Synthesis and properties of submicron albumin spheres

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Synthesis and properties of submicron albumin spheres
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123 leaves : ill. ; 29 cm.
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McCluskey, Richard A., 1961-
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Subjects / Keywords:
Serum Albumin -- drug effects   ( mesh )
Microspheres   ( mesh )
Drug Carriers   ( mesh )
Delayed-Action Preparations   ( mesh )
Pharmacology and Therapeutics thesis Ph.D   ( mesh )
Dissertations, Academic -- Pharmacology and Therapeutics -- UF   ( mesh )
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Thesis:
Thesis (Ph.D.)--University of Florida, 1987.
Bibliography:
Bibliography: leaves 120-122.
Statement of Responsibility:
by Richard A. McCluskey.
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Typescript.
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Vita.

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University of Florida
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SYNTHESIS AND PROPERTIES OF SUBMICRON ALBUMIN SPHERES


BY





RICHARD A. MCCLUSKEY










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



UNIVERSITY OF FLORIDA


1987

















Copyright 1987

by

Richard A. McCluskey












ACKNOWLEDGEMENTS


The author wishes to express his appreciation to his committee and especially to

Dr. Goldberg for their support and guidance in completing this work. Also, a sincere

thanks to Dr. Bill Longo, Dr. Y. Yaacobi, and Jan Stacholy for their advice and to

Satoshi Sakuri for his technical assistance.

Assistance was graciously provided with electron microscopy by Micro Analytical,

Inc., with photon correlation spectrometry by Dr. Shah, and with the Lewis lung

carcinoma model by Dr. Sitren.



















TABLE OF CONTENTS

PAQE

ACKNOW LEDGEMENTS ........................................................................................... iii

LIST OF TABLES .............................................................................................. ......... viii

LIST OF FIGURES ............................................................................................... ix

ABSTRACT ................................................................................................................ xii

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

2. MATERIALS AND METHODS ......................................................................9...

2.1 Synthesis of Albumin Spheres ...............................................................9...

2.1.1 Submicron (<1 pmrn) Albumin Spheres ..........................................9...

2.1.2 Polyanionic Submicrospheres / 20 % Polyglutamic
Acid (PGA) ................................................................................. 10

2.1.3 Magnetically Responsive Submicrospheres / 16 %
Magnetite ................................................................................... 11

2.1.4 Radiopaque Submicrospheres / 2 % Barium Sulfate .................12

2.1.5 Fluorescent, Maximum Cross-Link Density Sub-
microspheres / 4 % Fluorescein Isothiocyanate-
Albumin ..................................................................................... 12

2.1.6 Microspheres (>1 pm) ................................................................ 13

2.1.7 Polyanionic Microspheres / 20 % PGA .................................... 13












2.1.8 Fluorescent, Minimum Cross-Link Density Microspheres /
4 % Fluorescein Isothiocyanate-Albumin .............................14

2.1.9 Fluorescent, Intermediate Cross-Link Density Micro-
spheres / 4 % Fluorescein Isothiocyanate-Albumin .............15

2.1.10 Fluorescent, Maximum Cross-Link Density Micro-
spheres / 4 % Fluorescein Isothiocyanate-Albumin ..............16

2.1.11 Fluorescent, Supersaturation Cross-Link Density
Microspheres / 4 % Fluorescein Isothiocyanate-Albumin ......16

2.1.12 Polyanionic, Magnetically Responsive, Albumin Micro-
spheres / 22 % Polyglutamic Acid, 17 % Magnetite ............17

2.2 Characterization by Light and Electron Microscopy ........................17

2.2.1 Size Determination of Spheres ..............................................17

2.2.2 Effect of Size and Cross-Link Density on Hydration
Sw selling ................................................................................ 18

2.3 Glycine Quenching and 14-C Labeling of Reactive Aldehyde
G roups ............................................................................................ 19

2.4 Dry Storage of Submicrospheres .....................................................20

2.5 In Vitro Degradation of Albumin Microspheres .................................21

2.5.1 Effect of Size on the Rate of Enzymatic Degradation of
Albumin Submicrospheres .....................................................21

2.5.2 Effect of Cross-Link Density on the Rate of Degradation
of Albumin Spheres ............................................................... 22

2.5.3 Effect of Enzyme Concentration on the Rate of Degradation
of Albumin Spheres ............................................................... 23

2.6 In Vivo Degradation of Albumin Microspheres .................................24

2.6.1 Intramuscular Degradation of Maximum Cross-Link
Density Microspheres ............................................................ 24

2.6.2 Intramuscular Degradation of Minimum Cross-Link
Density Microspheres ............................................................ 24

v












2.6.3 Intratumor Degradation of Minimum Cross-Link
Density Microspheres ............................................................25

2.7 Postsynthesis Drug Loading by Submicrospheres ...........................26

2.7.1 Primaquine Loading ................................................................26

2.7.2 Adriamycin Loading ................................................................27

2.7.3 cis-Platinum (ll)diaminedichloride (cis-Platinum)
Loading .................................................................................. 27

2.7.4 Mitomycin-C Loading .............................................................28

2.7.5 5-Fluorouracil Loading ...........................................................29

2.7.6 Hydroxyurea Loading .............................................................29

2.7.7 Methotrexate Loading ............................................................30

2.8 Presynthesis Loading of Fluorescein-Labeled Carboxymethyl
Cellulose ......................................................................................... 30

2.8.1 Fluorescein Labeleing of Carboxymethyl Cellulose ...............30

2.8.2 Presynthesis Loading of Microspheres with Fluorescein-
Labeled Carboxymethyl Cellulose ........................................31

2.9 Drug Release from Submicrospheres .............................................32

2.9.1 Diffusional Release of Primaquine from Submicrospheres ......32

2.9.2 Combined Diffusional and Enzymatic Degradative Release
of Primaquine from Submicrospheres .................................33

2.9.3 Combined Diffusional and Enzymatic Degradative Release
of Fluorescein-Labeled Carboxymethyl Cellulose from
Minimum Cross-Link Microspheres .....................................33

2.9.4 Combined Diffusional and Degradative Release of
Fluorescein-Labeled Carboxymethyl Cellulose from
Maximum Cross-Link Microspheres ....................................34











2.10 In Vitro Uptake of Albumin Submicrospheres by Rabbit
Blood Cells ..................................................................................... 35

2.10.1 Silver Labeling of Albumin Submicrospheres ....................... 35

2.10.2 In Vitro Uptake of Silver-Labeled Submicrospheres by
Rabbit Blood Cells ................................................................ 35

2.11 Systemic Clearance of Albumin Submicrospheres in
Rabbits .................................................................................... 37

3. RESULTS AND DISCUSSION ................................................................. 83

3.1 Submicrospheres .............................................................................. 83

3.2 Submicrosphere Size Distribution ..................................................... 86

3.3 Storage and Resuspension .............................................................. 87

3.4 Surface Modification ......................................................................... 88

3.5 Controlled Cross-Link Density Microspheres ................................... 90

3.6 Hydration and Swelling of Microspheres .......................................... 91

3.7 Degradation of Microspheres by Papain ......................................... 93

3.8 Drug Uptake by Submicrospheres ................................................... 96

3.9 Drug Release ................................................................................... 99

3.10 In Vivo Degradation .......................................................................... 102

3.11 In Vitro Uptake of Submicrospheres by Rabbit Blood Cells .............107

3.12 Systemic Clearance of Submicrospheres ........................................ 108

4. CONCLUSIONS ....................................................................................... 115

REFERENCES ................................................................................................... 119

BIOGRAPHICAL SKETCH ................................................................................1... 23














LIST OF TABLES

TABLE PfiE

1 Effect of Drying Media on Resuspension of Albumin
Submicrospheres from 4 mg/ml Dispersion .........................................39

2 Primaquine Uptake and Release By Albumin Submicrospheres ...........40

3 Distribution of 14-C Glycine Quenched Submicrospheres
(0.33 um ) with Tim e ............................................................................. 41













LIST OF FIGURES


FIGURE

1 Size Distribution of Dry Submicrospheres as Determined by TEM ...........42

2 Transmission Electron Micrograph of Dry Albumin Submicro-
spheres Showing Size Distribution ........................................................43

3 Transmission Electron Micrograph of a 25 nm Albumin
Submicrosphere .....................................................................................44

4 Scanning Electron Micrograph of Albumin Submicrospheres ................45

5 Transmission Electron Micrograph of Albumin Submicrospheres
Containing 20 wt % Polyglutamic Acid ...................................................46

6 Transmission Electron Micrograph of Albumin Submicrospheres
Containing 16 wt % Magnetite ...............................................................47

7 Scanning Electron Micrograph of Albumin Submicrospheres
Containing 2 wt % Barium Sulfate .........................................................48

8 Transmission Electron Micrograph of Albumin Submicrospheres
Containing 4 wt % Fluorescein-Albumin ................................................49

9 Optical Micrograph of Albumin Microspheres Containing 22 wt %
Polyglutamic Acid ...................................................................................50

10 Light Micrograph of Intermediate Cross-Link Density Albumin
Microspheres Before (right, 33 gim) and After (left, 55 gim)
Hydration Swelling .................................................................................51

11 Light Micrograph of Maximum Cross-Link Density Albumin
Microspheres Before (left, 38 um and 33 gIm) and After (right,
49 jpm and 44 pm) Hydration Swelling ...................................................52

12 Variation of Hydration Swelling with Sphere Size: Percent
Increase in Diameter on Hydration vs Dry Diameter ..............................53

13 Surface Modification of Albumin Submicrospheres with Glycine
and Silver ............................................................................................... 54














14 Variation of the Rate of In Vitro Degradation with Size: Degradative
Release of Soluble Fluorescein-Labeled Protein from Maximum
Cross-Linked Spheres (0.3 lim and 20 gim) by Papain (0.024
units/mg spheres) .................................................................................. 56

15 Variation of the Rate of In Vitro Degradation with Cross-Link
Density: Degradative Release of Soluble Fluorescein-Labeled Protein
by Papain (0.024 units papain/mg spheres) from 20 uim Spheres ..........58

16 HPLC Chromatogrph of Degradation Products from Minimum and
Maximum Cross-Link Density Submicrospheres ..................................59

17 HPLC Molecular Weight Calibration for Analysis of In Vitro
Enzymatic Degradation Products: Retention Time vs Molecular
Weight (Polyethylene Glycol Reference) .............................................. 60

18 Variation of the Rate of In Vitro Enzymatic Degradation of
Maximum Cross-Unked 20 Jim Albumin Microspheres ........................62

19 Optical Micrograph of Mouse Muscle 3 Days after Injection with
20 pm Maximum Cross-Link Density Spheres ......................................63

20 Optical Micrograph of Mouse Muscle 10 Days after Injection with
20 jim Maximum Cross-Link Density Spheres ......................................63

21 Optical Micrograph of Mouse Muscle 44 Days after Injection with
20 p.m Maximum Cross-Link Density Spheres .....................................64

22 Optical Micrograph of Mouse Tumor Tissue immediately after
Injection of 1 jpm Minimum Cross-Link Density Spheres .....................65

23 Optical Micrograph of Mouse Tumor Tissue 3 Days after
Injection of 1 jim Minimum Cross-Link Density Spheres .....................65

24 Optical Micrograph of Mouse Tumor Tissue 9 Days after
Injection of 1 i.m Minimum Cross-Link Density Spheres .....................66

25 Optical Micrograph of Mouse Tumor Tissue 16 Days after
Injection of 1 pm Minimum Cross-Link Density Spheres .....................66

26 Structures of Drugs Tested for Incorporation into Submicrospheres ......67













27 Primaquine and Adriamycin Spectrophotometric Calibration
C urve at pH 4 ........................................................................................ 69

28 Variation of Primaquine Uptake with pH in Submicrospheres ..............70

29 cis-Platinum and Fluorescein-Labeled Carboxymethyl Cellulose
Spectrophotometric Calibration Curve at pH 4 .....................................71

30 Variation of Adriamycin Uptake with pH in Submicrospheres ..............72

31 Variation of cis-Platinum Uptake with pH in Submicrospheres ............72

32 Structure of Fluorescein-Labeled Carboxymethyl Cellulose
Incorporated into Microspheres During Synthesis ................................73

33 Diffusional Release of Primaquine from Albumin Submicrospheres .......74

34 Variation of Release of Primaquine with Enzymatic Degradation
from Maximum Cross-Link Density Albumin Submicrospheres ..............76

35 Variation of the Rate of Enzymatic Degradative Release of
Fluorescein-Labeled Carboxymethyl Cellulose (fl-CMC)
with Enzymatice Degradation from 20 gim Minimum Cross-Link
Density Albumin Spheres ..................................................................... 78

36 Variation of Enzymatic Degradative Release of Fluorescein-Labeled
Carboxymethyl Cellulose (fl-CMC) with Enzyme Concentration
from 20 gm Maximum Cross-Link Density Albumin Spheres ..............80

37 Transmission Electron Micrograph of Silver-Labeled Albumin
Submicrospheres Taken Up by a Rabbit White Blood Cell after
6 Hours Incubation In Vitro .................................................................. 81

38 Clearance of 14-C Glycine Quenched Albumin Submicrospheres
from the Circulation of Rabbits ............................................................. 82

39 Transmission Electron Micrograph of Maximum Cross-Linked
Albumin Submicrospheres Showing Degradation after 6 Months
in W ater at 40 C ...................................................................................1... 11

40 Variation of the Cross-Link Density of Albumin Microspheres ..............112

41 Hydration Swelling of Albumin Microspheres ........................................ 113

42 Enzymatic Degradation of Albumin Microspheres ................................114

xi















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



SYNTHESIS AND PROPERTIES OF SUBMICRON ALBUMIN SPHERES

By

Richard A. McCluskey

August 1987




Chairman: Dr E. P. Goldberg
Major Department: Department of Pharmacology and Therapeutics


There has been considerable research into the use of human serum albumin

microspheres as insoluble drug carriers for controlled delivery and sustained release

of drugs. Albumin submicrospheres synthesized by methods reported to date are

hydrophobic and require surfactants for preparation of injectable aqueous

dispersions. Hydrophilic albumin microspheres have been recently synthesized in

this laboratory and partially characterized but have been restricted in size to greater

than 1 p.m.

A primary objective of this study was the development of a method for preparing

hydrophilic albumin submicron spheres (submicrospheres). Round solid

submicrospheres suitable for investigation of systemic administration were readily

prepared by this method and their properties investigated.

xii












Electron microscopy indicated that the synthesis is capable of producing submicro-

spheres in a wide size range (10 nm 1 lpm). A variety of soluble and particulate

substances, such as polyglutamic acid, magnetite grains, barium sulfate, and

carboxymethyl cellulose, were successfully incorporated by entrapment during

synthesis in a variety of concentrations.

The hydrophilic nature of the submicrospheres reported here allows the

postsynthesis incorporation of high concentrations of drugs. For example,

submicrospheres containing up to 48 wt % of adriamycin were synthesized.

Submicrospheres containing other drugs were also prepared and drug release

properties were measured in vitro by a static flow technique.

In vitro uptake of submicrospheres by rabbit blood cells and systemic clearance of

isotope-labeled submicrospheres in rabbits was studied. A low level of uptake by

white cells was observed in vitro and 98 % of intravenously administered

submicrospheres were removed from the circulation in two hours.

Hydrophilic albumin microspheres in the 1 20 p.m size range were also

synthesized to study their biodegradation. Enzymatic degradation was examined with

respect to the effect of size, enzyme concentration, and cross-link density on the rate

and extent of degradation. The effects of size and cross-link density on soft tissue

degradation were also studied in vivo following intramuscular and intratumoral

injection in mice.

The results of this study indicate that albumin microspheres and submicrospheres

have significant potential as drug carriers for localized delivery and sustained release

of drugs.
















CHAPTER 1
INTRODUCTION



The design of a drug delivery system to obtain a higher therapeutic index for a

drug may be achieved by altering the physiological disposition and pharmacokinetics

of the drug. The purpose of this research was to study the synthesis and properties of

an insoluble particulate drug delivery system, microspheres, which may be capable of

providing localized and controlled release of drugs, thereby affording greater

versatility in methods of administration as well as increasing the therapeutic index of

the drug.

The concepts of drug targeting and controlled release were understood by the

practitioners of the Ayurveda system of medicine in India as early as 6000 BC (1). At

least one of their techniques, that of incorporating drugs into oil particles, is similar

to techniques still used today for prolonging drug activity and for forming drug depots

parenterally. Direct injection of a drug oil emulsion into a target tissue, an

apparently simple method of localization and controlled release, has been shown to

prolong the retention time of drugs in tumors (2). The increase in retention time of

bleomycin in solid rat tumors, from a few minutes for free drug to about an hour for

the drug oil emulsion, significantly improved the antitumor activity of the drug.

Another consequence of drug localization would be to decrease the systemic

concentrations of the drug. Aminoglycoside antibiotics such as gentamicin and

1












tobramycin and anticancer drugs such as adriamycin, methotrexate, and cisplatin,

are notable for their toxic side effects. Reduction of the toxicities of these drugs by

reducing the systemic concentrations would enhance their clinical value.

With such benefits in mind, the use of insoluble systemically administered drug

carriers for localization and release of drugs, capable of parenteral administration,

are attracting increased attention. Particulate carriers may minimize toxic side

effects and protect labile agents against degradation in the blood since the drug is

protected from the surrounding medium. Many carrier systems have been examined,

principally synthetic liposomes and polymeric microspheres.

Liposomes are vesicles consisting of concentric polar phospholipid bilayers

(commonly a mixture of phophatidylserine and phosphatidylcholine ), varying in size

from 10 nm for unilamellar vesicles to several microns for multilamellar vesicles.

Liposome carriers can encapsulate a large volume of drug (3), but suffer from the

problem of rapid clearance by the reticuloendothelial system, regardless of size and

surface charge, e.g., a maximum circulatory half-life of 16 hours with <1jmn

unilamellar liposomes consisting of sphingomyelin and cholesterol (4,5). The larger

liposomes are preferentially retained in the lungs and the smaller liposomes in the

liver and spleen (6). Although liposomes have proved to be proficient at localizing

drugs in the lungs (7), they offer little advantage for drugs whose target areas are

elsewhere in the body. Other disadvantages of the liposome carriers include the

spontaneous leakage of the entrapped agent, a relatively inert surface limiting further

surface chemistry, and poor stability of the vesicle.












Microspheres, unlike the capsular liposomes, consist of porous matrices throughout

the entire volume of the carrier, with the loaded agent dispersed through the matrix of

the carrier or bound to the surface.

Solid polymeric microspheres consisting of polyalkylcyanoacrylates (8,9),

polystyrene (10), polymethylmethacrylate (11,12), and others have been studied as

drug carriers in biological systems. Despite having other specialized uses (13), all

have serious inadequacies as drug carriers because of resistance to biodegradation,

inability to load significant quantities of drug, inert surfaces, or inherent

hydrophobicity. To date, the only microspheres combining hydrophilicity, ability to

load a large quantity of drug, and exhibit biodegrability have been microspheres of

cross-linked dextran or serum albumin.

Both dextran and albumin are degraded in vivo. Biodegrability of cross-link

microspheres is a function of cross-linking density and accessibility to degradative

enzymes (14). Serum albumin is a natural circulatory drug carrier (15,16).

Equilibrium binding to various agents depends on hydrophobic and electrostatic

interactions (17), thereby minimizing the need for covalent binding between drug and

carrier and facilitating drug release.

Hydrophilic albumin microspheres in the size range of 1-200pmn have been

prepared and studied by Longo et al. (18). Some drug uptake and in vitro release

parameters have been characterized. An additional refinement has been the

incorporation of polyglutamic acid to promote ion-exchange as the primary

mechanism of drug binding and release (19). Albumin spheres containing 20%












polyglutamic acid by weight can bind adriamycin from solution to a concentration of

40% of the final loaded weight of the sphere ( double the amount absorbed by albumin

spheres without polyglutamic acid). Drug release is then dependent upon the ionic

strength and the rate of fluid turn over of the surrounding medium.

The dominant mechanism of removal of spheres from the circulation following

intravenous or intraarterial injection is, as with liposomes, size dependent filtration

in capillary beds, primarily in the lungs, liver, and spleen (20). The kinetics of

removal from circulation have been well characterized for polystyrene spheres with

a diameter of at least 1 p.m (10,20,21). For spheres less than 10 pm diameter, there

appears to be no difference between intraarterial and intravenous administration

when injected into beagle dogs. The circulatory half-life of 3.4 .rn spheres is

reported to be 1.6 and 1.7 min from the venous and arterial circulation, respectively.

Microspheres >8p1m were preferentially retained in the lungs, while smaller

spheres gradually cleared the lungs and were retained by the liver and spleen (22).

The unavoidable mechanical filtration that occurs in the reticuloendothelial system

has relegated microspheres >1 m to delivery of drugs to the lungs and liver or for

establishing an immobilized drug depot, e.g., via intramuscular injection, or to

non-parenteral routes such as oral and topical administration, and has spurred

development of smaller microspheres, called nanoparticles, for improved

systemically administered drug delivery systems.

Nanoparticles reported to date are primarily of polymethylmethacrylate (12) and

polyalkylcyanoacrylates (23). Polymethylmethacrylate nanoparticles (80 and

150nm diameter) have been injected i.v. in rats and mice and the elimination and












distribution of the particles examined (12,22). For both sizes, not more than 0.3%

of the administrated dose remained in circulation after 30 minutes. The particles

were initially localized primarily in the lungs and liver, but were redistributed with

time. After several days, the number of spheres decreased in the lungs, greatly

increased in the liver and slightly increased in the spleen and bones. Since

emulsifying agents appear to play a role in the removal of lipid substances from the

blood stream, it was anticipated that adsorbed surfactants could influence the

clearance of nanoparticles. Leu et al. (22) studied polymethylmethacrylate

nanoparticles coated with albumin and Poloxamer 188 (an emulsifying agent) and

found that the Poloxamer coated particles were retained in the blood longer, but still

only 1% remained after 2 hr. Although albumin has been shown to prevent platelet

adhesion and to enhance the biocompatibility of some materials, albumin coating of the

particles did not appear to significantly alter circulatory life. Hydrophilic albumin

nanoparticles have not been reported in the literature as yet. Preparation of

hydrophilic albumin nanoparticles analogous to the Goldberg et al. (19) microspheres

would be important to enable investigation of the interactions of such nanoparticles

with blood components and possible enhancement of circulatory lifetimes.

Despite the indicated problems with available nanoparticles, some in vivo

applications have been explored. Split infuenza vaccines have been bound to

polymethylmethacrylate nanoparticles (80-200nm diameter) (24). When injected

into mice and guinea pigs, the polymer adjuvant yielded higher antibody titers than

aluminum hydroxide adjuvant or fluid vaccines (25). In addition, the particulate












polymer vaccines were more stable against temperature inactivation than were

vaccines with aluminum hydroxide or without adjuvants (26).

Strategies for targeting spheres depend on the intended application, e.g., direct

local depot injection of spheres larger than 1 Ilm (18) or coating the spheres with

antibodies. Another interesting approach is the incorporation of magnetic iron oxide

into the particles and then using an extracorporeally applied magnetic field to localize

the particles. Such a technique was designed for use with microspheres >1lim by

Widder et al. (27). The spheres studied, however, were produced by heat denaturation

of aqueous albumin dispersed in cotton seed oil. Due to the suspending medium and the

method of denaturing the albumin, this method of synthesis produces hydrophobic

spheres 1-1 O1m in diameter. Spheres of this type are rapidly removed from

circulation and all spheres not trapped in the first pass through the magnetic field are

lost to the reticuloendothelial system.

In one study, adriamycin-bearing magnetic spheres were infused into the caudal

vein directly below a tumor target area and were targeted by placing a permanent

magnet over the tumor. Up to 90% of the spheres were retained in the target area and

80% tumor regression was obtained in non-metastatic solid sarcomas of the upper

tail segments of rats (28). However, this model was designed for optimal retention of

the spheres in the target area and such a situation would rarely exist naturally.

Sugibayashi, et al. (29) used the same technique for a deeper tissue model ( lung

metastases in rats) and showed only a slight increase in drug concentration and no

regressions of the tumors. Powerful electromagnets have been constructed capable of

focusing a sufficient magnetic field at any depth of the body, but the short circulation












time of currently available microspheres virtually necessitates direct cannulation

and infusion of the target area with the spheres. Nanoparticles with a longer

circulatory retention should be able to circulate until trapped in a magnetic field and

localize in more inaccessible tumors.

In view of the foregoing, it appears that opportunities for improved drug delivery

systems as well as adjuvants and diagnostics exist using hydrophilic microsphere

compositions. The fact that albumin is itself a natural circulatory protein and lends

itself to microsphere synthesis, and that such systems could even utilize individual

patient albumin specimens under certain therapeutic or diagnostic circumstances,

makes further investigations of the synthesis and behavior of albumin microspheres a

potentially fruitful research topic.

This study concerns the development of a new method for preparing hydrophilic

albumin submicron spheres (submicrospheres). The method developed is based on a

modification of the Longo (18) steric stabilization technique. Electron microscopy and

photon correlation spectroscopy were used to determine size distribution and

morphology.

Albumin microspheres synthesized in this study were used to bind drugs such as

adriamycin, primaquine, and cis-platinum. Drug release properties were measured

in vitro. Incorporation of insoluble material was also studied by incorporation of

barium sulfate and magnetite.

Microspeheres were synthesized with varying cross-link densities to study the

effect of cross-link density on the rate and extent of enzymatic degradation. The effect

of enzymatic degradation on the release of primaquine and a high molecular












weight drug model, carboxymethyl cellulose, was also studied. The effect of size and

cross-link density on in vivo degradation was studied following intramuscular and

intratumoral injection of microspheres into mice.

In vitro phagocytosis of submicrospheres by rabbit blood cells was compared by

TEM to the phagocytosis of 10 mr microspheres. Circulatory clearance of 14-C

labeled submicrospheres following intravenous administration in rabbits was

examined to investigate the suitability of systemic administration of hydrophilic

albumin submicrospheres.

















CHAPTER 2
MATERIALS AND METHODS



2.1 Syntheses of Albumin Spheres



2.1.1 Submicron (<1 prm) Albumin Spheres

Human serum albumin (0.6g) (Sigma, recrystallized and lyophilized) was

dissolved in 2.0 ml of distilled water. This solution was added to a 300 ml Labconco

lyophilization flask containing 50 ml of a 2 wt % solution of cellulose acetate butyrate

(CAB) (MW 65,000) (Polyscience) in dichloroethane (Fisher Scientific). The

suspension was dispersed by ultrasonification (Heat Systems-Ultrasonics Inc. Model

W-375 with a Q-horn probe) for 10 min at 375 watts while cooling the dispersion

flask with ice. The dispersate was added to a 250 ml flask containing 110 ml of 2 %

CAB in dichloroethane and stirred rapidly with a magnetic stirrer (Corning, Model

PC-351).

Aqueous glutaraldehyde, 10 ml, (25 wt %) (Polyscience) and 50 ml of toluene

were combined in a 100 ml centrifuge tube. The two phases were dispersed by

ultrasonification (15 sec at 200 W). The dispersion was allowed to phase separate and

40 ml of the toluene phase, saturated with glutaraldehyde, was combined with the

rapidly stirred albumin dispersate. After stirring rapidly for 2 hrs at room












temperature, the dispersate was transferred to six screw-cap 50 ml test tubes and

the cross-linking reaction was completed while being gently mixed with a rotary

mixer (Labquake Industries) for 12 hrs at room temperature.

The resulting sphere suspension was recombined in a 1 liter beaker and diluted to

400 ml with acetone and washed to remove the CAB dispersant by centrifuging at

50,000g for 30 min (Beckman J2-21 Centrifuge with a JA-17 Rotor) and

resuspending the pellet in 40 ml of acetone. After washing the spheres four times in

this way, the spheres were fractioned by size by being suspended in 95 % ethanol and

centrifuged at 3500g for 10 min. The pellet was discarded and the supernatant was

centrifuged at 50,000g for 30 min and washed twice with distilled water. The

product was a light brown suspension obtained in a 50 % yield (0.301 g). The yield

was determined by placing a 200 dl aliquot of spheres on a tared microscope slide,

drying, and weighing the residue. The number average diameter was 82 nm and the

volume average diameter was 92 nm as determined by transmission electron

microscopy in Section 2.2.1 (Figures 1-4).

After the last centrifugation, the spheres were resuspended in 10 % dextran

(Sigma, mw.500,000). The spheres were then lyophilized and stored at room

temperature in a dessicator.



2.1.2 Polvanionic Albumin Submicrospheres / 20% Polvalutamic Acid (PGA\

Human serum albumin (0.48 g) and polyglutamic acid (0.12 g) (Sigma) were

dissolved in 2.0 ml of distilled water. This solution was added to 50 ml of 2 wt %












CAB in dichloroethane and dispersed by ultrasonification at 375 W for 10 min. After

ultrasonification, the dispersate was added to a rapidly stirred solution containing

110 ml of 2 wt % CAB in dichloroethane and 40 ml of the toluene-glutaraldehyde

solution described in Section 2.1.1. The solution was stirred rapidly for 2 hrs, then

transferred to a rotary mixer and mixed overnight at room temperature. The spheres

were then washed with acetone as in Section 2.1.1, and stored at room temperature in

acetone.

The product was a light brown suspension obtained in a yield of 46 % (275 mg).

The spheres had an average diameter of 83 nm (Figure 5) as determined by TEM in

Section 2.2.1.



2.1.3 Magnetically Responsive Submicrospheres / 16% Magnetite

Human serum albumin (0.5 g) was dissolved in 1.6 ml of distilled water.

Magnetite (y-Fe304)(0.48 g of a 21 wt % suspension in 0.1 wt % bovine serum

albumin) (Bioclinical Group, Inc.) was added and mixed to homogeneity. The

suspension was dispersed in 50 ml of 2 wt % CAB in dichloroethane by

ultrasonification at 375 W for 10 min. After ultrasonification, the dispersate was

added to a rapidly stirred solution containing 110 ml of 2 wt % CAB in dichloroethane

and 40 ml of the toluene- glutaraldehyde solution described in Section 2.1.1. The

dispersate was cross-linked at room temperature, washed free of polymer as in

Section 2.1.1, and allowed to settle overnight. The precipitate was discarded and the

supernatant stored at room temperature in acetone.












The product was a light brown suspension in a 40 % yield (238 mg) The spheres

had an average diameter of 82 nm (Figure 6) as determined by TEM in Section 2.2.1

and contained 16 wt % magnetite, as confirmed by analysis of the iron content by

inductively coupled plasma.

2.1.4 Radiopaque Submicrospheres / 2% Barium Sulfate

Human serum albumin (0.588 g) was dissolved in 1.9 ml of distilled water.

Barium sulfate (0.012 g) (Sigma) was added and mixed to homogeneity. The

suspension was dispersed in 50 ml of 2 wt % CAB in dichloroethane by

ultrasonification at 375 W for 10 min. After ultrasonification, the dispersate was

added to a rapidly stirred solution containing 110 ml of 2 wt % CAB in dichloroethane

and 40 ml of the toluene- glutaraldehyde solution described in Section 2.1.1. The

dispersate was cross-linked at room temperature, washed free of polymer, and sized,

as in Section 2.1.3, and stored at room temperature in acetone.

The final product was a light brown suspension obtained in a 32 % yield

(231 mg), containing 2 wt % barium sulfate, with an average diameter of 90 nm

(Figure 7) as determined by TEM in Section 2.2.1.



2.1.5 Fluorescent. Maximum Cross-Link Density Submicrospheres/ 4% Fluorescein
Isothiocyanate-Albumin

Bovine serum albumin (0.475 gm) and fluorescein isothiocyanate bovine albumin

(0.025 gm) (Sigma) were dissolved in 1.667 ml of distilled water. This solution was

dispersed into 30 ml of 2 wt % CAB in dichloroethane by ultrasonification at 375 W

for 10 min. After ultrasonification, 7.5 ml of the toluene-glutaraldehyde solution












described in Section 2.1.1 was added and the suspension mixed rapidly at room

temperature for 20 hrs. The dispersate was washed and sized as in Section 2.1.1 and

stored at room temperature in acetone.

The final product was a light yellow suspension obtained in a 51 % yield

(0.255 gm) with an average diameter of 89 nm (Figure 8) as determined by TEM in

Section 2.2.1 and with maximum cross-link density.



2.1.6 Microspheres (>1 pm)

Human serum albumin (1.0 gm) was dissolved in 3.333 ml of distilled water and

dispersed in 30 ml of 2 wt % CAB in dichloroethane by vortex mixing in a 50 ml

centrifuge tube at maximum power for 10 min (Vortex-Genie, Scientific Industries,

Inc.). After dispersing, 15 ml of the toluene-glutaraldehyde solution described in

Section 2.1.1 was added and the dispersate mixed overnight on a rotary mixer. The

polymer was removed by suspending the spheres in acetone and centrifuging at 1300g

for 10 min. After washing four times in this manner, the spheres were resuspended

in acetone and stored at room temperature.

The product was a light brown suspension obtained in a yield of 92 wt % (0.92 gm)

with an average diameter of 14 pm as determined by light microscopy in Section

2.2.1.



2.1.7 Polyanionic Microspheres / 20 % PGA

Human serum albumin (0.8 gm) and PGA (0.2 gm) were dissolved in 3.333 ml of

distilled water. The solution was dispersed in 30 ml of 2 wt % CAB in dichloroethane












by vortex mixing in a 50 ml centrifuge tube at maximum power for 10 min. After

dispersing, 15 ml of the toluene-glutaraldehyde solution described in in Section 2.1.1

was added and the dispersate mixed overnight on a rotary mixer. The spheres were

washed as in Section 2.1.6 and stored at room temperature in acetone.

The product was a light brown suspension obtained in a 97 % yield (0.97 gm). The

spheres had an average diameter of 12 g.m as determined by light microscopy in

Section 2.2.1.



2.1.8 Fluorescent. Minimum Cross-Link Density Microspheres / 4 % Fluorescein
Isothiocyanate-Albumin

Bovine serum albumin (0.96 gm) and fluorescein isothiocyanate- albumin

(0.04 gm) were dissolved in 3.333 ml of distilled water. The suspension was

dispersed in 30 ml of 2 wt % CAB in dichloroethane by vortex mixing in a 50 ml

centrifuge tube at maximum power for 10 min. After dispersing, 2 ml of the

toluene-glutaraldehyde solution described in Section 2.1.1 (0.28 mmoles

glutaraldehyde) was added and the dispersate mixed overnight on a rotary mixer. The

polymer was removed by suspending the spheres in acetone and centrifugung at 1300g

for 10 min. After washing the spheres four times in this manner, the spheres were

suspended in 30 ml of distilled water and centrifuged for 15 sec at 260g. The pellet

was discarded and the supernatant was centrifuged at 520g for 30 sec. This pellet

was resuspended in acetone, had an average diameter of 20 pm (10- 30 p.m

diameter), and stored at room temperature. The supernatant was centrifuged at 1000g












for 30 sec. The pellet was discarded and the supernatant was centrifuged at 1300g for

10 min. The pellet was resuspended in acetone, had an average diameter of 1 p.m

(0.5-3.0 pm diameter), and stored at room temperature. Sizing was by light

microscopy as in Section 2.2.1.

The products were light yellow suspensions obtained in yields of 41 % (0.41 gm)

for the 20 pim spheres and 17 % (0.17 gm ) for the 1 p.m spheres, and with the

minimum cross-link density needed for sphere formation.




2.1.9 Fluorescent. Intermediate Cross-Link Density Microspheres /4 % Fluorescein
Isothiocyanate-Albumin

Bovine serum albumin (0.96 gm) and fluorescein isothiocyanate- albumin were

dissolved in 3.333 ml of distilled water. The suspension was dispersed in 30 ml of

2 wt % CAB in dichloroethane by vortex mixing in a 50 ml centrifuge tube at

maximum power for 10 min. After dispersing, 4 ml of the toluene-glutaraldehyde

solution described in Section 2.1.1 (0.56 mmoles glutaraldehyde) was added and the

dispersate mixed overnight on a rotary mixer. The spheres were washed and sized as

in Section 2.1.8 and stored at room temperature in acetone.

The products were light yellow suspensions obtained in a 47 % yield (0.47 gm)

for the 20 pm spheres and a 21 % yield (0.21 gm) for the 1 pm spheres, and with

twice the minimum cross-link density needed for sphere formation.












2.1.10 Fluorescent. Maximum Cross-Link Density Microspheres / 4 % Fluorescein
Isothiocyanate-Albumin

Bovine serum albumin (0.96 gm) and fluorescein isothiocyanate-albumin

(0.04 gm) were dissolved in 3.333 ml of distilled water. The solution was dispersed

in 30 ml of 2 wt % CAB in dichloroethane by vortex mixing in a 50 ml centrifuge

tube at maximum power for 10 min. After dispersing, 15 ml of the toluene-

glutaraldehyde solution described in Section 2.1.1 (2.1 mmoles glutaraldehyde) was

added and the dispersate mixed over night on a rotary mixer. The spheres were washed

and sized as in Section 2.1.8 and stored at room temperature in acetone.

The products were light yellow suspensions obtained in a 40 % yield (0.40 gm)

for the 20 pm spheres and a 22 % yield (0.22 gm ) for the 1 p.m spheres and with all

possible cross-link sites saturated.



2.1.11 Fluorescent. Supersaturation Cross-Link Density Microspheres / 4 %
Fluorescein Isothiocyanate-Albumin

Bovine serum albumin (0.96 gm) and fluorescein isothiocyanate-albumin

(0.04 gm) were dissolved in 3.333 ml of distilled water. The solution was dispersed

in 30 ml of 2 wt % CAB in dichloroethane by vortex mixing in a 50 ml centrifuge tube

at maximum power for 10 min. After dispersing, 20 ml of the toluene-glutaraldehyde

solution described in Section 2.1.1 (2.8 mmoles glutaraldehyde) was added and the

dispersate mixed overnight on a rotary mixer. The spheres were washed and sized as

in Section 2.1.8 and stored in acetone at room temperature.

The products were light yellow suspensions obtained in a 40 % yield ( 0.40 gm)

for the 20 pm spheres and a 29 % yield ( 0.29 gm) for the 1 pmrn spheres and with












1.3 times more cross-linking agent added than the amount that was designated a

saturating concentration.


2.1.12 Polvanionic. Magnetically Responsive. Albumin Microspheres /
22% Polvolutamic Acid. 17 % Magnetite

Human serum albumin (0.19 gm) and polyglutamic acid (0.06 gm) were dissolved

in 0.8 ml of distilled water. Magnetite (0.24 gm of a 21 wt % suspension) was added

and the suspension mixed to homogeneity. The suspension was dispersed in 20 ml of

2 wt % CAB in dichloroethane by vortex mixing in a 50 ml centrifuge tube at

maximum power for 10 min. After dispersing, 5 ml of the toluene glutaraldehyde

solution described in Section 2.1.1 was added and the dispersate mixed overnight on a

rotary mixer. The spheres were washed as in Section 2.1.6 and resuspended in acetone

and stored at room temperature.

The product was a dark brown suspension obtained in a yield of 98 % (0.268 gm).

The spheres had an average diamete of 14 l.m (Figure 9) as determined by light

microscopy in Section 2.2.1.





2.2 Characterization by Light and Electron Microscopy



2.2.1 Size Deteremination of Spheres

The microspheres (>1 p.m) produced in Sections 2.1.6-2.1.12 were sized while in

water. The microspheres were suspended in distilled water, placed on a microscope












slide, and covered with a glass cover slip. The spheres were examined on a Nikon light

microscope and size was measured with an ocular micrometer. At least 100 spheres

were examined.

While not used for sizing, microspheres were also prepared for scanning electron

microscopy by suspending the spheres in acetone, placing a drop on an aluminum SEM

stub, and drying at room temperature under vacuum. A 200 A coating of

gold-palladium was applied with a sputter coaster (Hummer V). Samples were

examined with a JOEL JSM-35CF scanning electron microscope at 20 kV.

Submicrospheres (<1plm) produced in sections 2.1.1 2.1.5 were suspended in

acetone, dried on carbon coated copper grids (200 mesh), and examined on a JOEL

JEM-200CX transmission electron microscope at 80 kV. At least 100 spheres were

photographed and measured.

The Z-average diameter of wet submicrospheres from Section 2.1.1 2.1.5 was

measured by analysis with a Photon Correlation Spectrometer (Brookhaven) at

514.5 nm. The spheres were suspended in distilled water and analyzed at 250C.



2.2.2 Effect of Size and Cross-Link Density on Hydration Swelling

Spheres synthesized in Section 2.1.8, with minimum cross-link density, were

suspended in acetone and a few drops of the suspension applied to a glass microscope

slide. The slide was air dried for 5 minutes and covered with a glass cover slip. Using

a Nikon light microscope, a cluster of five to ten spheres was selected and the

diameters of the spheres measured using an ocular micrometer. A drop of 0.9% saline












was placed on the edge of the cover slip and allowed to diffuse across the slide, under

the cover slip, while the cluster of spheres was maintained in view. When the saline

reached the spheres, the spheres were observed to swell. The spheres were allowed to

rehydrate for 5 minutes and were sized again with the ocular micrometer. This

procedure was repeated until at least 50 spheres had been examined (Figure 10-11).

The percent increase in diameter due to hydration swelling was calculated and plotted

versus the dry diameters of the spheres (Figure 12).

The above procedure was repeated with intermediate cross-link density spheres

from Section 2.1.9 and with maximum cross-link density spheres from Section

2.1.10. The above procedure measured spheres of chosen diameters.





2.3 Glvcine Quenching and 14-C Labeling of Reactive Aldehyde Groups



Submicron spheres (130 mg) synthesized in Sections 2.1.1 2.1.5 and stored in

acetone were centrifuged at 50,000g for 30 min. The pellet was resuspended in

20 ml of distilled water, centrifuged at 50,000g for 30 min and resuspended again in

4 ml of water. The suspension was added to 10 ml of 2 M glycine (Sigma) in a 50 ml

test tube and mixed on a rotary mixer for 18 h at room temperature.

The spheres were washed four times by centrifuging at 50,000g for 30 min and

resuspending the pellet in 20 ml of water. After the last centrifugation, the spheres

were resuspended and stored at room temperature in acetone. In this way, reactive

surface aldehyde groups were capped with glycine (Figure 13).












Alternatively, the 4 ml sphere solution was added to 0.4 ml of 14-C(U)-glycine

(100 .Ci/0.86 Ipmole/ml water) (New England Nuclear) in a 5 ml vial and mixed on

a rotary mixer for 18 hrs at room temperature.

The spheres were repeatedly washed with distilled water by centrifuging at

50,000g for 30 min and resuspending the pellet in 20 ml of distilled water, until the

total free 14-C glycine in the supernatant was less than 5 % of the total remaining

14-C label, as determined by oxidation and liquid scintillation (see Section 2.10.2).

After the last centrifugation, the 14-C labelled spheres were resuspended in 4 ml of

distilled water and stored at 4 oC In this way, reactive surface aldehydes were capped

with glycine and the spheres were labeled with 14-C.





2.4 Dry Storage of Submicrosoheres



Submicrospheres (20 mg) synthesized in Section 2.1.1 were resuspended in 5 ml

of 0.1%, 1.0% and 10.0% solutions of dextran (100,000 MW, Sigma) in distilled

water. Each suspension was divided in half; one half remained at room temperature

while the other half was frozen at -200C and lyophilized to dryness. The dried

suspension was reconstituted with 2.5 ml of distilled water, gently mixed by hand, and

visually compared to the equivalent suspension that had not been dried. The solutions

were scored on the following basis: completely reconstituted, ** mostly

reconstituted, *** mostly unreconstituted, and **** completely unreconstituted.












This procedure was repeated with 0.1%, 1.0%, and 10.0% aqueous solutions of

polyethylene glycol (6000 MW, Sigma), bovine serum albumin (Sigma),

polyoxyethylene (20) sorbitan mono-oleate (Fisher Scientific), and sodium chloride

(Table 1).

2.5 In Vitro Degradation of Albumin Microspheres



2.5.1 Effect of Size on the Rate of Enzymatic Degradation of Albumin Microspheres

Cysteine (80 mg)(Sigma) and 50 mg of submicron spheres (29 mg/ml)

synthesized in Section 2.1.5, with maximum cross-link density and containing

4 wt % fluorescein isothiocyanate-albumin, were added to 10 ml of the reaction

buffer in a 50 ml test tube and distilled water added to bring the final volume to 12.5

ml. The reaction buffer consisted of sodium tetraa) ethylenediamine tetraacetate

(0.719 gm) (Fisher Scientific) and tris(hydroxymethyl)amino- methane

(0.605 mg) (Sigma) dissolved in 100 ml of distilled water. This buffer was used for

all degradation reactions. The pH was adjusted to 7.5 with either 0.1 N HCI or 0.1 N

NaOH. Papain (300 units/ml) (Sigma) was diluted 1:100 with reaction buffer.

While the mixture was stirred with a magnetic stirrer, 1.0 ml of diluted papain

(3 units/ml) was added. After the enzyme was added, the solution was stirred for

20 hrs; a 0.65 ml aliquot was removed every 15 min for 2 hrs and once every 6 hrs

after that. The samples were centrifuged at 30,000g for 5 min and the supernatants

(0.55 ml) were diluted with 1.0 ml of distilled water and assayed spectro-

photometrically at 495 nm. Absorbance at 495 nm was plotted versus time to

compare rates of degradation (Figure 14).












The above procedure was repeated with 50 mg of 20 gm spheres(25 mg/ml)

synthesized in Section 2.1.10 with maximum cross-link density and containing

4 wt % fluorescein-labeled albumin.



2.5.2 Effect of Cross-Link Density on the Rate of Degradation of Albumin Spheres

Cysteine (80 mg) (Sigma) and 50 mg of 20 um spheres (31 mg/ml), synthesized

in Section 2.1.8, with minimum cross-link density and containing 4 wt % fluorescein

isothiocyanate-albumin, were added to 10 ml of the reaction buffer (Section 2.5.1) in

a 50 ml test tube and the volume brought to 12.5 ml with distilled water. The pH was

adjusted to 7.5 with either 0.1 N HCI or 0.1 N NaOH. While the mixture was stirred

with a magnetic stirrer, 0.4 ml of diluted papain (3 units/ml) (Sigma) was added.

After the enzyme was added, the solution was stirred for 20 hrs; a 0.65 ml aliquot

was removed every 10 minutes for 2 hrs and once every 6 hrs after that. The aliquots

were centrifuged at 2,000g for 1 min and the supernatants (0.55 ml) were diluted

with 1.0 ml of distilled water and assayed spectrophotometrically at 495 nm. The

absorbance at 495 nm was plotted versus time to compare rates of degradation

(Figure 15).

The above procedure was repeated with 50 mg of 20 pmn intermediate cross-link

density spheres (22 mg/ml) synthesized in Section 2.1.9, with 50 mg of 20 pm

maximum cross-link density spheres (25 mg/ml) synthesized in Section 2.1.10, and

with 50 mg of spheres synthesized in Section 2.1.11 using a supersaturating

concentration of glutaraldehyde, all containing 4 wt % fluorescein-labeled albumin.












The final degradation products from the minimum and maximum cross-link density

spheres were saved for molecular weight characterization. The samples (10 gil) were

analyzed by high pressure liquid chromatography (Perkin-Elmer, Model 3B) with

Shodex GPC A-800P and GPC A-80M columns in series. The solvent was distilled

water at a flow rate of 0.1 ml/min and absorbance was measured at 220 nm. The

retention times of the degradation products (Figure 16) were compared to

polyethylene glycol standards (Figurel7).



2.5.3 Effect of Enzyme Concentration on the Rate of Degradation of Albumin Spheres

Cysteine (80 mg) (Sigma) and 50 mg of 20 gIm spheres (25 mg/ml), synthesized

in Section 2.1.10, with maximum cross-link density and containing 4 wt %

fluorescein labeled-albumin, were added to 10 ml of the reaction buffer (Section

2.5.1) in a 50 ml test tube and the volume brought to 12.5 ml with distilled

water. The pH was adjusted to 7.5 with either 0.1 N HCI or 0.1 N NaOH. While the

mixture was stirred with a magnetic stirrer, 0.2 ml of diluted papain (3 units/ml)

(Sigma) was added. After the enzyme was added, the solution was stirred for 20 hrs; a

0.65 ml aliquot was removed every 10 minutes for 2 hrs and once every 6 hrs after

that. The aliquots were centrifuged at 2,000g for 1 min and the supernatants

(0.55 ml) were diluted with 1.0 ml of distilled water and assayed spectro-

photometrically at 495 nm. The absorbance at 495 nm was plotted versus time to

compare rates of degradation (Figure 18).

The above procedure was repeated with 0.4 ml and 0.6 ml of diluted papain

(3 units/ml).












2.6 In Vivo Degradation of Albumin Microspheres



2.6.1 Intramuscular Degradation of Maximum Cross-Link Density Microspheres

Maximum cross-link density 20 ipm microspheres synthesized in Section 2.1.10

(50 mg) were resuspended in 1.0 ml of distilled water. Twenty-five female white

mice (CFW strain, 25-30 gm) were anesthetized with Halothane and were injected

intramuscularly in both hind legs with 0.1 ml of the 20 p.m sphere solution. Three

mice from each group were sacrificed by cervical dislocation 1, 3, 10, 19, 26, 36,

44, and 50 days postinjection.

The injected leg muscle was excised, fixed in 10 % formalin for 48 hrs,

dehydrated embedded in paraffin, and 1 p.m sections were obtained. The sections were

mounted on glass microscope slides, stained with haematoxylin and eosin, and

examined by light microscopy (Figure 19-21).

The above procedure was repeated with 50 mg of the 1 pm maximum cross-link

spheres synthesized in Section 2.1.10.



2.6.2 Intramuscular Degradation of Minimum Cross-Link Density Microspheres

Minimum cross-linked 20 pm microspheres synthesized in Section 2.1.8

(50 mg) were resuspended in 1.0 ml of distilled water. Twenty-two female white

mice (CFW strain, 25-30 gm) were anesthetized with Halothane and were injected

intramuscularly in both hind legs with 0.1 ml of the 20 p.m sphere solution. Three












mice from each group was sacrificed by cervical dislocation 1,3, 5, 9, 12,16, and

20 days postinjection. The injected leg muscle was excised and processed for

microscopy as in Section 2.6.1.

The above procedure was repeated with 50 mg of the 1 p.m minimum cross-link

spheres synthesized in Section 2.1.8.



2.6.3 Intratumor Degradation of Minimum Cross-Link Density Microspheres

Six female white mice (CFW, 25 30 gm) with solid subcutaneous tumors (Lewis

lung carcinoma) approximately 1 cm in diameter on their flanks were donated by Dr.

Sitren (30). Minimum cross-link density microspheres synthesized in Section 2.1.8

(50 mg of 1 p.m spheres) were resuspended in 1.0 ml distilled water to give a

50 mg/ml solution. The mice were anesthetized with ether and were injected with

0.3 ml of the spheres suspension using a 30 g needle. The injection was actually three

separate injections of 0.1 ml each, each injection being 1200 from the others and

aimed toward the center. The suspension was slowly injected into the tumor; each

injection taking about 30 seconds.

One animal was sacrificed at the time of injection and 1, 3, 9, 12, and 16 days

after injection. The tumors were excised and processed for microscopy as in Section

2.5.1 (Figure 22-25).












2.7 Postsvynthesis Drug Loading by Submicrospheres



2.7.1 Primaquine Loading

Albumin submicrospheres (maximum cross-linked) synthesized in Section 2.1.1

(70 mg) were resuspended in 35 ml of primaquine solution (2 mg/ml) (Sigma)

(Figure 26) and evenly distributed into seven test tubes. The pH of the tubes was

adjusted to 4 through 10 with 1 N NaOH. The test tubes were capped and mixed on a

rotary mixer at 4C for 18 hrs.

The test tubes were centrifuged at 1300g for 5 min and the supernatants decanted

and saved. The spheres were washed once with distilled water and the supematants

decanted, adjusted to pH 5 with 10 N HCI, and saved. An aliquot from each of the

unabsorbed drug and wash supernatants was diluted 200-fold with distilled water and

assayed for drug concentration spectrophotometrically at 353 nm. The absorbance

values were plotted on a standard concentration curve and the percent drug absorbed

was calculated. A typical standard concentration curve is shown in Figure 27. The

maximum drug absorption was at pH 9, and this yielded spheres that contained

36 wt % primaquine (Figure 28, Table 2).

The above procedure was repeated with 70 mg of 20 % PGA submicrospheres

synthesized in Section 2.1.2. At the optimum pH of 9, the spheres bound 41 wt %

primaquine. Submicrospheres synthesized in Section 2.1.1 and Section 2.1.2 (20 %

PGA) and glycine quenched in Section 2.3 were also loaded with drug. At the optimum

pH of 9, the spheres contained 32 wt % primaquine for the non-PGA spheres and 35

wt % for the 20 % PGA spheres.












2.7.2 Adriamvcin Loading

Submicron albumin spheres synthesized in Section 2.1.1 (70 mg) were

resuspended in 35 ml of adriamycin solution (2 mg/ml) (Farmitalia) (Figure 26)

and evenly divided into seven test tubes. The pH of the tubes was adjusted to 4 through

10 with 1 N NaOH as in Section 2.7.1, and the tubes mixed in the dark on a rotary

mixer at 40C for 18 hrs.

The spheres were centrifuged and washed and the supematants diluted 200-fold

with distilled water as in Section 2.7.1. The concentration of adriamycin in the

supernatants was measured spectrophotometrically at 480 nm and the percent drug

absorbed was calculated (Figure 27). Maximum drug absorption was at pH 8 and

yielded spheres that contained 33 wt % adriamycin (Figure 30).

The above procedure was repeated with 70 mg of 20 % PGA submicrospheres

synthesized in Section 2.1.2. At the optimum pH of 8, the spheres contained 48 wt %

adriamycin. Submicrospheres synthesized in Section 2.1.1 and Section 2.1.2

(20 % PGA) and glycine quenched in Section 2.3 were also loaded with drug. At the

optimum pH of 8, the spheres bound 32 wt % adriamycin for the non-PGA spheres and

47 wt % adriamycin for the 20 % PGA spheres.



2.7.3 cis-Platinum(II)diaminedichloride (cis-Platinum) Loading

Submicron albumin spheres synthesized in Section 2.1.2 (70 mg) containing

20 % PGA were resuspended in 35 ml of cis-platinum solution (2 mg/ml) (Siqma)

(Figure 26) and evenly divided into seven test tubes. The pH of the tubes was adjusted












to 4 through 10 with 1 N NaOH as in Section 2.7.1, and the tubes mixed in the dark on

a rotary mixer at 40C for 18 hrs.

The spheres were centrifuged and washed and the supernatants diluted 200-fold

with distilled water as in Section 2.7.1. The concentration of cis-platinum in the

supernatants was measured spectrophotometrically at 198 nm and the percent drug

absorbed was calculated (Figure 29). Maximum drug absorption was at pH 6 and

yielded spheres that contained 1.5 wt % cis-platinum (Figure 31).

The above procedure was repeated with 70 mg of submicrospheres synthesized in

Section 2.1.1. and with submicrospheres synthesized in Section 2.1.1 and Section

2.1.2 (20 % PGA) and glycine quenched in Section 2.3. There was no measurable

drug uptake at any pH.



2.7.4 Mitomvcin-C Loading

Submicron albumin spheres synthesized in Section 2.1.1 (70 mg) were

resuspended in 35 ml of mitomycin solution (2 mg/ml) (Bristol Laboratories)

(Figure 26) and evenly divided into seven test tubes. The pH of the tubes was adjusted

to 4 through 10 with 1 N NaOH as in Section 2.7.1, and the tubes mixed in the dark on

a rotary mixer at 40C for 18 hrs.

The spheres were centrifuged and washed and the supernatants diluted 200-fold

with distilled water as in Section 2.7.1. The concentration of mitomycin in the

supernatants was measured spectrophotometrically at 281 nm and the percent drug

absorbed was calculated. There was no measurable drug loading at any pH.












The above procedure was repeated with 70 mg of 20 % PGA submicrospheres

synthesized in Section 2.1.2. and with submicrospheres synthesized in Section 2.1.1

and Section 2.1.2 (20 % PGA) and glycine quenched in Section 2.3. There was no

measurable drug uptake at any pH.



2.7.5 5-Fluorouracil Loading

Submicron albumin spheres synthesized in Section 2.1.2 (70 mg) containing

20 % PGA were resuspended in 35 ml of 5-fluorouracil solution (2 mg/ml) (Sigma)

(Figure 26) and evenly divided into seven test tubes. The pH of the tubes was

adjusted to 4 through 10 with 1 N NaOH as in Section 2.7.1, and the tubes mixed in the

dark on a rotary mixer at 4C for 18 hrs.

The spheres were centrifuged and washed and the supematants diluted 200-fold

with distilled water as in Section 2.7.1. The concentration of 5-fluorouracil in the

supernatants was measured spectrophotometrically at 268 nm and the percent drug

absorbed was calculated. There was no measurable drug loading at any pH.



2.7.6 Hydroxyurea Loading

Submicron albumin spheres synthesized in Section 2.1.2 (70 mg) containing

20 % PGA were resuspended in 35 ml of hydroxyurea solution (2 mg/ml) (Sigma)

(Figure 26) and evenly divided into seven test tubes. The pH of the tubes was adjusted

to 4 through 10 with 1 N NaOH as in Section 2.7.1, and the tubes mixed in the dark on

a rotary mixer at 4C for 18 hrs.












The spheres were centrifuged and washed and the supematants diluted 200-fold

with distilled water as in Section 2.7.1. The concentration of hydroxyurea in the

supernatants was measured spectrophotometrically at 191 nm and the percent drug

absorbed was calculated. There was no measurable drug uptake at any pH.



2.7.7 Methotrexate Loading

Submicron albumin spheres synthesized in Section 2.1.2 (70 mg) containing

20 % PGA were resuspended in 35 ml of methotrexate solution (2 mg/ml) (Sigma)

(Figure 26) and evenly divided into seven test tubes. The pH of the tubes was adjusted

to 4 through 10 with 1 N NaOH as in Section 2.7.1, and the tubes mixed in the dark on

a rotary mixer at 40C for 18 hrs.

The spheres were centrifuged and washed and the supernatants diluted 200-fold

with distilled water as in Section 2.7.1. The concentration of methotrexate in the

supernatants was measured spectrophotometrically at 299 nm and the percent drug

absorbed was calculated. There was no measurable drug uptake at any pH.





2.8 Presynthesis Loading of Fluorescein-Labeled Carboxymethyl Cellulose



2.8.1 Fluorescein Labeling of Carboxymethyl Cellulose

Carboxymethyl cellulose (500 gm)(55,000 MW, Sigma) was dissolved in 25 ml

of distilled water. Nitrogen was bubbled through the solution for 15 min and 2 ml of












pyridine were added. Fluorescein isothiocyanate (0.2 gm)(Sigma) was dissolved in

the solution and mixed for 48 hrs at room temperature in the dark.

The pH of the solution was adjusted to 7 with 0.1 N HCI and it was dialyzed with

membrane dialysis tubing (Spectrapor #1, 6,000-8,000 MW cutoff) against

4 liters of distilled water, with frequent changes of the water, for 3 days at 40C. The

fluorescein-carboxymethyl cellulose (Figure 32) was then dried under vacuum at

800C and stored in a desiccator at room temperature.


2.8.2 Presynthesis Loading of Microspheres with Fluorescein-Labeled
Carboxymethyl Cellulose

Bovine serum albumin (0.95 gm) and fluorescein-carboxymethyl cellulose

(0.05 gm) were dissolved in 3.333 ml of distilled water. The suspension was

dispersed in 30 ml of 2 wt % CAB in dichloroethane by vortex mixing in a 50 ml

centrifuge tube at maximum power for 10 min. After dispersing, 15 ml of the

toluene-glutaraldehyde solution described in Section 2.1.1 (2.1 mmoles

glutaraldehyde) was added and the dispersate mixed overnight on a rotary mixer. The

spheres were washed as in Section 2.1.1 and stored at room temperature in acetone.

The product was a bright orange suspension obtained in a 97 % yield (0.97 gm).

The spheres were in the 10-30 p.m size range and contained 5 wt % fluorescein-

carboxymethyl cellulose and had maximum cross-link density.

The above procedure was repeated using 2 ml of the toluene-glutaraldehyde

solution (0.28 mmoles glutaraldehyde) as the cross-linking agent. The product was a

bright orange suspension obtained in a 91 wt % yield of spheres in the 10 30 p.m












size range, containing 5 wt % fluorescein-carboxymethyl cellulose and with

minimum cross-link density.





2.9 Drug Release from Submicrospheres



2.9.1 Diffusional Release of Primaquine from Submicrospheres

Submicrospheres synthesized in Section 2.1.1 (80 mg) were suspended in 40 ml

of primaquine (2 mg/ml) and the pH adjusted to 9. The suspension was mixed

overnight at 40C. The spheres were centrifuged out of suspension and washed twice

with distilled water. Spectrophotometric assay of the supernatant at 353 nm

confirmed the drug loading as 36 wt %. Loaded submicrospheres (20 mg) were

transferred to 500 ml of isotonic saline (Fisher) (pH 7.4) and gently stirred with a

magnetic stirrer at room temperature for 16 hrs. A 1.0 ml aliquot was removed

every 20 min for 6 hrs, and once every 6 hrs after that. The sample was centrifuged

at 50,000g for 10 min and the supernatant decanted and assayed for drug

concentration as in Section 2.7.1. The concentration of drug (mg primaquine/mg

spheres) was plotted against time to compare release rates (Figure 33).

The above procedure was repeated with submicrospheres synthesized in Section

2.1.1 and quenched as in Section 2.3, with submicrospheres synthesized in Section

2.1.2 containing 20 % PGA, and with submicrospheres synthesized in Section 2.1.2

containing 20 % PGA and quenched as in Section 2.3.












2.9.2 Combined Diffusional and Enzymatic Dearadative Release of Primaquine from
Submicrospheres

Submicrospheres synthesized in Section 2.1.2 with maximum cross-link density and

containing 20 % PGA were loaded with primaquine as in Section 2.9.1. Cysteine

(80 mg) and 5 mg of loaded spheres (10 mg/ml) were added to 20 ml of degradation

buffer (Section 2.5.1) and the pH adjusted to 7.4. While the mixture was gently stirred

with a magnetic stirrer, 1.0 ml of diluted papain (3 units/ml) was added. The solution

was stirred for 20 hrs and an aliquot was removed every 30 min for 6 hrs, and once

every 6 hrs after that. The samples were centrifuged at 50,000g for 10 min and the

supernatant decanted and assayed for drug concentration as in Section 2.7.1. The

concentration of drug (mg primaquine/mg spheres) was plotted against time to compare

release rates (Figure 34).

The above procedure was repeated without enzyme and with 2.0 ml of the 3 unit/ml

papain dilution.


2.9.3 Combined Diffusional and Enzymatic Degradative Release of Fluorescein-Labeled
Carboxymethyl Cellulose from Minimum Cross-Link Microspheres

Cysteine (80 mg) (Sigma) and 50 mg of 20 p.m spheres (51 mg/ml), synthesized in

Section 2.8.2, with minimum cross-link density and containing 5 wt % fluorescein-

carboxymethyl cellulose, were added to 10 ml of the reaction buffer (Section 2.5.1) in a

50 ml test tube and the volume brought to 12.5 ml with distilled water. The pH was

adjusted to 7.5 with either 0.1 N HCI or 0.1 N NaOH. While the mixture was stirred

with a magnetic stirrer, 0.02 ml of diluted papain (3 units/ml) (Sigma) was added.

After the enzyme was added, the solution was stirred for 20 hrs and a 0.65 ml aliquot was












removed every 10 min for 2 hrs and once every 6 hrs after that. The aliquots were

centrifuged at 2,000g for 3 min. The supernatants (0.55 ml) were diluted with 1.0 ml of

distilled water and assayed spectrophotometrically at 495 nm (Figure 29). The

concentration of fluorescein carboxymethyl cellulose was plotted against time to

compare rates of release (Figure 35).

The above procedure was repeated without enzyme and with 0.2 ml and 0.4 ml of the 3

unit/ml papain dilution.


2.9.4 Combined Diffusional and Enzymatic Degradative Release of Fluorescein-Labeled
Carboxvmethyl Cellulose from Maximum Cross-Link Microspheres



Cysteine (80 mg) (Sigma) and 50 mg of 20 pm spheres (53 mg/ml), synthesized in

Section 2.8.2, with maximum cross-link density and containing 5 wt % fluorescein-

carboxymethyl cellulose, were added to 10 ml of the reaction buffer (Section 2.5.1) in a

50 ml test tube and the volume brought to 12.5 ml with distilled water. The pH was

adjusted to 7.5 with either 0.1 N HCI or 0.1 N NaOH. While the mixture was stirred

with a magnetic stirrer, 0.4 ml of diluted papain (3 units/ml) (Sigma) was added. After

the enzyme was added, the solution was stirred for 20 hrs and a 0.65 ml aliquot was

removed every 10 min for 2 hrs and once every 6 hrs after that. The aliquots were

centrifuged at 2,000g for 3 min. The supernatants (0.55 ml) were diluted with 1.0 ml of

distilled water and assayed spectrophotometrically at 495 nm (Figure 29). The

concentration of fluorescein carboxymethyl cellulose was plotted against time to

compare release rates (Figure 36).












The above procedure was repeated without enzyme and with 1.2 ml and 2.0 ml of the 3

unit/ml papain dilution.





2.10 In Vitro Uptake of Albumin Submicrospheres by Rabbit Blood Cells



2.10.1 Silver Labeling of Albumin Submicrospheres

Methenamine (3 gm) (Polysciences), silver nitrate (0.25 gm) (Fisher Scientific),

and sodium tetraborate (0.6 gm) (Fisher Scientific) were dissolved in 11 ml of distilled

water. Submicrospheres (1 gm) synthesized in Section 2.1.1 were added and the

suspension was gently mixed in the dark for 20 hrs at 600C. After 20 hrs, the color of

the suspension changed from light brown to black and the spheres were washed twice with

20 ml of 5 % sodium thiosulfate (Fisher Scientific) and three times with isotonic saline.

The spheres were stored in saline at 40C until used.

The above procedure was repeated with 1 gm of spheres consisting 0.05 gm of 10 gIm

microspheres from Section 2.1.6 and 0.95 gm of submicrospheres from Section 2.1.1.



2.10.2 In Vitro Uptake of Silver-Labeled Submicrosphere by Rabbit Blood Cells

A female New Zealand white rabbit (2.7 kg) was anesthetized by intramuscular

injection of Ketaset (0.8 ml) and Rompun (0.8 ml) and 60 ml of blood was withdrawn by

cardiac puncture. A 15 ml sample was placed in 30 ml of 5 % glutaraldehyde (EM grade,

Polysciences) in isotonic saline and the remainder of the blood (45 ml) was placed in a












50 ml screw cap polypropylene tube containing 20,000 units of heparin (Sigma)

dissolved in 1 ml of isotonic saline and 500 mg of submicrospheres labeled with silver in

Section 2.10.1. The cells in glutaraldehyde were gently mixed at room temperature for

1 hr and then stored at 40C.

The cells were incubated at 39C with gentle mixing in an Isotemp incubator (Fisher

Scientific) and 15 ml aliquots were removed after 1 hr, 6 hrs, and 24 hrs of incubation.

As each sample was removed, it was placed into 30 ml of 5 % glutaraldehyde and fixed at

room temperature for one hour. After each sample was fixed, it was stored at 4C until all

the samples were collected. At the 24 hrs time point, 0.1 ml of cells was diluted 50 times

with isotonic saline, stained with Trypan Blue (Eastman Kodak Co.), placed on a

hemacytometer, and examined by light microscopy to estimate the percentage of viable

white cells. After 24 hrs of incubation, 10-15 % of the white cells were viable.

After all the samples were collected, they were washed three times in isotonic saline

and serially dehydrated in 25 %, 50 %, 75 %, and 100 % ethanol.The cells were

transferred to 100 % acetone, and embedded in Spurr's resin (Polysciences). Thin sections

(300 lim) were cut on an ultra-microtome (LKB, Inc), mounted on 300 mesh copper

grids, and poststained with uranyl acetate and lead citrate. The sections were examined on

a Hitachi HU-11 E electron microscope at 75 kV. The above procedure was performed

twice with the submicrosphere suspension and twice with the combined 10 Rm-

submicrosphere suspension labelled with silver in Section 2.10.1. Microspheres and

submicrospheres were identified by the clearly visible silver grains on their surfaces

(Figure 37).












2.11 Systemic Clearance of Albumin Submicrospheres in Rabbits



Three female New Zealand white rabbits (2.7 kg) were anesthetized with Rompun

(0.8 ml) and Ketaset (0.8 ml) injected intramuscularly. A 25 g butterfly needle was

placed in a vein in the right ear and 15 mg of submicrospheres (Section 2.1.1) were

injected into each rabbit. The submicrospheres (15 mg), labelled with 14-C in Section

2.3 (0.3 gCi of activity), were suspended in 1 ml of isotonic saline and were injected

slowly over 1 minute. Anesthesia was maintained by periodic injections of Ketaset. At 15,

30, 45, 60, and 120 min after the injection, 10 ml of blood was drawn via heart

puncture from each rabbit, using 100 units of heparin per ml of blood as anticoagulant.

After 2 hrs, the rabbits were sacrificed with sodium pentobarbital and the lungs, spleens,

and livers removed.

Small pieces of the organs (0.1 0.5 gm) were placed into ashless paper cones and

burned on a tissue oxidizer (Packard Inst. Co., Model 306). The samples were burned for

2 min and the 14-C -labeled CO2 generated was collected in 6 ml of Carbosorb and 12 ml

of Permafluor V (Packard Inst. Co.). Blood samples (0.5 ml) were placed on ashless gauze

pads in paper cones and oxidized. The blood cells were also centrifuged and samples of

serum oxidized separately as above. The oxidation products were counted on a scintillation

counter (Perkin-Elmer) three times for 10 min each. The activity of each sample was

normalized by mass and plotted against time.

The above procedure was repeated with three more rabbits, taking blood samples 1, 2,








38



4, and 6 days after injection. The urine and feces were also collected. After 6 days, the

animals were sacrificed and the blood, organs, urine, and feces were assayed for isotope as

above. The equivalent mass of submicrospheres was calculated and plotted versus time

(Figure 38, Table 3).










39








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Figure 12. Variation of Hydration Swelling With Sphere Size:
Percent Increase In Diameter On Hydration vs Dry Diameter

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Figure 13 Surface Modification of Albumin Submicrospheres with Glycine and Silver


















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Figure 20. Optical Micrograph of Mouse Muscle 10 Days after Injection with
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Figure 22. Optical Micrograph of Mouse Tumor Tissue Immediately after
Injection of 1 pm Minimum Cross-Link Density Spheres


Figure 23. Optical Micrograph of Mouse Tumor Tissue 3 Days after
Injection of 1 jpm Minimum Cross-Link Density Spheres

































Figure 24. Optical Micrograph of Mouse Tumor Tissue 9 Days after
Injection of 1 pm Minimum Cross-Link Density Spheres
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Figure 25. Optical Micrograph of Mouse Tumor Tissue 16 Days after
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unquenched 20 % PGA spheres 0 quenched 20 % PGA spheres




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unquenched spheres 0 glycine quenched spheres

unquenched 20 % PGA spheres 0 glycine quenched 20 % PGA
spheres


















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Submicrospheres Taken Up By a Rabbit White Blood Cell After
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CHAPTER 3
RESULTS AND DISCUSSION




3.1 Submicrospheres



A method was developed in this study for preparing hydrophilic submicron albumin

spheres (submicrospheres). Figure 1 shows the dry size distribution of dry

submicrospheres typically produced by this method (average diameter 82 nm).

Figures 2,3, and 4 are electron micrographs of these submicrospheres. The

submicrospheres were easily suspended in a variety of aqueous media such as distilled

water, physiological saline, and phosphate buffer without the need for surfactants.

The method developed was a modification of the Longo steric stabilization method

(18). Briefly, an aqueous albumin solution is dispersed by sonication into an organic

polymer solution and the polymer adsorbs to the surface of the aqueous droplets. In

general, as smaller droplets form, they have a tendency, due to surface tension, to

recombine into larger droplets. However, when adsorbed onto the surface of the

droplets, the polymer molecules acted as spacers to keep the droplets from

recombining, i.e., the droplets are sterically stabilized. When the suspension has been

dispersed to the desired droplet size, glutaraldehyde is added to cross-link the

83












albumin molecules. The glutaraldehyde reacts with primary amine groups to form

Schiff base (imine) linkages (Figurel3). Once the albumin droplets are completely

cross-linked, the adsorbed polymer is washed off.

The glutaraldehyde cross-linking agent is relatively insoluble in the suspending

solvent, and is therefore first dissolved in toluene. The concentration of glutaraldehyde

in toluene was previously shown to be 0.14 mmoles/ml of toluene (18).

Previous techniques for the synthesis of albumin microspheres have used vegetable

oil as the hydrocarbon dispersing medium. Although partially successful in stabilizing

the droplets, the oil was not readily washed off, creating a hydrophobic surface on the

spheres prepared by this method (4). The technique developed by Longo and adapted

here for submicrosphere synthesis yielded spheres with a hydrophilic surface by

using polymer-solvent steric stabilization

In addition to producing hydrophobic microspheres, the vegetable oil method also

limited the minimum size of the spheres. Attempts to reduce the size of the droplets

below 1 pm by vigorous stirring or sonication were unsuccessful and denatured the

protein prematurely. Development of a less viscous dispersing medium was also

important for the synthesis of smaller spheres, achieved in this study.

The albumin matrix of the spheres can entrap small insoluble particles and soluble

macromolecules. Fluorescein-labeled albumin was incorporated into microspheres so

that enzymatic degradation could be followed by release of fluorescein-labeled

fragments of albumin. Submicrospheres containing up to 4 wt % of fluorescein -

albumin, pictured in Figure 8, were synthesized. Poly L glutamic acid (PGA)












(Figure 26) was also incorporated into the spheres during synthesis to enable drug

uptake and release by ion exchange. Submicrospheres containing up to 20 wt % PGA

(Figure 5) were synthesized. Both fluorescein albumin and PGA were dissolved in

the aqueous phase for synthesis.

Insoluble, particulate material was also entrapped in the matrix of the spheres.

Barium sulfate was incorporated into the spheres during synthesis as a radio-opaque

contrast agent. Submicrospheres containing up to 2 wt % barium sulfate (Figure 7)

were synthesized, although the radio-opacity of the spheres was not tested. Magnetite

(Fe304) was also incorporated into the spheres during synthesis to make the spheres

magnetically responsive. Submicrospheres containing up to 16 wt % magnetite

(Figure 6) were synthesized. The magnetite grains were generally between 5 nm and

25 nm in diameter. The grains were not homogeneously distributed, but most

submicrospheres had about three magnetite grains apiece. The grains of barium

sulfate were too small and of too low contrast to be seen, but the magnetite grains were

clearly visible by TEM.

Microspheres containing 9 wt % magnetite were also synthesized in the 1 pm -

20 p.m range (Figure 9) using a variation of the submicrosphere synthesis; the

aqueous suspension was dispersed less energetically, thereby making larger droplets

and producing larger spheres.












3.2 Submicrosphere Size Distribution



The submicrosphere synthesis produced an initial size distribution ranging from

10 nm to 5 pm. A subset of the initial size distribution (about 50 wt %), separated

out by centrifugation, had a dry size range of 10 nm to 200 nm and was designated the

submicrosphere subset. The size distribution of dry submicrospheres, as determined

by electron microscopic examination of at least 100 spheres (Section 2.2 and Figure

2) is shown in Figure 1. The number average diameter was 82 nm.

Dry submicrospheres were also hydrated in water and examined by photon

correlation spectroscopy which yielded a Z-average diameter of 333 nm.* Within the

limits of the apparatus, the hydrated suspension appeared monodisperse, allowing a

direct comparison of the dry number average and hydrated Z-average diameters. The

percent increase in diameter on hydration was 416 %, from 82 nm to 333 nm.











Dnnjdj Dj njdj

at monodisprsity Dn= Dz
at monodispersity Dn= Dz












3.3 Storage and Resuspension



Perhaps the most obvious characteristic of submicrospheres when viewed by TEM

(Figures 4-7), is that the spheres tend to form into clusters when dried. As the

spheres are dried on the carbon coated copper grids used for TEM, they are forced

closer together until contact between them is made. Van der Waal's forces which

become dominant at this point produce clusters of spheres as seen in the micrographs.

For this reason, submicrospheres were stored in water or acetone, and not allowed to

dry.

Submicrospheres stored in acetone for 14 months, either at room temperature or

4C, showed no signs of degradation when examined by transmission electron

microscopy. However, spheres stored in distilled water at 4C showed almost complete

degradation in about 4 months (Figure 39). A means of storing the spheres in a dry

state that would allow for simple resuspension in water was therefore sought. This was

accomplished by coating the spheres with macromolecules that would prevent

aggregation during drying. A variety of coatings were studied, ranging from simple

surfactants to macromolecules (Table 2). In each case, spheres were suspended in the

chosen solution and lyophilized to dryness. Concentrated solutions (10 %) of

macromolecules such as dextran (500,000 MW), polyethylene glycol (6000 MW),

and bovine albumin (67,000 MW) allowed complete aqueous resuspension with only

gentle manual shaking. The effect of these macromolecules was analogous to the effect

of cellulose acetate butyrate, which sterically stabilized the aqueous droplets during




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