared with each group.
41 Figure 2 1 Window chamber installed mouse model (A) Dorsal skinfold wi n dow chamber installed athymic (nu/nu) mouse (B) A photograph of the 4T1 tumor s growing at the implant ation site in the window chamber indicated by the dashed circle, 5 days after implantation.
42 Figure 2 2 Hyperspectral imaging system. (A) The Sorg lab imaging system. (B) Window chamber installed mouse placed on the heating pad and anesthetized throug h the nose cone.
43 Figure 2 3 Hyperspectral imaging to create HbSat map (A) 3 D hyper spectral image cube represents a single  ), (B) I mage processing method to obtain HbSat map from the stack of images that each image is acquired at different wavelength through hyperspectral imaging method.
44 Figure 2 4. Experimental set up for in vitro DPH measurements. The sample cuvette in temperature controller at under physiological temperature is exposed by 0.08W halogen lamp.
45 Figure 2 5. In vivo DPH measurement setup with 4T1 tumor mass be aring mouse model. Each dialysate sample is collected at 0, 6, 12, and 24 hrs after the photo activated SSRBCs or free calcein injection via tail vein.
46 CHAPTER 3 INTR AVITAL MICROSCOPY IMAGING OF MACROPHAG E LOCALIZATION TO IMMUNOGENIC PARTICLE S AND CO LOCALI ZED TISSUE OXYGEN SA TURATION Polymer biomaterial particles have the potential to be used as drug delivery vehicles. Both hydrophilic and hydrophobic drugs can be loaded into polymer particles and administered by either th e parenteral or oral route [148 156] Several therapeutics, such as plasmid DNA, peptides, proteins and low molecular weight compo unds, have been particle loaded [148, 150, 154] We are interested in particl e based methods of vaccination for optimizing the response of antigen presenting cells, such as macrophages and dendritic cells, and minimizing adverse tissue responses to biomaterial polymer particles and encapsulated drugs or adjuvants [157 160] One advantage of biodegradable vaccine carriers is that they can be designed to target antigen presenting cells with differential timedrelease profiles with the potential to preclude th e need for booster doses [148, 150, 151] In the design of biomaterial particle based vaccines it is important to consider the adaptive and innate immune responses to the vaccine and any potential adverse effects of immune response on local tissue at the vaccination site. For example, macrophages are antigen presenting cells that can modulate both adaptive and innate immune responses to biomaterials, and activation of macrophages after phagocytosis of biomaterial microparticles (MPs) can result in an inflammatory response leading reorgani zation of the tissue matrix  Phagocytosis by macrophages can also induce a burst of reactive oxygen species (ROS), and excessive ROS produ ction can lead to apoptosis  In terms of macrophage function, it would be desirable for a biomaterial particle based vaccine to stimulate the antigen presenting behavior of macrophages
47 while inhibiting excessive ROS production and other inflammatory functions that are potentially damaging to local tissue. We are interested in using intravital microscopy for investigation of immune cell localization and function an d co localized tissue effects in response to various biopolymer particle based vaccine formulations, including encapsulated drugs, cytokines and adjuvants. Intravital microscopy has enabled investigations of various aspects of cell function in vivo for cel ls such as tumor cells and i mmune cells [127, 163] Intravital microscopy with rodent dorsal skin window chamber preparations enables in vivo serial observation s in the same animal over time [127, 163, 164] and these models have been used to study angiogenesis and macrophage response to implan ted biomaterial scaffolds  To our knowledge, however, there have been no reports in which intravital microscopy has been used to document real time immune cell localization and co localized tissue effects that give insight into potentially harmful immune cell function, especially in terms of endogenous biomarkers of inflammation with potential for clinical application. Prev iously we have used spectral imaging of mouse window chambers to investigate tissue microvessel oxygenation changes from endogenous HbSat signals in response to tumor growth and development [145, 165, 166] In the present proof of principle study, we used fluorescence and spectral imaging intravital microscopy of mouse window chambers to investigate macrophage accumulation and co localized tissue microvessel HbSat changes in response to an inflammatory stimulus from MPs. Lipopolysaccharide (LPS), a coating found on the exterior of Gram negative bacteria, was encapsulated in poly ( D,L lactide co glycolide ) (PLGA) MPs to stimulat e an innate
48 immune response  Intravital fluorescence imaging was used to monitor localization of systemically administered fluorescent macrophages to fluorescent PLGA MPs and spectral imaging was u sed to measure the co localized microvessel HbSat This technique may enable investigations of specific immune cell responses to MP vaccine formulations while simultaneously monitoring potentially harmful tissue effects in order to optimize MP vaccine form ulations. Materials and Methods Microparticles PLGA (Lactel, AL) with 50:50 composition in hexafluoroisopropanol and inherent viscosity of 0.55 0.75 dl g 1 was used to generate MPs of 1 m diameter using a standard water oil water so lvent evaporation techn ique  The PLGA polymer was dissolved in methylene chloride at 20% (w/v) concentration. A primary emulsion was generated by mixing a fluorescent dye solution with the PLGA solution with or without LPS. A 100 l aliquot of 5 mg ml 1 fluorescein isothiocyanate (FITC) solution (Invitrogen) in phosphate buffered saline (PBS) was mixed with 1000 l of 20% PLGA solution using a tissue miser homogenizer (Fisher Scientific) at a speed of 26,500 rpm for 60 s. To create MPs containing LPS, 100 l of 1 mg ml 1 LPS from Escherichia coli 026:B6 (Sigma Aldrich) in PBS was added to the primary emulsion prior to homogenization. After the first homogenization, the primary emulsion was added to 10 ml of 5% polyvinyl alcohol (PVA, molecular weight ~100,000 g mol 1 ) (Fisher Scientific) solution in PBS and the homogenizing was continued at 19,500 rpm for 60 s to form the secondary emulsion. The secondary emulsion was then added dropwise to 50 ml of 0.5% PVA solution to produce precip itate of particles. The precipitated particles were agitated using a magnetic stirrer (Fisher Scientific) for 3 h to evaporate residual methylene chloride. The
49 remaining solution was then centrifuged at 10,000g for 10 min to collect MPs. The supernatant wa s discarded and an equivalent amount of PBS was added. The MPs were resuspended in PBS by vortexing. This process of centrifugation and resuspension of particles was performed three times. The PBS was aspirated from the centrifuged MPs, which were then fla sh frozen in liquid nitrogen and kept under vacuum in dry ice overnight to evaporate any water trapped in the MPs. The dried MPs were stored at 20 C until use. The MPs were manufactured using methylene chloride which is toxic to living organisms and thus helps insure sterility of the particles during manufacturing of MPs. Sterility was preserved by using sterile solutions, glassware, etc. during the manufacturing process. In order to assess the amount of endotoxin present on the non degraded blank MPs the chromo Limulus amebocyte lysate (chromo LAL) assay was solution of 410 7 particles ml 1 Quantification of endotoxin levels by chromo LAL revealed that the particle endotoxin levels were below the detection limit of 0.050 endotoxin units ml 1 The MPs were characterized by scanning electron microscopy and were determined to be spherical with an 890 nm diameter and smooth morphology. LPS loading did not affect the fluorescence s ignal from the MPs as determined by fluorescence microscopy imaging of solutions of known amounts of particles in square profile microcapillary tubes (VitroCom, Mountain Lakes, NJ). The fluorescence signals measured in vitro and normalized to particle numb er for LPS containing and non LPS MPs were 0.5430.092 vs. 0.547 0.101, respectively (relative fluorescence intensity,
50 mean standard deviation). No statistically significant difference (t test, p > 0.655) was found between MPs with and without LPS in ter ms of the fluorescence signal. Macrophage Cell Line Macrophage murine RAW 264.7 cells were cultured in 35 mm tissue culture treated polystyrene dishes (Corning). The macrophage medium consisted of 10% fetal bovine serum (Bio Whittaker) and 1% penicillin st reptomycin (Hyclone) in DMEM/F12 (1:1) with L glutamine (Cellgro, Herndon, VA). The cells were incubated at 37C in a 5% CO 2 atmosphere and passaged every 3 days with a 1:3 split during passage. Macrophages were fluorescently labeled by incubation with the fluorescent dioctadecyl tetramethylindodicarbocyanine, 4 chlorobenzenesulfonate salt (DiD) (Invitrogen) at a concentration of 5 g ml 1 in a serum free solution of 2 mM dextrose in PBS for 30 min at 37C. The cells were cen trifuged for 5 min at 330g, washed with PBS, and then suspended in 200l of PBS. The cells were delivered systemically at a concentration of 8000 l 1 and a total volume of 100 l via the tail vein 2 h after surgery. Research suggests that a brief inflammato ry response over a 2 h period could be expected after window chamber surgery due to exposure to the titanium window chamber structures  thus macrophage s were injected 2 h after implantation of the windows. For the purposes of this study, the brief recovery time was expected to be enough to observe localization of the administered labeled macrophages to the MPs sufficiently above a response to factors ass ociated with surgery given the strong induction of an innate immune response provoked by LPS. Animal Model All in vivo procedures were conducted under a protocol approved by the University of Florida Institutional Animal Care and Use Committee and conforme d to the NIH
51 Guide for Care and Use of Laboratory Animals. Ten female athymic (nu/nu) nude mice, weighing 20 26 g (Charles River Laboratories, Raleigh, NC), were surgically implanted with titanium dorsal skinfold window chambers while under anesthesia cons isting of ketamine (100 mg kg 1 ) and xylazine (10 mg kg 1 ) injected intraperitoneally. Nude mice were used in this study for the following reasons: we have extensive experience with window chambers in this model from research with xenograft tumors; in this proof of principle study the nude mouse was an acceptable stand in for the genetically modified mice we may use in future experiments with adoptive transfer of various immune cells; and nude mice have previously been used in immune cell research for certa in purposes when a T cell deficient host was desired, such as for the adop tive transfer of leukocytes  In our study, the lack of T cells in the nude mice was not critical since we were primarily interested in tracking the administered RAW 264.7 macrophages. During surgery one layer of dorsal skin was carefully and completely removed in a circular area of about 12 mm diameter and covered with a glass coverslip. Spectral and fluorescence imaging were performed f or up to 6 days after administration of fluorescently labeled macrophages. After surgery animals were housed in an environmental chamber maintained at 33C and 50% humidity with free access to food and water and standard 12 h light/dark cycles. A total of 10 mice were placed in one of four different groups: control mice (n = 3), which received saline injections instead of MPs; LPS( ) mice (n = 3), which received MPs without LPS, LPS(+) mice (n = 3), which were administered MPs with LPS at a concentration of 400,000 particles in 20l; and LPS(++) mice (n = 1), which were administered MPs with LPS at a concentration of 2,000,000 particles in 20l. The 20l
52 total dose of MPs was injected in two 10l volumes in two different locations in the window chamber. Intr avital Fluorescence and Spectral Imaging A Zeiss microscope (Carl Zeiss, Incorporated, Thornwood, NY) was used as the imaging platform. A 100W tungsten halogen lamp was used for transillumination of the window chamber. Spectral image datasets were acquired with a monochrome scientific grade CCD camera thermoelectrically cooled to 20C (DVC Company, Austin Texas) and fluorescence images were acquired with an ANDOR iXon electron multiplying CCD (EMCCD) camera thermoelectrically cooled to 50C (ANDOR Technol ogy, South Windsor, CT). A 2.5 objective (Carl Zeiss, Inc., Thornwood, NY) was utilized for imaging. Spectral imaging of microvessel HbSat was perf ormed as done previously [145, 165] Briefly, customized LabView software (National Instruments, Austin, Texas) was used to automatically acquire spectral images. Spectral image data were collected using a liquid crystal tunable filter (CRI, Cambridge, MA) with a 400 720 nm pass band and 10 nm nominal bandwidth. Band li mited images were acquired from 500 to 575 nm at 5 nm intervals to create HbSat pseudocolor maps from pure oxy and deoxy Hb reference spec tra as described previously  Fluorescently labeled macrophages and PLGA MPs were imaged using the EMCCD camera with a FITC filter set (Chroma Technology Corp., Rockingham, VT; excitation: 480 nm, with 20 nm bandwidth; emission: 550 nm, with 30 nm bandwidth) and a Cy5 filter set (Chroma Technology Corp., Rockingham, VT; excitation: 640 nm, with 20 nm bandwidth; emission: 680 nm, with 30 nm bandwidth), respectively. Image processing and statistical analysis were performed using Matlab (The Mathworks, Incorporated, Natick, Massachusetts) and
53 SPSS software (SPSS for Windows; SPSS Inc., Chicago, Illinois, USA), respectively. Spectral and fluorescence imaging were performed up to 6 days after surgery. Results After administration of macrophages via tail vein injection, two different large regions of interest (ROIs) corresponding to the two regions where MPs were injected were chosen to investigate the interaction between MPs and localized macrophages, and adjacent microvessel HbSat during a posterior analysis phase. Macr ophage localization to MPs was presumably due to a combination of macrophage accumulation and extravasation. H yperspectral and Fluorescence Imaging Macrophage accumulation and HbSat changes over time for the different groups (control saline injected, LPS( ), LPS(+) and LPS(++)) are shown in Figs. 3. 1 3. 4. Images in the rows are transmitted light, microvessel HbSat, distribution of MPs and macrophage accumulation. Images were acquired at 0, 6, 12, 24, 48, 72 and 144 h after the injection of labeled macropha ges for control, LPS( ), and LPS(+) mice, and at 0, 1, 2, 4, 6, 8, 12, and 24 h for LPS(++) mice. There were perturbations in tissue microvessel HbSat over time in all of the different groups. As shown in Figs. 3 1 and 3 2, the macrophage accumulation appe ars similar for both the control group, administered the saline carrier alone, and the LPS( ) group, with unloaded MPs, demonstrating a lack of appreciable inflammation response to the unloaded MPs. There is greater macrophage accumulation in the LPS(+) gr oup as compared to the control group, demonstrating appreciable inflammation due to LPS release (Fig. 3 3). Note that variatio n in the signals of MPs in LPS( ) and LPS(+) groups was observed (Figs. 3 2 ~ 4). This may be due to several reasons. More active interaction of macrophages with
54 LPS could have caused wider dispersion of MPs in the LPS(+) and LPS(++) groups than in the LPS( ) group. Additionally, some FITC dye quenching may have occurred in acidic MPs and cell endosomes, but when dye was released int o neutral pH tissue, the fluorescence increased  It was observed that there was significantly larger and more rapid macrophage accumulation in the LPS(++) m ice than in any of the other groups (Fig. 3 4). Additionally, there was a very large decrease in tissue microvessel HbSat in the LPS(++) mice that initiated in the vicinity of the MPs and spread out over time. Six hours after injection of macrophages, a si gnificant amount of macrophages had accumulated in the window chamber, and there was extensive microvessel disruption and tissue necrosis. Tissue alterations leading to necrosis could be observed 1 h after injection of macrophages (arrows in Fig. 3 4). Qua ntitative A nalysis for Hemoglobin Saturation Map Microparticles and Macrophages The results of quantitative regional data analysis of Figs. 3 1~3 4 are shown in Figs. 3 5 ~3 7. At least 25 different locations on arterioles and venules near MP implantation sites for each mouse were chosen as ROIs to quantitatively analyze HbSat and macrophage signals. Note that macrophage fluorescence signals in the ROIs were due to total accumulated DiD loaded macrophages in the ROIs, with no distinction between those in bl ood vessels and ones that extravasated. ROIs on arterioles and venules that encompassed the immediate vicinity of the microvessels for LPS(++) (25 ROIs), LPS(+) (26 ROIs per mouse), LPS( ) (28 ROIs per mouse) and saline control (30 ROIs per mouse) were ana lyzed for all of the imaging time points. Numerical HbSat data and macrophage signals in the ROIs were plotted at various imaging time points as mean standard deviation and are shown in Figs. 3 5 and 3 6,
55 respectively. The HbSat in arterioles and venules of the LPS(+) mice was relatively stable and lower than in the LPS( ) and control groups, whereas the macrophage signals were higher in the LPS(+) than in the LPS( ) and control groups. Statistical differences (p < 0.05) of HbSat data in LPS(+) from other groups were apparent between 24 and 12 h in Fig. 3 5. Similarly, the macrophage signals of the LPS(+) group at the same ROIs demonstrated statistical differences (p < 0.05) from 12 h (Fig. 3 6). In the LPS(++) mice an immediate and rapid decrease in vascu lar oxygenation and an increase in macrophage accumulation were observed. The fluorescence intensity representing the macrophage signal for the LPS(++) mice in Fig. 6 is the actual macrophage signal 5.5 so the signal could be superimposed for easy viewi ng with the other groups shown in the figure. Within each group there was no difference in the macrophage signal between ROIs on arterioles or venules. Statistical Analysis for A ccumulated Region of Interests D ata HbSat and macrophage signal measurements w ere averaged over all imaging time points, as shown in Fig. 3 7, and analysis of variance (ANOVA) followed by a assess the statistical significance of the differences between the groups. The HbSat an d macrophage data fr om the LPS(+ ) and LPS(++) groups were found to be significantly different (p < 0.05) vs. the LPS( ) and control groups, and there was a significant difference between LPS(++) and LPS(+). No significant difference between control and LPS( ) was observed. In summary, we tracked localization of a specific immune cell, systemically administered macrophages in our case, to inflammatory LPS loaded biomaterial MPs as a model vaccine/adjuvant delivery system. Simultaneously we measured from endogenous signals of Hb Sat changes in the co localized tissue microvessel
56 oxygenation due to the host innate immune response to the MPs. For our experimental groups we observed a general trend of greater and faster macrophage localization with stronger inflammatory stimuli in te rms of the amount of LPS concentrated in an area. With increased macrophage localization to MPs, we observed a trend of decreased microvessel oxygenation. Microvessel disruption and tissue necrosis occurred in the extreme case.
57 Figure 3 1. Hyperspect ral and fluorescence images for a control mouse with saline (carrier) injection without microparticles. Rows from top to bottom Transmitted light (brightfield), microvessel HbSat (HbSat), microparticles (MP), and macrophage distributions (Macrophages) ac quired at 0, 6, 12, 24, 48, 72, and 144 hours after the injection of labeled macrophages for a control mouse with saline (carrier) injection without microparticles. The window chamber area has a 12mm circular diameter. The color bar shown at bottom of the figure represents the oxygenation level in HbSat maps.
58 Figure 3 2 Hyperspectral and fluorescence images for a LPS( ) mouse with microparticles lacking LPS Rows from top to bottom Transmitted light (brightfield), microvessel HbSat (HbSat), micropart icles (MP), and macrophage distributions (Macrophages) acquired at 0, 6, 12, 24, 48, 72, and 144 hours after the injection of labeled macrophages for a LPS( ) mouse with microparticles lackin g LPS The window chamber area has a 12mm circular diameter. The color bar shown at bottom of the figure represents the oxygenation level in HbSat maps.
59 Figure 3 3. Hyperspectral and fluorescence images for a LPS(+) mouse with LPS Rows from top to bottom Transmitted li ght (brightfield), microvessel HbSat (HbSat), microparticles (MP), and macrophage distributions (Macrophages) acquired at 0, 6, 12, 24, 48, 72, and 144 hours after the injection of labeled macrophages for a LPS(+) mouse with LPS encapsulated microparticles chamber area has a 12mm circular diameter. The color bar shown at bottom of the figure represents the oxygenation level in HbSat maps.
60 Figure 3 4. Hyperspectral and fluorescence images for LPS(++) mouse with LPS encapsula Rows from top to bottom Transmitted light (brightfield), microvessel HbSat (HbSat), microparticles (MP), and macrophage distributions (Macrophages) acquired at 0, 6, 12, 24, 48, 72, and 144 hours after the injectio n of labeled macrophages for LPS(++) window chamber area has a 12mm circular diameter. The color bar shown at bottom of the figure represents the oxygenation level in HbSat maps. White arr owheads indicate the regions where tissue necrosis occurs.
61 Figure 3 5. Numerical HbSat data for each group of ROIs at various time points. The ROIs are selected on arterioles and venules separately. Solid lines represent HbSat in arteriol es and venules of control, LPS( ), and LPS(+) (mean S.D.) while dotted lines represent HbSat in the arterioles and venules of LPS(++) (mean S.D.). = LPS(+) HbSat in the arterioles and venules are statistically different (p <0.05) from control and LPS( ) after 24 a nd 12 hours post injection of macrophages, respectively. # = LPS(++) HbSat from the arterioles and venules are statistically different (p <0.05) from LPS(+) group after 6 hours post injection of macrophages.
62 Figurea 3 6. Numerical macrophage signals f or each group of ROIs at various time points. The ROIs are selected on arterioles and venules separately. Solid lines represent the macrophage fluorescence signals at arterioles and venules of control, LPS( ), and LPS(+) (mean S.D.) while dotted lines re present macrophage fluorescence signals at the arterioles and venules of LPS(++) (mean S.D.). Note that the LPS (++) macrophage signals are plotted as actual macrophage signals divided by 5.5. = LPS(+) macrophage signals at arterioles and venules are s tatistically different (p <0.05) from control and LPS( ) groups after 12 hours post injection of macrophages. # = LPS(++) macrophage signals in the arterioles and venules are statistically different (p <0.05) from LPS(+) group after 6 hours post injection of macrophages.
63 Figure 3 7. The accumulated ROI data overall time points are analyzed using ANOVA hoc test. The bars represent HbSat in arterioles and venules of each group (mean S.D.) while the dotted lines represent the macrophag e fluorescence signals at the arteriole and venule ROIs of each group (mean). = LPS(+) and LPS(++) groups data are significantly different from the control group (p < 0.05). # = LPS(++) group data are significantly different from LPS(+) (p < 0.05).
64 Discu ssion Antigens and therapeutic drugs encapsulated in MP delivery systems have been investigated clinically  Polymer based antigen delivery systems for vaccines have the potential to release antigens in a continuous manner such that a single dose of a biodegradable polymer vaccine can be substituted for the repeated doses required in some conventional vaccinations [150, 153] PLGA, a US Food and Drug Administration approved biomaterial, has been the most e xtensively investigated of the polymer based materials for controlled delivery of antigens and drugs  Numerous undesired effects, including impaired blood vessel function, tissue regeneration failure and organ failure, have occurred by incompati ble tissue response to biomaterials alone [171, 172] The combination of biomaterials, antigens and adjuvants in a biomaterial MP based vaccine formulation represents a complex system that can result in complex res ponses from various immune cells with undesired tissue effects. The adjuvant formulation is particularly important in order to optimally shape the adaptive immune response without inducing excessive inflammation  Therefore, the adequate evaluation of the biocompatibility and biodegradability of new biomaterial / drug combinations for MP based vaccines requires in vivo experiments to fully appreciat e the immune cell and tissue responses. Typically, a series of inflammatory responses occur immediately after implantation of biomaterials [145, 159, 165] It is desirable to tune the inflammatory response to MP ba sed vaccines in order to optimize the vaccination process. For example, macrophages actively react and respond to almost all the various biomaterials within minutes to hours after implantation because the inflammatory response is immediately initiated by i njury to vascularized connective tissue in the implantation procedure 
65 Macrophages mediate several inflammatory responses in host tissues by infiltrating biomaterial based implants, including those made of metal, ceramics, cements, polymers and protein constructs [167, 172] Because of the long lifetime of macrophages (from days to months)  and the key role they play in innate and adaptive immune responses and chronic inflammation [175, 176] it is particularly critical to control the response of macrophages to MP based vaccines. It would be desirable for MP based vaccines to stimulate the antigen presenting behavior of macrop hages while inhibiting tissuedamaging functions. In vivo imaging may be a useful tool to investigate the responses of various immune cells to MP based vaccine formulations and the subsequent tissue effects. Intravital microscopy techniques have been utiliz ed where continuous physiological monitoring and repetitive measurements on the same animal are needed [127, 163, 164] Recently, dorsal skin window chambers have been used with mice to investigate host tissue resp onse to implantable PLGA scaffolds over various imaging time points  Neovascularization and inflammatory response to PLGA and the correlation between macrophage activities and blood vessel permeability was observed in these model systems  In the present study, spectral and fluorescence intravital microscopy imaging was used with mouse dorsal skin window chambers to simultaneously measure localization of systemically administered macrophages and co localized tissue microvessel oxyge nation in response to implanted PLGA MPs with and without encapsulated LPS. We observed an increase in macrophage localization to LPS loaded MPs compared to unloaded MPs and control saline injections, which is in agreement with other results from the liter ature [156, 167, 177, 178] There was greater
66 and more rapid macrophage localization to a higher density of LPS loaded MPs. Additionally we observed a concomitant decrease in local microvessel oxygenation in both a rterioles and venules with increased numbers of macrophages. With a sufficiently large number of macrophages we observed an extreme decrease in microvessel oxygenation that was accompanied by tissue necrosis. The decrease in microvascular oxygenation that occurred with increased amounts of accumulated macrophages was expected and was probably due to several different factors. For example, it is known that oxygen is consumed by activated inflammatory cells at a high rate  and phagocytosis of particulates by macrophages induce s a burst of ROS production  LPS greatly increases RAW 264.7 macrophage phagocytosis  and phagocytosis of PLGA MP s by these cells results in an oxidative burst  Nitric oxide (NO) production in endothelial cells and macrophages stimulated by activated macrophages may also have decreased microvessel oxygenation. LPS activ ated macrophages produce NO with respect to microbicidal functions in a dosedependent manner [153, 177, 180 182] NO can bind tightly to Hb such that the NO bound Hb does not easily dissociate to carry and transpor t oxygen [153, 177] Also, high concentrations of NO or its derivatives can interact with mitochondrial or other respiratory chain complexes, leading to tissue necrosis  Rucker et al. [ 156] used mouse dorsal skin window chambers to observe macrophage response to PLGA scaffold constructs placed in the window chamber. The authors observed a moderate increase in numbers of rolling and accumulated leukocytes in response to PLGA scaffolds co mpared to controls without scaffolds. In our study, we saw no significant difference between saline control and empty PLGA MPs in
67 terms of localization of macrophages. This discrepancy may be due to several factors. Rucker et al. used BALB/c mice, while at hymic nude mice were used in this study. Also, in Rucker et al. employed a comparatively large scaffold (3 mm3 mm1 mm), while in this study a relatively small amount of material was used. Finally, in Rucker et al. the PLGA scaffolds were placed on the ti ssue in the window chamber, while in our study the MPs and control saline doses were injected intradermally. The act of injecting the saline and empty control MPs may have induced similar mild inflammatory reactions in our study, unlike the control case in Rucker et al., in which no tissue damage occurred in the control case. Spectral imaging intravital microscopy of microvessel Hb captured simultaneously with immune cell localization to biomaterial MPs may be useful in laboratory investigations of MP based vaccine development. For example, Rucker et al.  used caspase 3 staining to search for evidence of apoptotic cells after implantation of biomaterial scaffolds in mouse window chambers, but these measurements required animal sacrifice for tissu e histology sections. In our study, using serial observations in vivo, we found a decrease in HbSat around LPS(+) particles and an extreme decrease around LPS(++) MPs accompanied by apparent tissue necrosis. The drop in HbSat was measured prior to tissue c hanges indicative of necrosis that could be observed with the naked eye. Measurements of microvessel HbSat around an MP vaccine injection site may also be useful for clinical investigations as measurements of tissue HbSat may potentially serve as a biomark er for a harmful inflammatory reaction. For example, Stamatas and Kollias  used clinical spectral imaging of skin to measure bulk tissue response to inflammatory stimuli such as rhus dermatitis, which can occur in reaction to
68 poison ivy exposure. The authors found a moderate increase in deoxy Hb around skin inflammation sites. Using spectroscopy of tissue Hb Liu et al.  have shown that there is an increase in tissue blood supply in periodontal inflammation, though the increase is insufficient for the metabolic demand of the inflammation process and thus tissue oxygenation is lower than normal.
69 CHAPTER 4 PREFERENTIAL ACCUMUL ATION OF SICKLE RED BLOOD CELL IN TUMORI C ENVIRONMENT The ideal drug carrier has to possess two important features to overcome systemic toxicity causing s ignificant morbidity from conventional chemotherapy to treat distant metastases: tumor selective accumulation and controlled release of drug contents from carriers Many drug carriers including nanoparticles  polymer micelles  polymeric conjugate  lipid microemulsion  liposomes  and resealed erythrocytes  have been developed as drug carriers. Among those drug c arriers, resealed RBCs loaded with therapeutic agents has been exploited extensively for both temporally and spatially controlled chemotherapeutic carriers. Ihler et al. first used the erythrocyte as the carrier to deliver enzy me in human body to heal certain diseases successfully  and then various drugs and other bioactive agents have been tried owing to their remarkable degree of biocompatibility, biodegradability, prolonged life t ime in circulation and a series of other potential advantages as described in Chapter 1. Interestingly, tu mor preferential accumulation by SSRBCs due to a combination of adhesion receptor over expression and mechanical changes induced in the sickle cells u nder the deoxygenated tumor environment were shown previously by several case reports Rapid tumor specific accumulation of epinephrine induced SSRBC was demonstrated in a mouse model  intravenously administer ed SSRBCs from human SCD patients shown to preferentially accumulate in a rat brain tumor model  and extensive intravascular sickling of erythrocytes was observed in sickle trait patient with stage IIIB squamo us cell carcinoma of the cervix by clinical case report  Theoretically, tumor regions expressing hypoxic stress are suited to change morpholog ical properties in SSRBCs. The environments of some tumor regions are
70 characterized in terms of low pH, low oxygen tension, relatively low blood flow due to disorganized vasculature and higher blood viscosity, permeability and interstitial pressure  Therefo re, HbS polymerization can be initiated by these unique characteristics under tumoric conditions and changes the flexibility of red blood cells. Other characteristics of tumor vasculature such as tortuous, chaotic vasculature or abnormal vessel lining also enhance sickling or promote SSRBC aggregation  Moreover, matured SSRBCs express a number of adhesion receptors interacting to endothelial cells such as B CAM/Lu, LW ( ICAM 4 ) and CD47  Zennadi et al. and Kaul et al. showed that overexpressed LW on SSRBCs is a major receptor for endothelial v 3 integrins contributing to the adhesion in animal models [86 88] and v 3 integrin is one of the overexpressed counter receptors in t umor microvascular endothelial cells  Accordingly, SSRBCs may have a promising combination of molecularly and mechanical ly accumulat ed factors as a new tumor targeting carrier with preferential accumulation in tumor sites occlusion under hypoxic condition described above, this study focused on the accumulation of several types of SSRBCs in tumors using intravital microscopy imaging and video recording of sickle cell accumulation to and obstruction of microvessels in tumors grown in dorsal skin fold window chambers. This intravital microscopic imaging method was employed previously for the quantitative analysis of f luorescently labeled macrophage localization to immunogenic particles  In this e xperiment, various blood samples were prepared to compare with each other and summarized in Table 4 1. Briefly, they were from human SSRBCs with /
71 without hydroxyurea treatment, wildtype mouse ( C57BL/6 ), and two types of transgenic SCD mouse models such as Berkeley SCD mouse o ne of the most commonly used SCD mouse model [188, 189] and knock in SCD mouse ( Hba tm1Paz Hbb tm1Tow Tg(HBA HBBs)41Paz/J ) recently developed and c onsidered as the better SCD mouse model than Berk eley mouse [190, 191] Fluorescently labeled blood cells were prepared for intravital microscopic observation and administered into 4T1 tumor bearing immunodeficient mouse models. Involved tissue s from tumor, spl een and kidney were harvested to perform histological test to compa re the entrapment rate of blood cells Tissue specimens harvested for histology were s nap frozen method to preserve the sickle cell fluorescence signal. In this Chapter, the tumor selective accumulation of SSRBCs from human S CD patients and SCD murine models to the endothelium and vaso occlusion in the tumoric environment were explored. Controlled release of substance from SSRBCs as the se cond required feature for drug carriers is examined i n Chapter 5 [99 101, 192] Table 4 1. Categorized groups for preferential accumulation of SSRBCs to tumoric microvasculature experiment. Group Species Hydroxyurea treatment n C57 Wildtype mouse: C57BL/6 N 6 Hyd roxyurea on Human SCD patient Y 5 Hydroxyurea off Human SCD patient N 3 Knock in Knock in SCD mouse: Hba tm1Paz Hbb tm1Tow Tg(HBA HBBs)41Paz/J N 7 Berkeley Berkeley SCD mouse: N 7 Materials and Methods Tumor Cells 4T1 mouse mammary adenocarcinoma cells w ere generously provided by Dr. Mark W. Dewhirst (Duke University Medical Center, Durham, NC). The cells were
72 cultured as a monolayer in DMEM (Cellgro, Inc., 1X, 4.5 g/l glucose, L glutamine and sodium pyruvate) with 10% fetal bovine serum (Biowhittaker, In c.), 1% L glutamine (Clonogen, Inc.) and 1% penicillin streptomycin (Clonogen, Inc.). Single cell suspensions were prepared in DMEM without serum at room temperature and stored at 4C until its subcutaneous injection in the window chamber on dorsal skin of mouse models. Animal M odel All in vivo experiments were carried out under a protocol approved by the University of Florida Institutional Animal Care and Use Committee (IACUC) and conformed to the NIH Guide for Care and Use of Laboratory Animals. Female a thymic (nu/nu) nude and wild type (C 57) mice weighing between 20 and 26 g were obtained from Charles River Laboratories (Raleigh, NC) for this study. A total of 28 4T1 tumor bearing mouse models with window chamber were placed in this experiment; C ontrol group (n=6) administered with the labeled normal RBCs from C57 d onor mice, Berkeley group (n=7) administered the labeled SSRBCs from Berkeley donor mice Knock in group (n=7) administered the labeled SSRBCs from Knock in donor mice Hydroxyurea on group ( n=5) administered the labeled SSRBCs from hydroxyurea medicated human SCD patients, Hydroxyurea off group(n=3) administered the labeled SSRBCs from human SCD patients untreated with hydroxyurea Window C hamber I nstallation Double sided titanium skinfold wi ndow chambers were surgically implanted into dorsal skin flap under anesthesia attained by intraperitoneal injection of ketamine (100mg/kg) and xylazine (10mg/kg). The epidermal and dermal layers of dorsal skin were carefully but completely removed in a ci rcular area of about 12 mm diameter. A
73 single cell suspension of 1010 3 tumor cells in 10ul was injected subcutaneously and a glass coverslip was placed on the window chamber to cover exposed tissue. After surgery, animals were housed in an environmental c hamber maintained at 33 C and 50% humidity with free access to food and water and standard 12 hrs light/dark cycles. Preparation of Blood Cells Fresh human SSRBCs from SCD patients were prepared by Dr. Richard Lottenberg (M.D. Professor, Department of Me dicine, Division of Hematology/Oncology, University of Florida) and murine SSRBCs from the transgenic SCD mouse models were collected by Dr. Angela E. Rivers (M.D, Ph.D. Assistant Professor, Department of Pediatrics, Division of Hematology/Oncology, Univer sity of Florida). The Institutional Review Board (IRB) of University of Florida approved of obtaining patient blood sample for this study. Informed written consents were obtained from all patients in accordance with the Declaration of Helsinki. Some of hum an SCD patients were medicated by hydroxyurea and all blood samples a re categorized in Table 4 1. Murine SSRBCs were obtained from Knock in and Berkeley SCD mouse models by retro orbital bleeding using a heparinized glass pipette and normal RBCs were obtai ned by cardiac puncture from a donor C57BL/6 wildtype mouse. All donor mice were anesthetized by 1% 2% isofluorane in air and blood samples were collected about 1.5ml from each donor mouse. The prepared blood samples were stored in ethylenediaminetetraacet ic acid (EDTA) tubes at 4C until use. Obtained cells from donor patients and mice were fluorescently labeled with a 1mM stock solution of Carbocyanine dye 1,1' dioctadecyl 3,3,3',3' tetramethylindodicarbocyanine, 4 chlorobenzenesulfonate salt (DiD solid, Invitrogen, D 7757) in ethanol using a modified procedure by Unthank et al  Briefly, the RBCs
74 were washed via centrifugation at 4C and resuspend in Dulbecco's Phosphate Buffered Saline (DPBS with 1mM of Ca 2+ and 0.5mM of Mg 2+ without phen ol red, SH30264.01, HyClone) until its supernatant is clear. Isolated 200ul of cells w ere incubated with 200ul of DiD stock solution in 10ml of sterile DPBS at room temperature for 30min. To protect the sample from ambient light, the all tube s were covered by aluminum foil during all procedures. Unbound DiD dye w as carefully removed by repeated washing steps. DiD labeled murine RBCs in 200 l of DPBS (hematocrit [Hct] 0.5 [50%]) w ere i administered in mouse anesthetized by 1% 2% isofluorane in air by tail v ein when the tumors reached about 6mm in a diameter. In the present study, the quantity of human and murine RBCs did not exceed 12% of the total circulating RBCs assuming that the mouse blood volume is 1.7ml, thereby minimizing any possible rheologic effec ts attributable to increased Hct  Fluorescently labeled RBCs ( 100 l ) were saved at 4C to evaluate for the efficiency of the labeling method by flow cytometry test. Intravital Spectral and Fluorescence I maging Mice anesthetized by 1% 2% isofluorane in air w e re placed on the stage of an Axio microscope (Carl Zeiss, Inc., Thornwood, NY) and body temperature was maintained at 37C by a heating pad. The spectral imaging system, image acquisition, and image processing techniques to create Hb saturation map and int ravital fluorescence images for localized RBCs in microvasculature, normal and tumor tissues were explained in Chapter 2 and in previous studies [145, 187] Briefly, microscopy was employed as a basic platform for the imaging system. A 100W tungsten halogen lamp and 100W mercury lamp was used for transillumination and epi illumination of the window chamber respectively Spectral image datasets were
75 acquired with a monochrome scientific grade CCD camera thermoelect rically cooled to 20C using 2.5 Fluars objective for up to 7 days prior to treated RBCs injection. Fluorescence imaging and video recording for quantitative analysis of blood cell accumulation were acquired with an Andor iXon EM CCD camera thermoelectric ally cooled to 50C using 5 Fluars and a 20x LD Plan NeoFluar objectives, respectively Customized LabView software was used to automatically acquire spectral images. Spectral image data were collected using a LCTF with a 400 720 nm pass band and 10 nm n ominal bandwidth. Band limited images were acquired from 500 to 575 nm at 5 nm intervals to create Hb saturation pseudocolor maps from pure oxy and deoxy Hb reference spectra as described previously  DiD (excitatio n: 644nm, emission: 665nm) marked blood cells were depicted and recorded using the EMCCD camera with a Cy5 filter set (Chroma Technology Corp., Rockingham, Vermont, excitation 640nm with 20nm bandwidth, emission 680nm with 30nm bandwidth) in line with the illumination source of a Zeiss FluoArc mercury lamp. For fluorescence imaging with 5 Fluars objective, at least 6 regions of interest (ROIs) on postcapillary venules with up to 25m diameter per mouse model were chosen where large number of immobile RBCs were observed in good focus and analyzed quantitatively. Since fast moving RBCs in blood stream were appeared as gray lines with increased exposure time (up to 150ms), motionless blood cells were easily observed due to their brighter fluorescence signal. They were considered as accumulated cells to vessel wall and quantified by the image processing techniques using Matlab software (The Mathworks, Incorporated, Natick, Massachusetts) as done previously 
76 Additionally, fluorescently labeled blood cells were recorded via streaming video analysis using 20 LD PlanNeoFluar objective (Carl Zeiss, Inc., Thornwood, NY) for 5min of streaming video with 1500 frames (exposure time of 200ms) were saved at 0, 30, 60, 90, 180, 360 and 720min after administration of labeled blood cells. All data were acquired in kinetic mode with 22 binning with a shift speed of 0.564s. Labeled SSRBCs that appeared to be immobile over 1 min in streaming video were considered as accumulated cells to vessel wall and counted manually. The number of immobile RBCs was divided by vascular area for numerical analysis. Statistical analysis was performed SPSS software (SPSS for Windows; SPSS In c., post hoc test were used to assess the statistical significance of the differences between the groups. Histology Particular organs such as liver, spleen and tumor tissue from mice were harvested immediately after the last imaging acquisition at 12 hrs after RBCs injection under dissecting scope. These specimens were selected for examination regarding phagocytosys or accumulation rate of SSRBC by reticuloendothelial system (RES) macrophages in the spleen and liver In addition, the microvascular occlusions observed in human SCD consistently affected the lungs, liver, kidneys, bone marrow, and spleen of the transgenic SCD mice  The harvested tissues were fixed with 10% formalin, dehydrated with 15%~30% sucrose solution, embedded in optimal cutting temperature (OCT) solution (Tissue Tec OCT, Fisher Scientific, Pittsburgh, PA) in plastic cryomolds and snap frozen above pre cooled methylbutane with dry ice at about 55C. Specimens were sectioned by
77 microtome (Microm, HM550 microtome / cryostat, Microm International, Walldorf, Germany) at 2 18C and each section was placed on Superfrost Plus slide (Fisher Scientific, Pittsburgh, PA). The slides were merely air dried at room temperature overnight, then one slide was examined under fluorescenc e microscopy and consecutive slide was stained with hematoxylin and eosin (H&E). Five random, but same fields for two consecutive slides, were selected each tissue sample, then fluorescence and H&E stained images were acquired at a 20 magnification. Fluor escence intensities from labeled RBCs were quantified to compare a ccumulation ratio using Matlab softwa re Results Monitoring Hb Saturation Map Bright field and HbSat maps were periodically observed after initiation of single cell suspension of 4T1 in dors al window chamber to monitor the enhanced vascular development to deliver the excessive oxygen to grow ing tumor mass. Tumor size, microvascular structure and HbSat changes were monitored on 1 st 3 rd 5 th and 7 th day after 4T1 cell initiation (Fig.4 1) th en labeled RBCs were administered when tumor size reached about 6 mm in a diameter with supplying vascular network s In vivo Numerical Analysis of Accumulated Sickle Red Blood Cells using a Single Fluorescent Microscopic Imag e After administration of flu orescently labeled RBCs and SSRBCs, at least 6 locations including enough number of venules within 25 m in diameter near tumor and normal sites in window chamber for each mouse were chosen as ROIs to quantitatively analyze fluorescence signals from prefer entially accumulated SSRBCs to endothelium. Fluorescence and bright field Images were acquired after 0min, 30min, 60min, 90min,
78 3hrs, 6hrs, and 12hrs post injection of labeled cells and compared with each group. For example, accumulated SSRBCs from Knock i n group in fluorescence images were segmented and overlaid to the bright field images of normal and tumor sites ( Fig. 4 2 ) G reater number of the accumulated SSRBCs to microvasculature in tumor sites were determined as compared with in the normal sites. An accumulation of SSRBCs in hypoxic tumor sites w as anticipated showing the enhanced accumulation to endothelial cells due to its polymerization of HbS in SSRBCs under deoxygenated environment and overexpressed adhesion receptors interacting with endothelia l cells more than normal tissue sites These results were consistent with research by others [90, 196] The numerical analysis for all groups at all of the imaging time points were shown in Fig. 4 3. As shown in F igure 4 3 (A and B), Hydroxyurea o ff group showed more dynamic and active accumulation at both tumor and normal sites than all the other groups. On the other hand, Hydroxyurea o n group demonstrated relatively stable accumulated rates as compared with the H ydroxyurea o ff group and it showed much less accumulation in tumor sites as compared with murine models In addition, its accumulation to blood vessels was similar both of normal and tumor sites. This was consistent with current clinical evidence that the efficacy of hydroxyurea the only approved treatment of SCD contributes to incre ased fetal Hb, decreased sludging and vaso occlusion, decreased ischemia and necrosis, and decreased membrane damage  T here was a relatively higher accumulation rate in the Hydroxyurea o n group than murine blood groups in normal sites and this could possibly be explained by the larger volume of as compared with normal mouse 
79 Among murine blood sample groups, SSRBC signal s of K nock in and Berkeley groups in normal sites showed slightly lower intensity than the control group, whereas much higher SSRBC signal of K nock in and Be rkeley group s were seen in tumor site ROIs compared to the control group Relative fluorescence intensities were calculated as shown in Fig. 4 3 (C). Averaged values of fluorescence signal on ROIs in tumor sites divided by ones in normal sites after 12 hr s post administration of SSRBCs / RBCs was calculated and considered as relative fluorescence intensity. Statistical significance of the differences between the groups were calculated using ANOVA followed by a Bonferroni post hoc test and a p value below 0 .05 was considered significant ly different Relative fluorescence intensity of Knock in group and Berkeley group were significantly different not only from murine control group but also from human sample groups ( p <0.05). The highest relative fluorescence i ntensity from Knock in group indicates that it had more enhanced accumulation rate s to endothelium in tumor sites while much less in normal sites. In addition, Knock in SCD mouse model has been considered as better SCD mouse model due to the therapeutic ge ne replacement upon homologous recombination regarding SCD research  In vivo Numerical Analysis of Accumulated Sickle Red Blood Cells using Fluorescence Video Recording Fig. 4 4 shows supplementary microscop ic observation of SSRBCs flux and accumulation by video recording for 5mins using 20x LD Plan NeoFluar objectives It was conducted and analyzed at all of the same imaging time points but human sample groups including Hydroxyurea o n and o ff were not inclu ded in this analysis due to its limited supply. Fig. 4 4 (A ) show the bright field images of the interested microvasculature using 20 magnified objective and a single fluorescence image from
80 video recordings of localized SSRBCs in the same area at 12 hrs after post cells injection. Scale bar represents 50m. B ar graph in Fig. 4 4 (B ) shows relative fluorescence intensity for murine groups at all of the imaging time points and delivered similar results as in Fig. 4 3. The immobile SSRBCs over 1 min in strea ming video were considered as accumulated cells to vessel wall and counted manually It was divided by the number counted same way in normal sites to calculate the relative fluorescence intensities Final relative fluorescence intensities were compare d by both imaging methods with different magnification in Fig. 4 4 ( C ) and two different evaluation method showed similar patterns. Knock in group had the most enhanced accumulation rate compared with the other murine models and it was significantly different f rom C57 and Berkeley groups. Histological Analysis of Accumulated Sickle Red Blood Cells in Liver, Spleen and Tumor Tissues The pathophysiologies in human SCD cause avascular necrosis, abnormal blood cell endothelium interactions, inflammation, infarctio n and end organ failures [78 81] Specifically, the microvascular occlusions observed in human SCD consistently affected the lung and liver disorders, kidney failure, bone marrow, and splenic infarction of the tran sgenic SCD mice  In this experiment, typicall y damaged organs in human SCD patients such as liver and spleen were selected. In addition, tumor tissues were prepared by same way. They were stained by H&E method and a population of SSRBCs was monitored in each specimen using fluorescence imaging. Segmented fluorescence signals in green from RBCs and SSRBCs were superimp osed the H&E stained images (Fig. 4 5 (A)). Numerically analysis of the cell population was performed and displayed in the bar graphs in Fig. 4 5 (B). To calculate the relative fluorescence
81 signals, the number of SSRBCs observed in a specific site was divi ded by the number of RBCs observed in the same area because the C57 group was considered as a reference group. I nterestingly, the relative higher fluorescnece intensity was detected in Knock in group compared with Berkeley group and they were significantly different from each other ( p <0.05)
82 Figure 4 1. Hyperspectral images of dorsal skinfold window chamber. T ransmitted light (brightfield) and Hb saturation (HbSat) images of dorsal skin fold window chamber were acquired on 1, 3, 5, and 7 days after in itiation of 4T1 cells. The circular window chamber area has a 12mm diameter. The color bar shown at the bottom of the figure represents the oxygenation level in the HbSat maps.
83 Figure 4 2. Microscopic images of postcapillary venules with transmitted li ght and fluorescence images were acquired. For example, accumulated Knock in SSRBCs in capillary bed with red dot were overlaid on vascular images using 5X magnified objective. Top row: normal tissue, bottom row: tumor tissue; 1st column: after 1hr, 2nd co lumn: after 6hrs, 3rd column: after 12 hrs post injection of labeled SSRBC via tail vein. Scale bar represents 50 um.
84 Figure 4 3. Bar graphs show numerical fluorescence intensities on selected ROIs in each group (meanSD). (A) Fluorescence intensity on ROIs in normal sites after 0min, 30min, 60min, 90min, 3hrs, 6hrs, and 12hrs post administration of SSRBCs,(B) Fluorescence intensities on ROIs in tumor sites at the same imaging time points,(C) Relative fluorescence intensity (signal from tumor sites signal from normal sites) were compared from accumulated RBCs on ROIs for each group after 12 hrs post injection of RBCs and SSRBCs. p < 0.05 compared with the other groups p < 0.05 compared with the other groups
85 Figure 4 4. In vivo numerical ana lysis of accumulated RBCs and SSRBCs using video recording with 20x objective. (A) Microscopic images of postcapillary venules with transmitted light and fluorescence images were acquired. 1 st row: transmitted images for normal tissue s 2 nd row: fluorescen ce images for normal tissues, 3 rd row: transmitted light images for tumor tissues, 4 th row: fluorescence images for tumor tissue s ; 1 st column: C57 group 2 nd column: Berkeley group 3 rd column: Knock in group Scale bar represents 50 um. ( B ) Relative fluor escence intensity on ROIs after 0min, 30min, 60min, 90min, 3hrs, 6hrs, and 12hrs post administration of SSRBCs and RBCs ( C ) Comparison of relative fluorescence intensity from two different imaging techniques after 12 hrs post administration of blood cells p < 0.05 compared with the other groups the other groups
86 Figure 4 4. continued
87 Figure 4 5 Fluorescently labeled cells in tissue specimens including liver, spleen and tumor were compared. Each specimen was stained b y H&E method and segmented blood ce lls in red were superimposed to stained image ( A ) Rows from top to bottom: liver spleen, and tumor tissue; 1 st column : C57 group 2 nd column: Berkeley group 3 rd column: Knock in group Scale bar represents 50 um. ( B ) F luorescence signal from each organ infused by SSRBCs was divided by ones infused by RBCs. p < 0.05 compared with the other groups the other groups Scale bar is 50um
88 Discussion Sickle cell disease is the most common inherited h ematologic disease in the United States and it causes substantial morbidity and mortality. Hypoxia induced polymerization of HbS deforms the RBCs and causes massive cation loss as well as increased expression of adhesion receptors. The major pathologic pro cesses involved in SCD are vaso occlusion, inflammation, procoagulant processes, hemolysis and altered vascular reactivity. However, a natural SCD animal model was not existing so that transgenic mouse models of SCD have been generated to investigate the c omplex pathophysiology and to evaluate potential therapies in vivo and preclinical studies such as anti sickling gene therapy. In our experiment, two types of SCD mouse models (Berkeley and Knock in mouse) were employed according to following reasons. Berk eley (Knock out) mouse is one of the most commonly utilized SCD mouse model developed at Lawrence Berkeley National Laboratory in 1997  It expressed a very severe sickling with very low Hb concentration level (5 g/dL). Additionally, it survived for a mean of 14 months so that this relatively short life span was unfeasible to be employed for new therapies. However, it has demonstrated h ematologic as well as histopathologic similarities and differences compared with human SCD patients. The similarities include sickling, hemolysis, reticulocytosis, severe anemia (hematocrit, 10~30%), leukocytosis, elevations of inflammatory cytokines, defe cts in urine concentrating ability,organ infarcts, glomerulosclerosis, and pulmonary congestion  The differences include lower Hb concentration, thalassemic phenotype, smaller volume of SSRBC s and splenomegaly  Another donor mouse was Knock in SCD model. In this recently developed mouse model, the mouse globin genes was re
89 (HbF) and the human homologous genes. As a result, a high HbS expression in the fetus lacking HbF was decreased to avoid the fetal death between 3 and 6 months of age and increase a mean survival up to18 months with slightly higher Hb concentration (8.9 g/dL)  Therefore, Knock in mouse model has been known as a better SCD mouse model for the therapeutic gene replacement than Berkeley mouse model  The goal of this experiment was to find appropriate SCD mouse model presenting us more accumulation to endothelial cell due to overexpressed adhesion receptors and enhanced polymerization under tumoric environments characterized i n terms of low pH, low oxygen tension, relatively low blood flow due to disorganized vasculature and higher blood viscosity, permeability and interstitial pressure  Quantitatively analyzed results in Fig. 4 3 (C) demonstrated that relative fluorescence inte nsities for SSRBCs from murine SCD models had superior accumulative events to vessel walls as compared with human SSRBCs and C57 groups. Although absolute fluorescence signals of accumulated SSRBCs from Hydroxyurea off group in tumor sites were higher than any other groups, relative fluorescence signal was decreased by elevated level of localized cells in normal sites. However, these results from current experiment were consistent with previous studies about tumor preferential accumulations by SSRBCs under the hypoxic tumor environment [90, 91] SSRBCs from Knock in mouse model showed higher accumulation rates compared with Berkeley mouse model. Since the Berkeley mouse model had a more fragile membrane causing hemol ysis, anemia and shorter lifespan in response to lack of human HbF than Knock in mouse, relatively fewer accumulated SSRBCs from Berkeley were detected on endothelial cells [81, 200, 201] In fact, Knock in mouse m odel was recently developed by Wu et al. in 2006  so
90 that preceding in vitro studies may be recommendable to have better understanding on the subject of measurement of hematocrit, hemolysis rate, the percentag e of sickled cells or rheologic characterization in blood stream under similar condition as tumor sites between two groups. The experiments in this chapter demonstrate that blood from the mouse models of SCD can effectively replicate the tumor accumulation of human SSRBCs that was previously reported in animal models and in clinical cases.
91 CHAPTER 5 IN VIVO DELAYED PHOTOHE MOLYSIS MEASUREMENT FROM PHOTOSENSITIZED SSRBCS LOADED WITH CALCEIN IN 4T1 TUMOR BEARING MOUSE MODEL In the previous chapter, the tumor selective accumulation of SSRBCs by SCD human patients and mouse models w as effectively demonstrated O ur intravital microscopic imaging system was employed to monitor the alteration of tumor microvasculature and functional changes of HbSat F luorescence i mages acquired from our imaging system were applied for quantitative analysis of the accumulated SSRBCs in the blood vessels. As we assumed, SSRBCs from SCD patients and animal models expressed the natural tumor preferential accumulation and it partially v erified the potential of SSRBCs as a novel drug carrier. Among the SSRBCs groups, the Knock in mouse model demonstrated the highest relative fluorescence intensit ies in selected ROIs in tumor and normal sites Based on this result, Knock in SCD mouse blood was used with in vitro DPH measurement in a controlled manner, and in vivo experiment s using immum o deficient mice In previously published experiments with drug loaded normal RBCs, several osmosis based techniques including hypotonic hemolysis, hypotonic dilution, hypotonic dialysis, hypotonic preswelling, and osmotic pulse have been developed for drug loading and were possibly applicable to SSRBCs [41, 59 61] T here are several advantages of the hypertonic preswel ling method. T his method employs simpler and faster procedures than the dialysis method so that it causes minimal destruction of the cell membrane with relatively high entrapment efficiency [42, 62] A s a result of moderate loading condition, the resulting resealed RBCs in systemic circulation has comparable lifespan to that of the normal RBCs  H ence, hypotonic preswelling
92 method was selected and modified for SSRBCs based on previously reported method s fo r normal RBCs  C haracterization of In vitro DPH expe riment for normal RBCs was performed to examine the photohemolysis reactions in relation to various photosensitizer concentrations, incubation temperature s irradiation temperature s irradiation power and irradiation time similar to Al Akhras and Grosswein er et al [99 101] However, careful manipulation was required to handle the sensitive carrier s for temporally controllable dye releas e method from photosensitized SSRBCs with the photosensitizer protoporphyrin IX ( PpIX ). S ince lots of SSRBCs accumulation was observed around 12 hrs after administration of the cells via tail vein injection in the previous chapter, v arious parameters were modified and optimized for t50 at th is time in this experiment The fractional he molysis was determined from the amount of Hb released into the solution normalized to an equivalent solution of fully lysed red blood cells. The results from in vitro DPH experiment were analyzed and the theoretical time required for t50 was calculated by Gompertz function  Microdialysis methodology has been a powerful sampling technique capable of continuous monitoring the concentration of unbound drugs both for in vitro and in vivo investigation of both endogenous and exogenous substances [103, 104] I t has been applied and broadened for various pharmaceutical studies including the transdermal delivery of drugs  tissue pharmacokinetics  and tissue pharmacodynamics [108, 109] A dditionally, this monitoring technique has been employed in various tissue and organs of human or murine models such as liver  heart  skin  blood vessels  placenta  st omach  ear  and tumor tissue [116 118] T he
93 microdialysis methodology was employed for in vivo DPH measurement and fl uorescence intensities of the collected dialysates from tumors were compared to healthy muscle tissues by microplate reader at 12 hrs and 24 hrs post injection In this experiment, we mainly focused on the controlled dye release from resealed SSRBCs group Four different groups including free calcein, calcein loaded SSRBCs (from Knock in mouse) with or without photoactivation, and calcein loaded normal RBCs (from wildtype mouse) with photoactivation by 1min irradiation were performed and compared with each group. Materials and Methods Materials Dulbecco's Phosphate Buffered Saline ( DPBS with 1mM of Ca 2+ and 0.5mM of Mg 2+ without phenol red, SH30264.01, HyClone ), Phosphate Buffered Saline (PBS, HyClone), 10X Phosphate Buffered Saline (10X PBS, HyClone) and (for mammals, S77939, Fisher Scientifics) were obtained from Fisher Scientifics (Pittsburg, PA). Calcein (623Da, ex: 495nm and em: 520nm, 019K1223, Sigma) and Protoporphyrin IX (PpIX, 1127K1570, Sigma) were obtained by Sigma Aldrich (St. Louis, MO). Microdialysis probes (CMA 20 Elite Microdialysis probes, 10mm of membrane length, 20 kDa of cut off, 8010436, CMA Microdialysis) were commercially available from CMA Microdialysis (Solna, Sweden). Preparation of Blood Cells All in vivo experim ents were carried out under a protocol approved by the University of Florida Institutional Animal Care and Use Committee (IACUC) SSRBCs from Knock in mouse w ere prepared by Dr. Angela E. Rivers (M.D, Ph.D. Assistant Professor, Department of Pediatrics, Di vision of Hematology/Oncology, University of
94 Florida) Donor mice were anesthetized by 1% ~ 2% isofluorane in air and blood samples were obtained by retro orbital bleeding using a heparinized glass pipette N ormal RBCs from a donor C57BL/6 wildtype mouse w er e prepared by the author. D onor mice were anesthetized by intraperitoneal injection of ketamine (100mg/kg) and xylazine (10mg/kg) and normal RBCs were obtained by cardi ac puncture O btained blood samples were stored in ethylenediaminetetraacetic acid (EDTA ) tubes at 4C until use. Preparation of Loaded Blood Cells with Calcein To load fluorescent dye into SSRBCs, the hypotonic preswelling method one of the osmosis based loading methods into normal RBCs was selected and modified  It was derived from the fact that exposing RBCs in a hypotonic solution leads to an enhanced permeability of the membrane and enlargement of pores with diameters of 200 to 500 (20~50 n m) in the membrane when it reaches hemolysis  The SSRBCs in about 1ml of DPBS solution were washed via temperature controllable centrifugation at 1600 rpm at 4C for 7min and resuspend in DPBS The supernatant was discarded and this step was repeated until its supernatant was clear. Packed SSRBCs were split by 200l into several polypropylene tubes and 1.2 ml of hypotonic DPBS solution with osmolarity of 0.67 was added into each tube. The cell suspension was gently mixed for 5 min and centrifugated at 1600 rpm at 4C for 7 min to separate swollen cells. The supernatant was discarded and a 40l of aliquot of a hemolysate (SSRBCs:DIH2O = 1:1 (v:v)) was added gently above the swollen SSRBCs to compensate loss of Hb and intracellular components during this loading procedure. In addition, it led the decrement of the osmolarity shock between the swollen cells and a queous dye solution. Then 250l of aqueous hypotonic solution of calcein (77.8 g ml 1 ) was gently added in each tube and mixed for 5 min. When the transparency of
95 supernatant was suddenly increased and white ghost cells were observed between supernatant a nd packed cells, hemolysis point of the SSRBCs was achieved. At this point, 100l of hypertonic solution (10X PBS) was added rapidly in each tube to reseal the pores created during cell swelling and lysis procedures by hypotonic environment. Cell suspensio ns were mixed by gentle swirling for 5 min and incubated at 37C for 30min to reanneal the resealed cells. The SSRBCs were washed twice in DPBS to remove unloaded calcein and resuspended to give an expected SSRBCs concentration (8 x 10 8 cells ml 1 ) for furth er procedures. That concentration was acquired by repeated absorbance measurement of lysed Hb from intact to completely lysed SSRBCs using spectrophotometer (NanoDropTM 1000, Nanodrop, Wilmington, DE) until maximum lysis had occurred. Preparation of Photo sensitized Blood Cells by PpIX The 1ml of SSRBCs in the tube was centrifuged at 1600 rpm at 4C for 7 min and the supernatant was discarded. Concentrated SSRBCs were incubated with 6ml of PpIX dissolved in DPBS solution (20M) at 37C for 30min and it was swirled every 15min during the incubating period. The SSRBCs were washed twice in DPBS to remove unbound photosensitizer and resuspended in 3ml DPBS solution. Treated SSRBCs were store d at 4C until it use To protect the sample from ambient light, the all tube s were covered by aluminum foil during all procedures. In V itro Delayed Photohemolysis M easurement I n vitro DPH measurement was previously described in Chapter 2. Briefly calcein loaded and photosensitized 810 8 cells of normal RBCs from C57 wildtype mouse by 50 M of PpIX and SSRBCs from Knock in mouse by 25 M of PpIX in 3ml were prepared. T wo d ifferent types of blood sample were prepared according to its own purpose and
96 limited number of Knock in mice. Normal RBCs w ere expected to present various t 50 depending on the irradiation time ( t irr = 0, 9, 11, 13, 15, 17 and 19 min ). Therefore, it could provide baseline data regarding characteristics of DPH for SSRBCs E ach sample was transferred to a glass cuvette and was positioned in temperature controll ed cuvette holder. N ormal RBCs and SSRBCs suspension s were irradiated by 0. 33 W and 0.08W halogen lamp for various irradiation times ( t irr = 0, 9, 11, 13, 15, 17 and 19 min for normal RBCs and t irr = 1,5,10 and 20mins ) to initiate photochemical reaction by PpIX at room temperature. Different light doses were achieved by varying the irradiation time for constant incident irradiance. After irradiation t he thermal reaction was initiated by increasing temperature of cuvette holder up to body temperature (37C) in dark. In DPH method, photo hemolysis rate s were measured from the beginning of incubation in dark after irradiation period. W hen the temperature reached at 37C, 30l blood sample s w ere collected periodically The collected blood sample was split into pa cked SSRBCs and supernatant by immediate centrifugation and then 20l of supernatant was stored in conical tubes to measure the absorbance for fractio nal photohemolysis using a spectrophotometer T he fractional hemolysis was determined from the amount of H b released into the solution normalized to an equivalent solution of fully lysed red blood cells. Gompertz F unction Empirical results were quantitatively analyzed by kinetics model to calculate the dependence of the irradiation time by the Gompertz functi on. The original Gompertz equation was described in equation (1). Gompertz function was employed to calculate the two parameters a and b regarding fractional photohemolysis by PpIX in equation (2) and estimate theoretical time required for 50% fractional p hotohemolysis ( t 50 ) in
97 equation (3). This function parameters a b and t 50 were important predict of the average DPH rate and this model has been assessed to investigate the hemolysis rate of photosensitized RBC by PpIX  The empirical t 50 was measured and compared with the theoretical t 50 determined by equation (3) described in Chapter 1. R 2 values were computed for the fit of t he empirical t 50 to the theoretical values of t 50 by the Gompertz function. Tumor C ells 4T1 mouse mammary adenocarcinoma cells were generously provided by Dr. Mark W. Dewhirst (Duke University Medical Center, Durham, NC). The cells were cultured as a monolayer in DMEM (Cellgro, Inc., 1X, 4.5 g/l glucose, L glutamine and sodium pyruvate) with 10% fetal bovine serum (Biowhittaker, Inc.), 1% L glutamine (Clonogen, Inc.) and 1% pen icillin streptomycin (Clonogen, Inc.). Single cell suspensions were prepared in DMEM without serum at room temperature and stored at 4C until its injection into mouse models. Animal M odels All in vivo experiments were carried out under a protocol approv ed by the University of Florida Institutional Animal Care and Use Committee and conformed to the NIH Guide for Care and Use of Laboratory Animals. Female athymic (nu/nu) nude and wild type (C 57) mice between 20 and 26 g were obtained from Harlan Laborator ies for this study. Single cell suspensions of 110 5 4T1 cells in 100l were prepared in DMEM without serum at room temperature and injected subcutaneously over the left quadriceps muscle of athymic nude mouse under anesthesia by 1~ 2% isofluorane in air. After the tumor cell injection, all mice were housed in an environmental chamber
98 maintained at 33C and 50% humidity with free access to food and water and standard 12hrs light/dark cycles. When the tumor size re ached to 10 mm in diameter, a 20 0l bolus of treated RBCs in DPBS solution (Hct: 50% V/V) or calculated concentration of free calcein (5g ml 1 ) equal to the amount loaded into RBCs was administered via tail vein Four different mouse groups were prepared and summarized in Table 5 1. Table 5 1 Cate gorized groups for in vivo DPH measurement of SSRBCs to tumoric microvasculature experiment. Group Blood type Calcein PpIX t irr [min] N RBC calcein PpIX 1min Wildtype mouse ( C57BL/6 ) Y Y 1 3 SSRBC calcein PpIX 1min Knock in SCD mouse Y Y 1 3 SSRBC calce in Knock in SCD mouse Y N 0 3 Free calcein N/A Y N 0 3 F ree calcein (Free calcein group) calcein loaded SSRBCs with ( SSRBC calcein PpIX 1min group) and without ( SSRBC calcein group) photoactivation, and calcein loaded normal RBCs with phot oactivation ( RBC calcein PpIX 1min group ) by 1min irradiation were injected animal models when the injected tumor mass reached 10mm in a diameter. In vivo Delayed Photohemolysis using Microdialysis Tubing When the tumor size at injected areas reached to about 10 mm in diameter, manipulated blood cells were systemically administered by tail vein. F or in vitro DPH measurement experiment, photosensitized blood cells were photo activated by irradiation for 1 min ( t irr = 1min) prior to their administration. T o investigate the controlled release rates of calcein from photoactivated SSRBCs in ti ssues, microdialysis probes were in serted at the sites for collection of dialysate Microdialysis probes and tubing connectors were e use
99 respectively After that, the probes were perfused with bubbles in tubing for 30min at a flow rate of 2 l min 1 by syringe pump The probes were then placed 5mm below the surface for tumor tissue through a plastic gu ide cannula. A probe was placed into the quadriceps muscle as a control site through a small incision made on the skin of implantation site with a small scalpel blade. The probe were inserted in a similar way in tumor tissue and sutured to skin. Dialysate samples were 1 during 4hrs intervals from the tumor tissue and the quadriceps muscle. Each sample was collected at a variety of time points after the photosensitized SSRBCs cells or fre e calcein injection via tail vein. Fluorescence signal of calcein in the dialysate could be analyzed using microplate reader and compared Statistical analysis was performed using SPSS software (SPSS for Windows; SPSS Inc., Chicago, Illinois, USA) and anal assess the statistical significance of the differences between the groups. Results Efficacy of Calcein Loading Rate into Sickle Red Blood Cells Hypotonic preswelling method for loading calcein into SSRBCs from Knock in mouse was modified from the protocol for normal erythrocyte  and performed. Light microscope and fluorescence flow cytometry techniques were employed to analyze the fraction of SSRBCs loading calcein in Fig. 5 1. U ntreated SSRBCs, treated SSRBCs without calcein and tr eated SSRBCs with calcein samples by modified hypotonic preswelling method were set up for fluorescence flow cytometry test. Example microscopic image of SSRBCs loading calcein is shown in Fig. 5 1(A) and distribution of fluorescence signals by flow cytome try is shown in Fig. 5 1(B). Modified hypotonic
100 preswelling showed 85.46% of entrapping efficacy of calcein into SSRBCs and this result was consistent with the fractional data of various hypo osmotic lysis methods in Table 1 5. In vitro Delayed Photohemoly sis Measurement of Photosensitized Sickle Red Blood Cells by PpIX DPH measurement was performed using photosensitized normal RBCs from C57 wildtype mouse with PpIX first. Optical absorbance of th e fractional hemolysis rate s (0%, 20%, 40%, 60%, 80% and 100% ) from the amount of Hb released into the solution was determined by spectrophotometer at 415nm ( Fig. 5 2 (A) ). T he sample of various hemolysis rates was normalized and linearly fitted to an equivalent solution of fully lysed red blood cells (Fig. 5 2 (B)) N ormal murine RBCs were photosensitized by 50 M of PpIX and photoactivated by 0.33W halogen lamp based on various irradiation time ( t irr = 0, 9, 11, 13, 15, 17 and 19 min) at 24 C. DPH measurement was created when the temperature of sample solution reac hed at 37 C in dark. Supernatant from the centrifugated sample in cuvette was collected every 5 min up to 105min and determined by spectrophotometer. Various sigmoidal curves depending on different irradiation time are presented in Fig. 5 2 (C). Each DPH c urve showed a typical sigmoidal shape and f ractional Hb release rate were possibly controll ed by the various light energy dose according to the irradiation time. The re leased Hb concentration from photoactivated SSRBCs was acce lerated by increased exposure time Photohemolysis measurement s from non photoactivated RBCs were negligible (< 10%) at final measurement time point (105 mins) in dark (Fig. 5 2 (C)). I t should be noted that thermal reaction by itself without photochemical activation cause si gnificant photohemolysis. C ombined two procedure such as photochemical and thermal reactions generated DPH
101 Similar experiments were repeated for Knock in SSRBCs and in vitro DPH results are shown in Fig. 5 3. From the results in Chapter4, systemically adm inistered SSRBCs could substantially accumulate in tumor sit es about 12 hrs post injection Therefore, 50% fractional photohemolysis ( t 50 ) rate in the range of about 12~24 hrs was desirable. All applicable parameters (i.e., light dose, irradiation time, an d concentration of PpIX ) from normal RBCs experiment were modified and optimized to produce significant photohemolysis at desired time point. F or example, l ight dose (from 0.33 to 0.08W) irradiation time range (from 9~19min to 1 20min) and concentration of PpIX (50 M 25 M) were customize d. DPH measurement of SSRBCs by various t irr (1 5, 10, and 20min) and the results from Gompertz analysis are demonstrated in Fig. 5 3 and average value of r 2 was over 0.99. Analysis of i n vitro Delayed Photohemolysis M easurement by Gompertz Function In this experiment, t 50 could be adjusted by only one parameter ( t irr ), whereas t he other factors, such as concentration of PpIX, irradiation temperature, incubation temperature and light energy dose, were fixed. DPH measur ements from Fig. 5 3 were analyzed by Gompertz function. The calculated parameters ( a and b ) accompanied with t he best fit to empirical curves by equation (2) and e mpirical and theoretical time t 50 by equation (3) are summarized in Table 5 2 The results a re plotted in Fig. 5 4 and linear fitting model applied to each parameter ( a b and t 50 ) presented high r 2 values (>0.98) in addition, Gompertz function could effectively estimate theoretical t 50 compared with empirical t 50 ( r 2 = 0.9844). In vivo Controll ed Calcein Release Form Sickle Red Blood Cells Dialysate samples were of 1 l min 1 during 4hrs intervals from the tumor ti ssue and the quadriceps muscle. In
102 vivo DPH measurements from dialysate sa mple s collected at 12 hrs and 24 hrs post administration are shown in Fig. 5 5 (A). T he fluorescence signal of released calcein from photoactivated SSRBCs loaded with calcein group by 1min irradiation ( SSRBC calcein PpIX 1min group) collected from both sit es (tumor and normal sites) at 24hrs post injection demonstrated statistical difference ( p <0.05) from the other groups. H owever, that of 12hrs measurements from same group but only in tumor sites was statistically different ( p <0.05). T hat of the other grou ps including SSRBC calcein, RBC calcein PpIX 1min, and free calcein group had similar fluorescence intensity at 12 and 24 hrs post injection. R elative fluorescence intensity measurements showed consistent results T o calculate relative fluorescence intens ity, the fluorescence signals of released or existing calcein at tumor sites at 24hrs post injection was divided by that of normal sites. It is shown in Fig. 5 5 (B) and only SSRBC calcein PpIX 1min group was statistically different from the other groups ( p <0.05)
103 Figure 5 1. Light microscope and fluorescence flow cytometry techniques were employed to analyze the efficiency of hypotonic preswelling method to SSRBCs. A: Microscopic fluorescence image of calcein entrapped in SSRBCs was superimposed on the transmitted light image. Scale bar shows 1 5 m. B: Flow cytometric analysis of SSRBCs loading calcein. X axis represents the fluorescence intensity from entrapped calcein in the SSRBCs. Y axis represents size of cells. Left: normal SSRBCs, Middle: sham tre ated SSRBCs, Right: calcein loaded into SSRBCs.
104 Figure 5 2 Delayed photohemolysis in normal canine RBCs photosensitized with PpIX were measured at 415nm. (A) Supernatant of collected photoactivated blood sample was measured at 415nm using spectropho tometer. (B) Linearly fitted model for optical density of various hemolysis (0%, 20%, 40%, 60%, 80% and 100%) fraction of normal canine RBCs at 415nm as reference group. (C) Photosensitized normal canine RBCs by 50 M of PpIX were irradiated by 0.33W halog en lamp for various irradiation time ( t irr = 0, 9, 11, 13, 15, 17 and 19 min) and fraction of photohemolysis were acquired. Photoactivation by irradiation was performed at 24C followed by incubation in dark at 37C, respectively.
105 Figure 5 2 continu ed
106 Figure 5 2 continued
107 Fig ure 5 3 Fractional photohemolysis rates of photosensitized SSRBCs by 25 M of PpIX were measured. The cells were irradiated by 0.08W halogen lamp at 24C during various irradiation time (1, 5, 10, and 20min) and incubat ed in dark at body temperature (37C). Open square with error bar represents experimental data by in vitro optical density measurements and dotted line is fitting curves by Gompertz function. r 2 values were calculated.
108 Table 5 2 SSRBCs with 2 5 M PpIX irr adiated with 0.08W halogen lamp at various irradiation times at fixed irradiation and incubation temperature t irr [min] t 50 [min] a t 50 [min] b a b [min 1 ] T irr [C] T inc [C] 1 940 934.4300 4.0906 0.0019 24 37 5 777 809.6226 4.1142 0.0022 24 37 10 664 653. 4984 4.3192 0.0028 24 37 20 476 477.4097 4.6782 0.0040 24 37 a E mpirica l results obtained from data curve s. b Theoretical values calculated from E quation (3 )
109 Fig ure 5 4 Parameters for Gompertz function were calculated based on fractional DPH meas urement of SSRBCs carrying calcein with various irradiation time ( t irr ). (A) x axis: irradiation time [min], y axis: parameter a in Gompertz function. r 2 = 0.98575. (B) x axis: irradiation time [min], y axis: parameter b in Gompertz function. r 2 = 0.99695. (C) x axis: irradiation time [min], y axis: t 50 in experimental results with black solid line and estimated t 50 values by Gompertz function. r 2 = 0.98444.
110 Figure 5 4 continued
111 Figure 5 5 In vivo fluorescence signal measurement through systemic ad ministration of prepared blood sample groups, such as Free calcein, RBC calcein PpIX 1min, SSRBC calcein, and SSRBC calcein PpIX 1min to t umor bearing mouse model. (A) Fluorescence signal from the dialysate in each group were determined at 12 hrs and 24hrs post administration. Fluorescence signal from SSRBC calcein PpIX 1min group 24hrs post administration in tumor (*) and normal sites ( ) is significantly different (p<0.05) from the other groups collected at 24hrs post administration Fluorescence signal f rom SSRBC calcein PpIX 1min group 12hrs post administration in tumor site (#) is significantly different (p<0.05) from the other groups collected at 12hrs post administration. (B) Relative fluorescence intensities were determined at 24hrs post a dministrati on Relative fluorescence intensity from SSRBC calcein PpIX 1min group is significantly different from the other groups (p<0.05).
112 Discussion Cytotoxic chemotherapy is limited by systemic toxicity. T he possible scenario to improve the therapeutic index i s development of tumor specific targeting drug carriers and controlled release of drug from carriers. I n the present work, SSRBCs from Knock in SCD mouse model was employed as a new drug carriers and its natural tumor targeting under unique environment of tumor sites using mouse model was evaluated from previous experiment. According to the results, the minim um required time to observe the accumulation of SSRBCs in the tumor sites was empirically determined at about 12hrs ~24 hrs. Using this finding, tempor ally controlled release of the entrapped fluorescent dye (calcein) from resealed SSRBCs was attempted using the DPH technique. Drug release by photodynamic activation using photosensitizer could provide rapid and predefined release. However, it is one of conventional ly used techniques for topical photodynamic therapy for the treatment of skin cancer and it is only available for identified and accessible tumor sites, not for randomly distributed solid tumors due to difficulties identifying and irradiating m ultiple tumor sites in disseminated locations that may be inaccessible [8, 203] T herefore, the possibility of ex vivo activation for in vivo drug release at disseminated tumors with tumor specific targeting maybe enabled with the technique in the project Flynn et al. demonstrated the directly photo dependent release of entrapped thrombolytic agent brinase from photosensitized RBCs by exposure of radiation from a 10mW HeNe laser above the sample  I n addition, DPH of photoactivated RBCs was more precisely analyzed by Al Akhras et al.  Adapting techniques in the previously reported studies in this project the combined techniques were used to manipulate
113 SSRBCs with i n vitro DPH experim ents Temporally controlled t 50 was observed by combined effects such as photochemical and thermal reactions. T he efficiency of our SSRBCs drug carrier model was evaluated by i n vivo DPH experiment As shown in data in Fig. 5 5, concentration of released calcein from photoactivated SSRBCs in tumor sites were significantly higher than other groups. I n addition, the large difference of fluorescence signal from released calcein was observed between the measurements at 12 hrs and 24 hrs post administration T h ese results pointed out two most important facts that SSRBCs from Knock in mouse were preferentially accumulated in target tissues around desired time point and temporally controlled to release entrapped calcein from SSRBCs S ince photohemolysis was tempor ally programmed to disrupt SSRBCs relatively higher fluorescence signal was observed in normal sites from photoactivated SSRBCs than the other groups at 12 hrs post administration. P hotochemically inactivated SSRBCs and activated RBCs carriers showed mode rate release of calcein or even less than free calcein group at tumor and normal sites. Intact SSRBCs due to inactivated photohemolysis demonstrated less fluorescence signals than activated SSRBCs group. P hotoactivated RBCs group demonstrated less signal i ntensity compared with that of SSRBCs because normal RBCs were not affected by tumor environment and therefore did not preferentially accumulate in the tumors I n addition, they were not significantly different from free calcein. T herefore, the highest rel ative fluorescence intensity was calculated in photoactivated SSRBCs group The experiments in this chapter demonstrate that in vi tro DPH based drug release technique from photoactivated SSRBCs can be adapted to in vivo mouse model and effectively shows th e temporally controlled release of
114 entrapped calcein from SSRBCs in tumor locations This results suggest that similar results may be obtained for chemotherapeutic agents.
115 CHAPTER 6 CONCLUSION S AND FUTURE WORK S Summary and Conclusion C onventional c ytotox ic chemotherapy for cancer treatment has limited therapeutic efficacy. D espite remarkable progress in development of chemotherapeutic agents and numerous drug delivery systems, major challenges such as inadequate concentration of therapeutic agents in tumo r sites and systemic toxicity in healthy normal tissues remain. T o overcome the limitations, advanced strategy for tumor targeting and controlled drug release method is necessary. I n this dissertation, the potential of sickle red blood cells ( SSRBCs ) as a novel drug carrier with tumor targeting and temporally controlled drug release characteristics was investigated SSRBCs obtained from Knock in sickle cell disease ( SCD ) mouse model had more enhanced accumulation to endothelial cells in tumor microvasculatu re than Berkeley mouse model, one of the most popularly used transgenic mouse model for SCD research, using our hyperspectral imaging system. This finding suggests an appropriate transgenic SCD mouse model with prolonged lifespan that enables researchers d eveloping therapeutic agents for human patients with SCD and with advanced stage of solid tumors to investigate vaso occlusive crisis and natural tumor targeting. However, t he mechanism s responsible for more enhanced accumulation by Knock in mouse than Ber keley mouse are not yet known and further investigation is necessary. T he hyperspectral imaging system provides direct information regarding morphological changes of tumor microvasculature, functional alteration of hemoglobin saturation while tumor mass gr ows, and quantitative measurements for localized SSRBCs in tumor and normal sites.
116 Modified hypotonic swelling method wa s applied to load SSRBCs with calcein and result ed in high entrapping efficacy ( 85.46% ). T emporally controlled release of calcein from resealed SSRBCs wa s achieved by Delayed photohemolysis (DPH) method. T his technique has been employed throughout in vitro and in vivo experiment s and demonstrates suitable calcein release in a controlled manner. Gompertz based fitting model was used for an alysis of empirical and theoretical DPH trials. 4T1 tumor bearing mouse models we re prepared to observe the calcein release rate from SSRBCs in tumor sites and significantly elevated fluorescence signals compared with similarly treated normal RBCs with pho toactivation and SSRBCs without photoactivation wer e detected by microplate spectrophotometer. L ight activated SSRBCs has a potential t o be us ed as a novel drug carrier with natural tumor targeting and temporally controlled release characteristics. I t is not only an alternative drug delivery system by itself, but also it enables to develop new drug carriers u sing mechanobiological mimicry of SSRBCs to cure human patient with advanced stage of cancer Future Work s I n this research, only the absorbance from the released Hb was determined through in vitro DPH experiment. However, the released calcein volume based on DPH may or may not show similar pattern as Hb according to different molecular weight T he determination of released calcein concentration from t he resealed SSRBCs needs to be characterized with that of Hb simultaneously through in vitro DPH C onventional chemotherapeutic agents (i.e. doxorubicin or equivalent therapeutic agents ) could be employed to replicate the in vivo experiment so that tumor s ize in terms of apparent tumor area can be monitored to evaluate its efficacy. H istological
117 analysis by specialists in tumor s normal tissues and organs typically damaged by SCD are required to eval uate its systemic toxicity F urther investigation involv i ng accurate concentration measurement using quantitative techniques like high performance liquid chromatography (HPLC) is necessary.
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137 BIOGRAPHICAL SKETCH Se woon Choe was born in Gwangju, Republic of Korea. He received his Bachelor of Science degree in the Department of Electronic and Electrical engineering from the Hong Ik University in Seoul, Korea in 2001. He began his graduate studies in the Department of Electrical and Computer Engineering at University of Florida from 2002 and earned Master of Science degree in 2004. In 2006, he made a decision to move into the field of biomedical engineering when he was a second year doctoral student and he joined in the Biophotonics Imaging, Therapeutics, and Sensing Laboratory (BITS lab) in the J. Crayton Pruitt Family Department of Biomedical Engineering He received Master of Science degree under the guidance of Dr. Brian S. Sorg in 2008 and continued to pursue hi s doctoral degree. In the BITS lab he has investigated the potential of SSRBC s as a novel drug carrier with better tumor targeting and controlled releas e of entrapped substance His research interests include biophotonics, biomedical optics, cancer diagno stics and therapy intravital microscopy microcirculation, and tumor angiogenesis.