Microspheres for incorporation of therapeutic substances and methods of preparation thereof

Novel hydrophilic protein or polypeptide microspheres prepared by dispersing an aqueous solution or dispersion of protein or polypeptide in an organic solvent solution of a high molecular weight polymer to form a stabilized dispersion of microspheres and cross-linking said microspheres with a polyfunctional cross-linking agent.

BACKGROUND OF THE INVENTION 
Insoluble polypeptide or protein microspheres containing therapeutic agents 
which enable the controlled release thereof in biological systems have 
generated growing interest in recent years [Kramer, J. Pharm. Sci., 63, 
page 1646 (1976); Widder et al: Cancer Research, 40, page 3512 (1980) and 
Widder et al; J. Pharm. Sci., 68, page 79 (1979)]. Systems utilizing the 
microspheres have the potential advantage of prolonging effective drug 
concentrations in the blood stream or tissue when injected thereby 
reducing the frequency of administration; localizing high drug 
concentrations; reducing drug toxicity, and enhancing drug stability. 
Albumin is a preferred protein or polypeptide for the preparation of such 
microspheres since it is a naturally occurring product in human serum. 
Although it is usually necessary to cross-link the albumin when preparing 
microspheres according to conventional methods cross-linked albumin may 
still be degraded depending upon cross link density thereby enabling the 
use thereof for drug delivery systems, etc. 
Conventional methods for the preparation of albumin microspheres are 
generally of two types. In one method, aqueous dispersions of albumin are 
insolubilized in vegetable oil or isooctane or other hydrocarbon solvent 
by denaturing at elevated temperatures (110.degree.-165.degree. C.). 
Another method involves chemical cross-linking of the aqueous dispersion 
of albumin at room temperature. Typical of these two types of methods are 
those described in U.S. Pat. Nos. 4,147,767; 4,356,259; 4,349,530; 
4,169,804; 4,230,687; 3,937,668; 3,137,631; 3,202,731; 3,429,827; 
3,663,685, 3,663,686; 3,663,687; 3,758,678 and Ishizaka et al, J. Pharm. 
Sci., Vol. 20, page 358 (1981). 
These methods, however, result in the formation of relatively hydrophobic 
microspheres which usually require a surfactant in order to disperse a 
sufficient quantity thereof in water or other systems for administration 
to a biological system to ensure the delivery thereto of an effective 
amount of any biologically active agent entrapped therein. In addition, 
the hydrophobic nature of conventional polypeptide microspheres make it 
extremely difficult to "load" large quantities of water soluble 
biologically active agents or other material within the microspheres after 
synthesis. 
It is an object of the present invention to provide more hydrophilic 
polypeptide microspheres which will accept high "loadings" of biologically 
active substances or other materials especially by addition of such 
substances after microsphere synthesis, and to prepare such drug loaded 
microspheres which do not require the utilization of surfactants to enable 
the preparation of highly concentrated dispersions thereof. 
It is a further object of the invention to provide hydrophilic microspheres 
which may be more readily modified by aqueous chemical methods to 
covalently attach proteins, enzymes, antibodies, immunostimulants, and 
other compounds to alter and improve microsphere properties. 
It is a further object of the present invention to provide a novel method 
for the preparation of such hydrophilic microspheres. 
It is still a further object of the present invention to provide novel 
hydrophilic microspheres containing biologically active or other 
substances and a method for the preparation thereof. 
It is still a further object of the present invention to provide a 
composition for administration to an animal, including humans, comprising 
the novel hydrophilic polypeptide microspheres containing a biologically 
active substance. 
It is still a further object of the present invention to provide a novel 
method for administering a biologically active substance to an animal 
based upon a system comprising hydrophilic polypeptide microspheres 
containing a biologically active substance. 
SUMMARY OF THE INVENTION 
The foregoing and other objects of the invention are provided by novel 
hydrophilic microspheres prepared by a method comprising 
(a) providing a dispersion of an aqueous solution or dispersion of 
polypeptide or protein microspheres in an organic substantially water 
immiscible solvent solution of high molecular weight polymer wherein the 
organic solvent is substantially a non-solvent for the protein 
microspheres and the soluble polymer stabilizes the protein or polypeptide 
microsphere dispersion. 
(b) incorporating a polyfunctional cross-linking agent for the protein or 
polypeptide in the dispersion, and 
(c) allowing the cross-linking agent to react with the protein or 
polypeptide microspheres for a time sufficient to cross-link at least a 
portion of the microspheres and, thereby to render the microspheres 
substantially insoluble in water and to provide free reactive functional 
groups therein. 
The present invention also provides novel cross-linked hydrophilic 
polypeptide microspheres containing additional substances prepared by 
reacting the free reactive functional groups of the above-described 
cross-linked protein microspheres with substances containing at least one 
functional group reactive therewith to form a covalent or other type of 
bond between the cross-linked protein microspheres and the additional 
substance. 
The present invention also provides a composition in unit dosage form 
adapted for administration to a biological system comprising a 
biologically effective amount of the above-described cross-linked 
hydrophilic polypeptide microspheres bonded with a biologically active 
substance. 
The present invention also provides a method of administering a 
biologically active substance to a biological system comprising 
administering thereto a biologically effective amount of the 
above-described composition. 
DETAILED DESCRIPTION OF THE INVENTION 
The present invention is predicated on the discovery that more hydrophilic 
protein or polypeptide microspheres can be prepared than heretofore if an 
aqueous polypeptide or protein solution is first dispersed in organic 
solvent solutions of certain high molecular weight polymers wherein the 
organic solvent is a non-solvent for the aqueous polypeptide. The 
resulting dispersion comprises a polypeptide or protein solution dispersed 
in a high molecular weight polymer solution in an organic solvent external 
phase. This polymer solution functions to stabilize the aqueous protein or 
polypeptide dispersion and ensures the integrity of the individual 
microspheres. 
When a polyfunctional cross-linking agent is introduced into this 
dispersion, preferably in the form of an organic solvent solution, the 
cross-linking agent is presented initially to the external surfaces of the 
microspheres rather than to the interior thereof as in the conventional 
prior art methods for microsphere production, thereby creating a 
relatively higher cross-link density at the surfaces of the microspheres 
than in the interior. This phenomenon also gives rise to a high 
concentration of free reactive functional groups from the cross-linking 
agent at the surface of the microspheres thereby facilitating increased 
hydrophilicity thereof especially if the microspheres are allowed to react 
with an added reagent reactive therewith, e.g., an amino acid or amino 
alcohol. The availability of these free amino acid or amino alcohol 
functional groups for reaction with other substances containing functional 
groups reactive therewith also renders the microspheres capable of being 
much more highly "loaded" with additional substances such as 
therapeutically active agents, etc., than conventional microspheres. 
Moreover, the more hydrophilic nature of the microspheres enhances their 
dispersion in aqueous media, thereby enabling the safe administration 
thereof to animals, including humans, or other biological systems in much 
greater amounts than conventional hydrophobic microspheres, which require 
the presence of possibly biologically deleterious amounts of surfactants 
to achieve similar concentrations of administrable microspheres. 
It will be understood by those skilled in the art, having been exposed to 
the principles of the present invention, that any protein or polypeptide 
capable of forming a cross-linked microsphere may be employed in the 
practice of the invention. Suitable such proteins or polypeptides include 
serum albumin, poly-L-lysine, poly-L-arginine, poly-L-histidine, 
polyglutamic acid, and any water soluble protein with functional amine 
groups such as enzymes, immunoglobulins, etc. 
Furthermore, other polypeptides or macromolecules may be incorporated into 
the albumin microspheres even if they do not participate in the 
cross-linking reaction. These may be added to the albumin in the aqueous 
phase in concentrations ranging from 0.1 to 30%, by weight, or more and 
become entrapped during the cross-linking process. Such added 
macromolecules include, for example, polyglutamic acid, carboxymethyl 
dextran, carboxymethyl cellulose, polygalacturonic acid, cellulose, 
dextran, etc. 
Where microspheres are to be subsequently reacted with a biologically 
active substance to produce a composition suitable for administration to a 
biological system, it is preferred to utilize an albumin to form the 
microsphere since it is a naturally occurring substance in most biological 
systems. Moreover, albumin, which has been cross-linked with most 
polyfunctional reagents, may be degraded in vivo, depending upon the 
extent of cross-linking, after administration to a biological system. 
Although any form of albumin may be used in the practice of the invention 
it is preferred to match the albumin with the biological system to which 
microspheres prepared therefrom are administered, e.g., human serum 
albumin, bovine serum albumin, rabbit serum albumin, fatty acid free human 
serum albumin, dog serum albumin, egg albumin, horse serum albumin, etc. 
The aqueous solution or dispersion of protein or polypeptide is first 
dispersed in an organic solvent solution of a high molecular weight 
polymer. 
Any polymer capable of forming a stabilized dispersion of the protein or 
polypeptide may be utilized in the practice of the invention. The polymer 
solution should be one which stabilizes the resulting dispersion of 
microspheres in the organic solvent/aqueous external phase against 
coagulation, agglomeration, etc. Suitable such polymers include, for 
example, acrylic polymers, e.g., polymethylmethacrylate, etc., 
polyoxyethylenepolyoxypropylene block copolymers, cellulose 
acetatebutyrate, polycarbonates, e.g., bisphenol polycarbonate, etc., 
polysulfones, polyacrylamides, polyvinyl alcohols, polyacetals, 
polystyrene and copolymers thereof, polyesters, polyamides, etc. 
The organic solvent for the polymer should be a non-solvent for the aqueous 
protein or polypeptide solution and inert with respect thereto and capable 
of forming at least a 1-40%, by weight, solution of the polymer. Suitable 
solvents will depend, of course, upon the particular protein or 
polypeptide used to form the microspheres and the stabilizing polymer. 
Having been exposed to the principles of the present invention those 
skilled in the art will be able to determine suitable polymers and organic 
solvents without the exercise of inventive faculties or undue 
experimentation. Typical of useful organic polymer solvents are toluene, 
benzene, chloroform, ethylene dichloride, methylene chloride, etc., and 
mixtures thereof. 
The term "microsphere" is intended to include any small particles of 
protein or polypeptide or mixture thereof with other macromolecules of 
generally spherical shape which is formed upon dispersion of an aqueous 
solution of protein or polypeptide in an organic solvent solution of a 
stabilizing polymer. 
The stabilization of the microspheres is largely dependent upon the 
concentration and molecular weight (M.W.) of the polymer in the organic 
solvent. Generally, as the M.W. of the polymer is increased, the 
concentration of said polymer can be decreased. The upper limit of polymer 
concentration is governed by the ease of removal of the polymer after 
microsphere cross-linking. Generally, concentrations between about 10% and 
30%, by weight, depending upon the particular protein or polypeptide and 
polymer selected, will be sufficient to produce suitable dispersions. 
Average size and size distribution of the microspheres is usually a 
function of the shear forces and the time, i.e., energy, necessary to 
prepare the dispersions. Increasing either the shear forces or time of 
dispersion or both decreases the size of the microspheres. Generally, when 
producing large microspheres, a lower polymer concentration may be used. 
Smaller microspheres usually require higher polymer concentrations and 
more dilute protein dispersions. 
Microspheres having a size in the range of from about 0.05 micron to about 
500 microns may be prepared according to the method of the invention. 
Those skilled in the art having been exposed to the principles of the 
invention as described herein will be capable of selecting appropriate 
polymer concentrations and molecular weights and dispersion techniques to 
prepare microspheres of a desired size without the exercise of inventive 
faculties and undue experimentation. 
Suitable cross-linking agents include polyfunctional reagents capable of 
reacting with the functional groups, particularly the amino groups, 
present on the protein or polypeptide to cross-link the molecules thereof. 
The selection of a particular cross-linking agent will depend to a large 
extent upon the intended use of the microspheres. Generally, however, any 
polyfunctional reagent, including those used heretofore in prior art 
methods to cross-link proteins, may be utilized to form the microspheres 
of the present invention. Typical of such reagents are polyaldehydes such 
as glutaraldehyde and polyisocyanates such as 2,4-tolylenediisocyanate, 
1,6-diisocyanatohexane and activated polyfluoro compounds such as 
1,5-difluoro-2,4-dinitrobenzene and 
P,P'-difluoro-m,m'-dinitrodiphenylsulfone. 
The cross-linking agent is preferably presented to the microspheres by 
incorporating in the above-described dispersion of protein or polypeptide 
microspheres an organic solvent solution of the cross-linking agent. The 
organic solvent for the cross-linking agent is one that is compatible with 
the solvent for the organic polymer and is likewise a non-solvent for the 
microspheres. The cross-linker diffuses into the microspheres from the 
organic phase with consequent increased concentration at the surfaces 
thereof. As a result, the other surfaces of the microspheres are usually 
cross-linked to a greater extent than the exterior portions. 
This is in contrast to conventional prior art methods wherein the 
cross-linker is usually present in the aqueous protein dispersion thereby 
resulting in substantially uniform cross-linking throughout the 
microspheres. In addition, there is a larger concentration of free 
functional groups at the outer surfaces of the microspheres of the present 
invention as a result of some reactions between only one of the functional 
groups of the cross-linkder with amino groups of protein or polypeptide 
molecules. 
These free reactive functional groups (e.g., aldehyde groups, where the 
cross-linker is a polyaldehyde) at the surface render the microspheres 
more hydrophilic and readily susceptible to wetting and dispersion in 
aqueous media, especially when further reacted with polar compounds such 
as amino acids or amino alcohols or when oxidized to carboxylic groups. 
Suitable organic solvents for the cross-linking agent include any which are 
non-solvents for the microspheres and which are compatible with the 
solvent for the organic polymer and the cross-linking agent. Suitable 
solvents include those listed above as suitable for solubilizing the 
polymer. Those skilled in the art, having been exposed to the principles 
of the present invention, will be capable of selecting suitable solvents 
for the cross-linkers without the exercise of inventive faculties or undue 
experimentation. 
The cross-link density as well as the number of free functional groups at 
the surfaces of the microspheres can be controlled by regulating the 
concentration of the cross-linking agent. Generally, as the concentration 
of cross-linker in the final dispersion is increased, the cross-link 
density and the number of free functional groups at the outer surfaces of 
the microspheres are increased. For most applications, a sufficient 
quantity of cross-linker solution is added to the microsphere dispersion 
to yield a final cross-linker concentration therein of between about 0.1% 
and about 25%, by weight. It will be understood, however, that any 
concentration consistent with an efficient completion of the method and 
the intended use of the microspheres may be utilized. 
The following non-limiting examples are illustrative of the novel 
microspheres of the present invention and of methods for their preparation 
and use. 
Preparation of glutaraldehyde cross-linked human serum albumin(HSA) 
microspheres(MS)

EXAMPLE 1 
HSA (0.150 g) (Sigma, recrystallized and lyophilized) was dissolved in 0.5 
ml water in a 16.times.125 mm test tube (this size test tube was used 
throughout all of the following procedures except where noted). The 
solution was added drop wise to a 25 wt % solution of 
polymethylmethacrylate (PMMA) (Polyscience, intrinsic viscosity 1.4) in a 
mixture of 1.5 ml chloroform and 1.5 ml toluene in a screw cap test tube. 
The mixture was dispersed with a vortex mixer (Vortex Genie Scientific 
Industries, Inc.) for two minutes at a power setting of nine. Aqueous 
glutaraldehyde 1.0 ml (25 wt%) and 1.0 ml of toluene were combined in a 
13.times.100 mm test tube. The two phases were dispersed by 
ultrasonification (Heat Systems-Ultrasonics, Model W-375) with a microtrip 
power head attachment (20 sec. at 50 watts). The resulting toluene 
solution of glutaraldehyde, (0.14 mmoles) was allowed to phase separate, 
pipeted off, and combined with the albumin dispersion. After addition of 
the glutaraldehyde saturated toluene, the albumin dispersion was mixed 
with a rotary mixer (Labquate Labindustries) at room temperature (r.t.) 
for 8 hrs. The resulting cross-linked HSA/MS were washed to remove all 
PMMA dispersant by the addition of 10.0 ml of acetone, test tube briefly 
agitated, then centrifuged (2000 RPM.times.2 min.), the supernate 
discarded and the HSA/MS-U pellet re-suspended with an additional 10.0 ml 
of acetone. This wash procedure was repeated eight times. After the last 
wash, HSA/MS were allowed to air dry. The product was a brown powder, 
0.122 g, 81% yield. The average diameter of the HSA/MS was 29 .mu.m as 
determined by optical microscopy. 
EXAMPLE 2 
HSA (0.150 g) was dissolved in 0.5 ml of water in a test tube. This 
solution was added dropwise to a 25 wt % solution of 
polyoxyethylene/polyoxypropylene copolymer (Poloxmer 188 (BASF Wyandotte 
Corp., MW 8430)) in 4.0 ml chloroform in a screw cap test tube. The 
mixture was dispersed with a vortex mixer for two minutes at power setting 
nine. Glutaraldehyde was used for cross-linking and was prepared with 
chloroform by sonification as previously described in Example 1. After 
addition of the glutaraldehyde saturated chloroform, the albumin 
dispersion was mixed with a rotary mixer at r.t. for 6 hrs. The resulting 
cross-linked HSA/MS were washed to remove all Poloxmer 188 dispersant as 
described in Example 1. The MS product was a brown powder, 0.115 g, 77% 
yield. The average diameter was 20-40 .mu.m, as determined by optical 
microscopy. 
EXAMPLE 3 
HSA (0.150 g) was dissolved in 0.5 ml of water in a test tube. This 
solution was added dropwise to a 3.0 wt % solution of cellulose acetate 
butyrate (CAB Polyscience, MW 73,000) in 4.0 ml of ethylene dichloride in 
a screw cap test tube. The mixture was dispersed with a vortex mixer for 
two minutes at power setting nine. Glutaraldehyde was used for 
cross-linking and was prepared as described in Example 1. After addition 
of the glutaraldehyde saturated toluene, the dispersion was mixed with a 
rotary mixer at r.t. for 24 hrs. The resulting cross-linked HSA/MS were 
washed to remove all CAB dispersant and dehydrated as described in Example 
1. The MS product was a brown powder, 0.11 g, 73% yield. The average 
diameter was 25 .mu.m determined by optical microscopy. 
EXAMPLE 4 
HSA (0.156 g) was dissolved in 0.5 ml of water in a test tube. This 
solution was added dropwise to a 20 wt % solution of bisphenyl 
polycarbonate (General Electric, MW 32,000) in 4.0 ml of chloroform. 
Glutaraldehyde was used for cross-linking and was prepared as described in 
Example 2. After addition of the glutaraldehyde saturated chloroform, the 
mixture was mixed with a rotary mixer at r.t. for 16 hrs. The resulting 
cross-linked HSA/MS were washed to remove all polycarbonate dispersant by 
the addition of chloroform (8 X, 10.0 ml volumes), then acetone (8 X, 10.0 
ml volumes) and water (4 X, 5.0 ml volumes). After the water wash, MS were 
examined with an optical microscope and stored frozen at 0.degree. C. 
Average diameter of the MS were 10-50 .mu.m. 
EXAMPLE 5 
Human Serum Albumin (Fatty Acid Free (FAF) Microspheres-Unquenched 
HSA(FAF) (0.145 g, Sigma) was dissolved in 0.5 ml of water in a test tube. 
This solution was dispersed in the PMMA solution and cross-linked with 
glutaraldehyde for 16 hrs as described in Example 1. MS were washed to 
remove all PMMA dispersant by centrifugation with acetone (8X) then water 
(4X). After the last water wash the HSA(FAF)MS pellet was re-suspended in 
10.0 ml of water. Aliquots (0.5 ml) were removed from the well-shaken 
sample and pipeted into three pre-weighed 13.times.100 mm test tubes, then 
placed in a 100 C. oven (National) to remove all water. Test tubes were 
cooled to r.t. and weighed, average of the three weights being used to 
determine the weight of MS per ml of water in the 10.0 ml volume. This 
yield was 0.114 g, 79% of HSA(FAF)MS. The average diameter of the MS was 
32 .mu.m as determined by optical microscopy. 
EXAMPLE 6 
Bovine, Dog and Rabbit Serum Albumin/Microspheres-Unquenched 
Bovine serum albumin (BSA) (0.150 g), dog serum albumin (DSA) (0.150 g) and 
rabbit serum albumin (RSA) (0.150 g) obtained from Sigma (fraction V) were 
dissolved in 0.5 ml of water in test tube. The albumin/MS were then 
synthesized as described in Example 1 with a cross-linking reaction time 
of 16 hrs. The MS were washed with acetone, (8X, 10.0 ml volumes) to 
remove all PMMA, then with water (4X, 5.0 ml volumes). After the last 
water wash the brown pellets were re-suspended in 10.0 ml of water, and 
weight of albumin/MS per ml was determined as described above. The yield 
was 0.109 g, 73% (2) 0.103 g, 69% and (3) 0.128 g, 82%. Average diameter 
of MS were 28, 14 and 13 .mu.m, respectively, as determined by optical 
microscopy. The MS are readily formed and are similar to MS produced from 
human albumin. This demonstrates the versatility of the procedure and the 
ability to synthesize MS from other mammelian protein which would be 
beneficial for veterinary applications when MS containing therapeutic 
agents are required. 
EXAMPLE 7 
Polylysine (PLY)/Microspheres-Unquenched 
PLY (0.151 g, MS 11,000, 0.11 .mu.moles, Sigma) was dissolved in 0.5 ml of 
water in a test tube. This solution was dispersed in the PMMA mixture and 
PLY/MS were synthesized as described in Example 1 with a cross-linking 
reaction time of 2 hrs. The PLY/MS were washed with acetone to remove all 
PMMA, then with water. After the last water wash the yellow pellet was 
re-suspended in 10.0 ml of water and weight of the PLY/MS were determined 
as described above. This produced 0.120 g of PLY/MS in solution, 79% 
yield. 
Polylysine (PLY) is a cationic polypeptide that consists of repeating units 
of amino residues with a net positive charge. PLY may be incorporated into 
the HSA matrix at various wt. concentrations. It is also possible to make 
PLY/MS. Glutaraldehyde was able to cross-link the PLY molecules in the 
same manner as albumin. PLY/MS would be advantageous because of its 
ability to conjugate acidic drugs by the formation of a salt complex. 
EXAMPLE 8 
Preparation of diisocyanate cross-linked bovine serum albumin(BSA) 
microspheres(MS) 
BSA (0.161 g) was dissolved in 0.5 ml of water in a test tube. This 
solution was dispersed in the PMMA solution as described in Example 1. 
Tolylene 2,4-diisocyanate (TDI) (Aldrich) was used to cross-link the 
albumin. Aqueous TDT (80% 4.6 mmoles) 1.0 ml, and 1.0 ml of toluene were 
combined in a 2.0 ml volumetric flask. After mixing well, 1.0 ml of the 
TDI/toluene solution was added to the albumin dispersion. The dispersion 
was then mixed with a rotary mixer at r.t. for 21 hrs. BSA/MS were washed 
to remove all PMMA dispersant by centrifugation with acetone then water. 
After the water wash, the white pellet was frozen in liquid nitrogen and 
lyophilized yielding 0.246 g of MS as a dry, free flowing white powder. 
EXAMPLE 9 
BSA (0.163 g) was dissolved in 0.5 ml of water in a test tube. The solution 
was dispersed in the PMMA solution as described in Example 1. Aqueous 
1,6-diisocyanatohexane (DCH) (98% Aldrich), 1.0 ml (5.8 mmoles) and 1.0 ml 
toluene were combined in a 2.0 ml volumetric flask. After mixing well, 1.0 
ml of the resulting solution was added to the albumin dispersion and mixed 
at r.t. for 21 hrs. The BSA/MS were washed and dehydrated according to the 
procedure in Example 8. The MS product was a white powder, 0.023 g, 76% 
yield. 
EXAMPLE 10 
Synthesis of Sub-Micron Microspheres 
HSA, 0.164 g, was dissolved in 0.5 ml of water and added dropwise to 3.0 
percent CAB in 25 ml of ethylene dichloride in 25.times.150 mm screw cap 
culture tubes. The mixture was dispersed with a Brinkman Homogenizer (Pt 
10-35) connected to a PT 20/TS probe generation at a setting of 6.5 for 10 
mins. The dispersion was added dropwise to a 500 ml round bottom flask 
containing 100 ml of 3.0 percent CAB polymer solution and mixed at medium 
speed with a magnetic stirrer. A 4.0 ml glutaraldehyde saturated toluene 
solution was used for cross-linking (see Example 1) and the dispersion was 
allowed to react for 2 hrs at room temperature. The cross-linked MS were 
washed out with acetone and dehydrated as described in Example 1. This 
yield 0.094 g of dry MS powder. Average size of the MS, determined using 
the scanning electron microscope, was 0.9 .mu.m. The size distribution is 
shown in Table 1. 
TABLE 1 
______________________________________ 
Sub-Micron: HSA/MS Size Distributions 
in CAB Dispersant 
% Fraction 
Energy 6.5 
Size/.mu.m Time (10 mins) 
______________________________________ 
0-0.5 6 
0.5-0.75 49 
0.75-1.0 13 
1.0-1.25 19 
1.25-1.5 10 
1.5-1.75 3 
1.75-2.0 0 
2.0-4.5 0 
______________________________________ 
EXAMPLE 11 
Microspheres with Variations In Cross-Link Density and Hydration 
BSA samples (1) 0.150 g, (2) 0.151 g (3) 0.150 g, and (4) 0.149 g were 
dissolved in 0.5 ml of water in test tubes. These solutions were dispersed 
in the PMMA mixture as described in Example 1. Glutaraldehyde was used for 
cross-linking and was diluted with water in the following ratios 1:1, 1:5, 
1:10 and 1:20; this gave a final molar concentration of (a) 1.25 moles, 
(b) 0.5 mmoles, (c) 0.25 mmoles, and (d) 0.125 moles. The glutaraldehyde 
solutions were combined with toluene and dispersed as described in Example 
1. After addition of the glutaraldehyde saturated toluene solutions (a) 
through (d) to the BSA/PMMA dispersions 1 through 4, they were mixed with 
a rotary mixer at r.t. for 22 hrs. After the final acetone wash and 
dehydration, the BSA/MS samples had a dry weight of (1) 0.125 g, (2) 0.140 
g, (3) 0.135 g, and (4) 0.063 g. An aliquot of water (10.0 ml) was added 
to each sample and allowed to hydrate for 1.0 hr at room temperature. The 
dispersions were then centrifuged and all of the supernate was carefully 
removed with a pasteur pipet. BSA/MS were then re-weighed to determine 
water content and were as follows: (1) 0.579 g, (2) 0.881 g, (3) 1.55 g, 
and (4) 0.969 g, by dividing the net weight by the dry weight the water 
content per mg of BSA/MS was determined. Average diameter of the MS for 
both the dry state and wet state was determined by optical microscopy. See 
Table 2. 
TABLE 2 
______________________________________ 
Varying Cross-Link Density and Hydration of BSA/MS 
(Glutaraldehyde Cross-Linking) 
Mean Mean 
Glutaraldehyde 
Diameter Diameter mg Water % 
Concentration 
Dehydrated 
Hydrated Uptake/mg of 
Hydra- 
(mmoles) (.mu.m) (.mu.m) BSA/HSA tion 
______________________________________ 
1.25 14 17 4.6 82 
0.50 13 21 6.3 86 
0.25 10 23 11.5 92 
0.13 17 37 15.4 94 
______________________________________ 
EXAMPLE 12 
BSA samples (1) 0.151 g, (2) 1.157 g, (3) 0.148 g and (4) 0.150 g were 
dissolved in 0.5 ml of water in test tubes. These solutions were then 
dispersed in the PMMA mixture as described in Example 1. Dilutions of 
tolylene 2,4-diisocyanate (TDC) were used for cross-linking and were 
prepared as follows. In four 10.0 ml volumetric flasks; (a) 0.4 ml, (b) 
0.2 ml, (3) 0.1 ml. and (d) 0.07 ml of TDC were diluted with toluene. This 
gave final molar concentrations of (a) 0.18 mmoles (b) 0.092 mmoles (c) 
0.046 mmoles and (d) 0.031 mmoles. One ml of the TDC/toluene solutions (a) 
through (d) were added to the albumin dispersions 1 through 4. The 
dispersions were then mixed with a rotary mixer at r.t. for 22 hrs. After 
the final acetone wash and dehydration, the BSA/MS samples had a dry 
weight of (1) 0.176 g (2) 0.171 g (3) 0.149 g and (4) 0.150 g. After 
hydration and removal of excess water as described in Example 11, the 
BSA/MS samples were re-weighed; this yielded (1) 0.314 g (2) 0.643 g (3) 
1.950 g and (4) 3.664 g. The water content of the MS were then calculated 
for each sample as described in Example 11. Average diameter of the BSA/MS 
for both the dry state and wet state were determined by optical 
microscopy. See Table 3. 
TABLE 3 
______________________________________ 
Varying Cross-Link Density and Hydration of BSA/MS 
(TID Cross-Linking) 
Mean Mean 
Concentration 
Diameter Diameter mg Water % 
of TDI Dehydrated 
Hydrated Uptake/mg of 
Hydra- 
(mmoles) (.mu.m) (.mu.m) BSA/MS tion 
______________________________________ 
0.18 18 19 1.8 64 
0.092 18 22 3.6 78 
0.046 21 30 13 93 
0.031 16 40 24 96 
______________________________________ 
EXAMPLE 13 
BSA samples (1) 0.150 g (2) 0.166 g (3) 0.151 g (4) 0.149 g and (5) 0.160 g 
were dissolved in 0.5 ml of water in test tubes. These solutions were then 
dispersed in the PMMA mixture as described in Example 1. Dilutions of 
1,6-diisocyanatohexane (DCH) were used for cross-linking and were prepared 
as follows: In five 10.0 ml volumetric flasks; (a) 5.0 ml (b) 2.0 ml (c) 
1.0 ml (d) 0.4 ml and (e) 0.2 ml of DCH were added and diluted with 
toluene. This gave final molar concentrations of (a) 2.9 mmoles (b) 1.16 
mmoles (c) 0.58 mmoles (d) 0.23 mmoles and (e) 0.12 mmoles. One ml of the 
DCH/toluene solutions (a) through (e) were added to the albumin 
dispersions (1) through (5). The dispersions were then mixed with a rotary 
mixer at r.t. for 22 hrs. After the final acetone wash and dehydration, 
the BSA/MS samples had a dry weight of (1) 0.140 g (2) 0.150 g (3) 0.141 g 
(4) 0.129 g and (5) 0.144 g. After hydration and removal of excess water 
as described in Example 11, the BSA/MS samples were re-weighed; this 
yielded (1) 0.240 g (2) 0.593 g (3) 0.898 g (4) 2.006 g and (5) 2.854 g. 
The water content of the BSA/MS were then determined for each sample as 
described in Example 11. Average diameter of the BSA/MS samples for both 
the dry state and wet state were determined by optical microscopy. See 
Table 4. 
TABLE 4 
______________________________________ 
Varying Cross-Link Density and Hydration of BSA/MS 
(DCH Cross-Linking) 
Mean Mean 
Concentration 
Diameter Diameter mg Water % 
of DCH Dehydrated 
Hydrated Uptake/mg 
Hydra- 
(mmoles) (.mu.m) (.mu.m) BSA/MS tion 
______________________________________ 
2.9 18 20 1.7 63 
1.16 21 29 3.7 79 
0.58 13 28 6.4 86 
0.23 17 38 16.0 94 
0.12 16 50 20.0 95 
______________________________________ 
The free functional groups on the cross-linked microspheres are capable of 
reaction with a wide variety of substances containing functional groups 
reactive with those on the microspheres whereby the substance is 
covalently or otherwise boned to the microspheres. There is virtually no 
limit to the types of substances which can be bound to the microspheres in 
this manner. Thus, the microspheres may be reacted with aminoalcohols, 
e.g., 2-aminoethanol or amino acids, e.g., glycine, to enhance 
hydrophilicity; coupled with, e.g., amino group containing drugs 
(adriamycin) for administration to a biological system or covalently 
bonded to large protein molecules such as lectins, enzymes or antibodies. 
In addition, changes in surface functionability of the microspheres may be 
used to enhance tissue immobilization by covalent or physical binding for 
specific tissue targeting using biospecific affinity ligands, e.g., 
tumor-specific immune assay reagents. 
Whereas protein and polypeptide microspheres containing entrapped or 
encapsulated substances prepared according to prior art methods are 
relatively hydrophobic requiring surfactants for dispersion in aqueous 
media and make post-forming aqueous chemical modification difficult, those 
of the present invention are hydrophilic, capable of dispersion in aqueous 
media in relatively large amounts without the necessity for surfactants 
and are readily useful for post-forming absorption of biologically active 
agents or chemical modification in aqueous media. 
Where it is desired to couple the microspheres with a substance which does 
not contain a functional group reactible with any free functional group in 
the cross-linked microsphere, the latter may first be reacted with a 
linking or bridging agent which has at least one functional group capable 
of reacting with the free functional group in the microspheres and at 
least one additional functional group which will react with the desired 
surfactant to covalently bond it to the microsphere. 
Suitable substances for chemical or physical bonding to the microspheres, 
depending, of course, upon the nature of the free functional groups 
thereon or the ability of the drug to naturally bind thereto include 
antitumor agents, e.g., adriamycin, bleomycin, chlorambucil, mitomycin C, 
etc., antibiotics, e.g., streptomycin, gentamycin, tobramycin, formycin A, 
etc., steroids, e.g., hydrocortisone phosphate, progesterone and other 
contraceptive hormones, etc. 
The term "biological system" as employed herein, is intended to include any 
living system, e.g., lower animal, human, plant, etc., to which a 
biologically active substance may be administered for therapeutic, 
diagnostic or other biological purpose. 
The following non-limiting examples illustrate the embodiment of the 
invention wherein the microspheres are bonded physically or chemically 
(covalently) to substances. Physical binding or association of drugs 
occurs readily with albumin and is satisfactory for many types of 
albumin-drug microsphere systems, especially where rapid drug release is 
desired. Chemical binding occurs via available reactive functional groups. 
In the following examples, the term "quench" refers to the reaction of 
free functional groups in the interior and/or on the surface of the 
microspheres with a substance containing a functional group reactive 
therewith. 
EXAMPLE 14 
Measurement of Free Reactive Aldehyde Groups in Microspheres 
Tritiated Leucine 
HSA/MS (10 .mu.m average diameter) were prepared as described in Example 1. 
The cross-linked HSA/MS were divided into two samples. One was quenched as 
described hereinbelow with 0.5 ml of 2-aminoethanol while the other sample 
was left unquenched. Tritiated leucine (New England Nuclear) specific 
activity 134.2.mu. Ci/mmoles/ml, was diluted with a L-leucine carrier to a 
final activity of 5.mu. curie/50 mmoles/ml. One ml of the isotope solution 
was added to each of three samples of 7.4 mg/ml unquenched and three 
samples of 8.0 mg/ml quenched MS in test tubes. The samples were incubated 
for 40 mins in a table top sonicator (E/MC RA Research), then washed four 
times with water by centrifugation (1000 RPM.times.2 mins). MS pellets 
were re-suspended in 2.0 ml of a scintillation cocktail (Aquasol New 
England Nuclear). From each of the solutions 1.0, 0.5, and 0.25 ml 
aliquots were removed and added to scintillation counter containers. The 
final volumes were adjusted to 15.0 ml with additional cocktail solution. 
Activity was determined using a Beckman Model 230 scintillation counter 
and values plotted against prepared standards. See Table 5. 
TABLE 5 
______________________________________ 
Concentration of Reactive Aldehyde Groups 
for HSA/MS by Binding of Tritium Labeled Leucine 
Physical or 
Chemical 
Binding of Leucine (moles) 
# of Leucine 
# Reactive 
Leucine Bound/ml of MS 
10 .mu.m MS 
CHO/MS* 
______________________________________ 
HSA/MS- 8.8 .times. 10.sup.-2 
5.5 .times. 10.sup.7 
-- 
Quenched 
HSA/MS- 1.3 .times. 10.sup.-1 
7.8 .times. 10.sup.7 
-- 
Unquenched 
Chemically 4.0 .times. 10.sup.-2 
2.4 .times. 10.sup.7 
2.4 .times. 10.sup.7 
Reacted 
(MS-U - MS-Q) 
______________________________________ 
*Assume one bound leucine equals one reactive aldehyde group. 
EXAMPLE 15 
Tritiated Concanavalin A (Con-A) Binding As a Function of Particle Size 
HSA/MS samples were prepared as described in Example 1. This produced MS 
with an average diameter of 30 .mu.m, 12 .mu.m, and 5 .mu.m. The 
cross-linked MS were divided into two samples, quenched and unquenched. 
Tritiated Con-A (New England Nuclear Corp.) specific activity 
42.4.mu.Cl/mmole/ml, was diluted with a carrier (Con-A, Sigma) to a final 
activity of 1.0.mu.Ci/0.136.mu.moles/ml, in 0.01 molar sodium phosphate 
buffer at pH 6.9. Two ml of the isotope solution was added to one sample 
of each particle size for both unquenched and quenched in test tubes. The 
samples were incubated for one hr in a table top sonicator, washed and 
re-suspended in a scintillation cocktail as described in Example 14. 
Activity was determined using a scintillation counter and values plotted 
against prepared standards. See Table 6. 
TABLE 6 
______________________________________ 
Con-A Binding to HSA/MS as a Function of MS Size 
Con-A Con-A 
(.mu.moles) 
(.mu.moles) 
Reacted Con-A 
Mean Diameter 
Bound/mg of 
Bound/mg of 
.mu.moles/mg 
(.mu.m) HSA/MS-U HSA/MS-Q (U - Q) 
______________________________________ 
30 2.6 .times. 10.sup.-4 
2.6 .times. 10.sup.-4 
0 
12 3.7 .times. 10.sup.-4 
3.2 .times. 10.sup.-4 
0.5 .times. 10.sup.-4 
5 6.7 .times. 10.sup.-4 
4.0 .times. 10.sup.-4 
2.7 .times. 10.sup.-4 
______________________________________ 
EXAMPLE 17 
Physical Analysis of Surface Properties of Microspheres, Capillary Wetting 
as a Function of Quenching 
HSA/MS (30 .mu.m average diameter) were prepared as described in Example 1. 
The HSA/MS were divided into two samples. One was quenched with glycine, 
as described hereinbelow, and the other sample was left unquenched, HSA/MS 
were dehydrated with acetone and air dried. Pasteur pipets (10 cm.times.1 
mm I.D.) were used as the capillary column. By gently pushing the pipet 
through a glass fiber (3 cm diameter, Gelman Type A-E) a plug was formed 
in the end of the column. HSA/MS samples were loaded into the column and 
packed by holding the capillary tube vertically on the rubber tip of the 
vortex genie, then vibrating the tube for 20 to 30 seconds at a speed 
setting of one, Columns were packed with HSA/MS to a height of 3.0 cm from 
top of glass fiber plug. The capillary tube was mounted vertically and 
placed in a PLEXIGLASS tank (10 cm.times.10 cm.times.5 cm filled with 
water) to a depth of one cm. The height of the water rise up the column 
was measured as a function of time. Both quenched and unquenched samples 
were run and the data taken (Table 7) was compared to HSA/MS produced by 
the prior art vegetable oil method (hydrophobic) 
TABLE 7 
______________________________________ 
Hydrophilic Measurements of HSA/MS as a Function 
of Surface Properties by Capillary Rise 
HSA/MS HSA/MS HSA/MS 
Unquenched Quenched Hydrophobic 
Time (mins) 
Height (mm) Height (mm) 
Height (mm) 
______________________________________ 
15 2.4 9.4 0.1 
35 2.7 13.7 0.1 
45 2.9 17.2 0.1 
60 3.2 20.1 0.1 
75 3.4 22.5 0.1 
90 3.5 24.9 0.1 
105 3.7 26.8 0.1 
120 3.8 28.6 0.1 
______________________________________ 
EXAMPLE 18 
Human Serum Albumin/Microspheres-Quenched (Q) 
HSA/MS were synthesized and washed as described in Example 1. After the 
last acetone wash, the HSA/MS pellet was re-suspended with 5.0 ml of 
water, briefly agitated and centrifuged. This was repeated four additional 
times. After the last water wash, the HSA/MS pellet was re-suspended in 
5.0 ml of 1.0 molar glycine HCl to "quench" the residual reactive aldehyde 
groups. The HSA/MS glycine solution was mixed at r.t. for 22 hrs with a 
rotary mixer. MS were removed from the unreacted glycine by 
centrifugation. After decanting the glycine supernate the HSA/MS pellet 
was re-suspended in a 50 ml polypropylene centrifuge tube (Corning) with 
45.0 ml of water (pH 3.0), briefly agitated and centrifuged (2000 
RPM.times.2 min). This wash procedure was repeated three times. The 
process was repeated again with water at pH 7.0 After the last water wash, 
MS were dehydrated with acetone (4x, 100% acetone in 10.0 ml volumes) and 
allowed to air dry. The product was a yellowish brown powder, 115 g, 77% 
yield. The average size of the MS was 26 .mu.m in diameter as determined 
with optical microscopy. 
The following non-limiting examples illustrate the embodiment of the 
invention wherein other non-cross-linking substance (macromolecules) are 
entrapped in the albumin microsphere producing a composite of the albumin 
with the substance. 
EXAMPLE 19 
Human Serum Albumin/Microspheres Containing Polyglutamic Acid 
(PGA)-Unquenched 
PGA (60,000 MW, Sigma) was added into HSA/MS in concentrations of 12, 16 
and 22 wt %. The PGA-HSA/MS were synthesized, washed and dehydrated as 
described in Example 1. Samples were prepared with the following weight 
ratios of HSA and PGA: 
______________________________________ 
HSA PGA 
______________________________________ 
1. 0.134 g 0.017 g (0.3 .mu.moles) 
2. 0.129 g 0.025 g (0.4 .mu.moles) 
3. 0.119 g 0.032 g (0.5 .mu.moles) 
______________________________________ 
The yields of the resulting cross-linked HSA/PGA/MS were as follows: (1) 
0.117 g, 78% (2) 0.134 g, 87% and (3) 0.124 g, 82%. The average diameter 
of the microspheres were 34, 29, and 29 .mu.m, respectively, as determined 
by optical microscopy. 
EXAMPLE 20 
Human Serum Albumin/Microspheres Containing Polyglutamic Acid-Quenched 
HSA/PGA/MS were synthesized as described in Example 19. The amount of added 
PGA was 11, 15 and 19 wt %. The HSA/PGA/MS were then quenched with glycine 
to "cap" residual aldehyde groups. Samples were prepared with the 
following weight ratios of HSA and PGA: 
______________________________________ 
HSA PGA 
______________________________________ 
1. 0.133 g 0.016 g (0.30 .mu.moles) 
2. 0.127 g 0.022 g (0.38 .mu.moles) 
3. 0.121 g 0.029 g (0.48 .mu.moles) 
______________________________________ 
The yields were as follows: (1) 0.115 g, 77% (2) 0.128 g, 85% and (3) 0.117 
g, 78%. The average diameter of the microspheres were 23, 28, and 28 
.mu.m, respectively, as determined by optical microscopy. 
Modification of Albumin Microspheres Cross-Linked with Tolylene 
2,4-Diisocyanate (TDI) 
EXAMPLE 21 
Bovine Serum Albumin with 14% Polyglutamic Acid-Unquenched 
BSA (0.131 g) and PGA (0.022 g) were combined and dissolved in 0.5 ml of 
water in a test tube. The albumin solution was dispersed in the PMMA 
mixture as described in Example 1. A 0.046 mmolar solution of TDI was 
prepared from 80% aqueous TDI and toluene. After mixing well, 1.0 ml of 
the solution was added to the albumin dispersion and mixed at r.t. for 12 
hrs with a rotary mixer. BSA/MA were washed to remove all of the PMMA 
dispersant with acetone and dehydrated as described in Example 1. The 
product was a white powder, 0.146 g, 95% yield. 
EXAMPLE 22 
Bovine Serum Albumin with 14% Polyglutamic Acid Microspheres-Quenched 
BSA (0.132 g) and PGA (0.021 g) were combined and dissolved in 0.5 ml of 
water in a test tube. The albumin solution was dispersed in the PMMA 
mixture, synthesized, washed and quenched as described in Example 18. The 
product was a white powder, 0.067 g, 44% yield. 
EXAMPLE 23 
Bovine Serum Albumin Microspheres Containing 11.7% Carboxymethyldextran 
(CMD)-Unquenched 
CMD was synthesized using the procedure of Pitha et al, J. Natl. Cancer 
Inst., Vol. 65, p. 5 (1980). Dextran (40,000 MW, Sigma) (5.0 g) was 
dissolved in 5.0 ml of water. This solution was added to 38 ml of 40% 
sodium hydroxide and 27 g of chloroacetic acid in a 125 ml Erlenmeyer 
flask. The suspension was stirred for 12.0 hrs at room temperature. After 
this process was repeated twice, the solution was extensively dialyzed 
against water using membrane tubing (Spectropor) with a 6,000-8,000 MW 
cutoff, inside diameter of the tubing was 25.5 mm. The modified dextran 
was frozen in liquid nitrogen and lyophilized. Yield of product was not 
recorded. The carboxylic content was 4.4 .mu.moles of carboxyl groups per 
mg of material. 
BSA (0.135 g) and CMD (0.018 g) were combined and dissolved with 0.5 ml of 
water in a test tube. The protein/dextran solution was dispersed in the 
PMMA mixture and the CMD-BSA/MS were synthesized as described above. The 
product was a white powder, 0.092 g, 60% yield. 
EXAMPLE 24 
Bovine Serum Albumin Microspheres Containing 15% 
Carboxymethyldextran-Quenched 
BSA (0.139 g) and CMD (0.024 g) were combined and dissolved in 0.5 ml of 
water in a test tube. The protein/dextran solution was dispersed in the 
PMMA mixture and the CMD-BSA/MS were synthesized as described in Example 
23. The CMD-BSA/MS were washed to remove all of the PMMA dispersant, 
quenched and dehydrated as described in Example 18. The product was white 
powder, 0.049 g, 30% yield. 
The incorporation of PGA into HSA/MS increased anionicity. For applications 
(e.g. experimental treatment of Brucellosis in cattle) involving large 
scale production of anionic/MS, the use of PGA might be limited because of 
its high cost. A viable alternative to PGA is carboxymethyldextran (CMD). 
The modified dextran is inexpensive and has the physical properties (high 
content of functional carboxyl groups) required to increase anionicity of 
the albumin/MS. 
EXAMPLE 25 
18 wt % Adriamycin-Human Serum Albumin/Microspheres-Unquenched 
Adriamycin in HCL (AD) (52.3 mg) (Farmitalia Carlo ERBA) was dissolved in 
25.0 ml of water, 5.0 ml of the clear dark red solution (10.46 mg AD) was 
combined with 9.99 mg of the HSA/MS-U (synthesized in Example 1) in a 
screw cap test tube. The pH of the cloudy red mixture was adjusted from 
4.00 to 5.70 by the addition of 0.1N NaOH and mixed with a rotary mixer at 
4.degree. C. in the dark for 11 hrs. The mixture was centrifuged (2000 
RPM.times.2 mins) and the light red supernate was carefully removed with a 
pasteur pipet and saved for analysis. The dark red pellet was re-suspended 
in 10.0 ml of water, briefly stirred, centrifuged and the supernate saved 
for analysis. This was repeated five times. After the last wash, the 
AD-HSA/MS-U were dehydrated with acetone and allowed to air dry. The MS 
product was a dark red powder 11.6 mg, 97% yield, containing 18 wt % AD. 
In Vitro AD Release: Dynamic Column Elution Method 
A dynamic flow column was used to measure in vitro drug release: 2.0 ml of 
water was added to the dry AD-HSA/MS-U (11.61 mg), the resulting red 
slurry was pipeted into a 140 mm.times.7 mm glass column. The ends of the 
column were modified with chromatography caps packed with glass wool and 
attached to threaded zero-volume collectors that were connected to 1.0 mm 
I.D. Teflon tubing. Care was taken to ensure that all the AD-HSA/MS-U were 
transferred into the column. The column was then placed in a circulating 
water bath at 37.degree. C. Physiological saline was pumped through the 
column at 0.4 ml/min with an HPLC pump (ALTEX model 110A). Fractions were 
collected every 30 mins for 15 hrs at 4.degree. C. Wash in each fraction 
was analyzed at 480 nm by UV/VIS to determine the AD concentration eluted 
from the AD/HSA/MS-U. All subsequent dynamic flow in vitro release studies 
were performed as just described unless otherwise noted. See Table 8. 
TABLE 8 
______________________________________ 
AD Release From 18 wt % AD-HSA/MS-U (29 .mu.m) 
11.61 mg AD-HSA/MS-U (2.09 mg AD) 
Time (hrs) 
Drug Release 
2.0 4.0 6.0 8.0 10.0 12.0 14.0 
______________________________________ 
Wt AD (mg) 
0.40 0.09 0.03 0.01 0.01 0.0 0.0 
Cumulative 
0.40 0.49 0.52 0.53 0.54 0.54 0.54 
Wt AD (mg) 
% Released 
19 24 25 25 26 26 26 
______________________________________ 
EXAMPLE 26 
33 wt % Adriamycin-Polyglutamic Acid (12%)-Human-Serum 
Albumin/Microspheres-Unquenched 
A volume of 5.0 ml of the stock AD solution (10.46 mg AD) prepared in 
Example 28 was combined with 11.39 mg of PGA (12%)-HSA/MS-U (synthesized 
in Example 19) in a screw cap test tube. The pH of the cloudy red mixture 
was adjusted from 4.45 to 5.84 by the addition of 0.1N NaOH and mixed with 
a rotary mixer at 4.degree. C. in the dark for 11 hrs. The AD-PGA 
(12%)-HSA/MS-U were removed from the drug free solution and the bound 
concentration of AD was determined as described above. The product was a 
dark red powder, 15.6 mg, 91% yield. Concentration of the bound AD to PGA 
(12%)-HSA/MS-U was 5.57 mg or 33 wt %. 
In Vitro Release 
The in vitro release of the free AD from AD-PGA 2%)/MS-U (15.6 mg) was 
measured and the results set forth in Table 9. 
TABLE 9 
______________________________________ 
AD Release from 33 wt % AD-PGA(11%)-HSA/MS-U (34 .mu.m) 
15.62 mg AD-PGA(11%)-HSA/MS-U (5.15 mg AD) 
Time (hrs) 
Drug Release 
2.0 4.0 6.0 8.0 10.0 12.0 14.0 
______________________________________ 
Wt AD (mg) 
1.11 0.41 0.16 0.08 0.08 0.07 0.05 
Cumulative 
1.11 1.52 1.68 1.76 1.84 1.91 1.96 
Wt AD (mg) 
% Released 
22 30 33 34 36 37 38 
______________________________________ 
EXAMPLE 27 
39 wt % Adriamycin-Polyglutamic Acid (16%)-Human Serum 
Albumin/Microspheres-Unquenched 
A volume of 5.0 ml of the stock AD solution (10.46 mg AD) prepared in 
Example 25 was combined with 10.40 mg of PGA (16%)-HSA/MS-U (synthesized 
in Example 19) in a screw cap test tube. The pH of the cloudy red mixture 
was adjusted from 4.32 to 5.80 by the addition of 0.1N NaOH and mixed with 
a rotary mixer in the dark at 4.degree. C. for 11 hrs. 
AD-PGA(16%)-HSA/MS-U were washed out of the free drug solution, and 
concentration of the bound AD was determined as described above. The 
product was a dark red powder, 11.1 mg, 65% yield. Concentration of the 
bound AD was 6.70 mg or 39 wt %. 
In vitro Release 
The in vitro release results of free AD from AD-PGA(16%)-HSA/MS-U (11.1 mg) 
are set forth in Table 10. 
TABLE 10 
______________________________________ 
AD Release From 39 wt % AD-PGA(16%)-HSA/MS-U (29 .mu.m) 
11.08 mg AD-PGA(16%)-HSA/MS-U (4.33 mg AD) 
Time (hrs) 
Drug Release 
2.0 4.0 6.0 8.0 10.0 12.0 14.0 
______________________________________ 
Wt AD (mg) 
0.85 0.29 0.19 0.14 0.12 0.11 0.09 
Cumulative 
0.85 1.14 1.33 1.47 1.59 1.70 1.79 
Wt AD (mg) 
% Released 
20 26 31 34 37 39 41 
______________________________________ 
EXAMPLE 28 
46 wt % Adriamycin-Polyglutamic Acid(22%)-Human Serum 
Albumin/Microspheres-Unquenched 
A volume consisting of 5.0 ml of the stock AD solution (10.46 mg AD) 
prepared in Example 25 was combined with 10.1 mg of PGA(22%)-HSA/MS-U 
(synthesized in Example 19) in a screw cap test tube. The pH of the cloudy 
red mixture was adjusted from 4.43 to 5.80 by the addition of 0.1N NaOH 
and mixed with a rotary mixer in the dark at 4 C for 11 hrs. 
AD-PGA(22%)-HSA/MS-U were washed out of the free drug solution, dehydrated 
and concentration of the bound AD was determined as described above. The 
product was a dark red powder 14.7 mg, 78% yield. Concentration of the 
bound AD was 8.61 mg or 46 wt %. 
In Vitro Release 
The release of the free AD from the AD-PGA(22%)-HSA/MS-U (14.7 mg) produced 
in this procedure was measured and the results set forth in Table 11. 
TABLE 11 
______________________________________ 
AD Release from 46 wt % AD-PGA(22%-HSA/MS-U (29 .mu.m) 
8.41 mg AD-PGA(22%)-HSA/MS-U (3.86 mg AD) 
Time (hrs) 
Drug Release 
2.0 4.0 6.0 8.0 10.0 12.0 14.0 
______________________________________ 
Wt AD (mg) 
1.01 0.24 0.26 0.14 0.11 0.08 0.07 
Cumulative 
1.01 1.25 1.51 1.65 1.76 1.84 1.91 
Wt AD (mg) 
% Released 
26 32 39 43 16 48 50 
______________________________________ 
EXAMPLE 29 
18 wt % Adriamycin-Human Serum Albumin/Microspheres-Quenched 
AD (50.5 mg), was dissolved in 25.0 ml of water in a volumetric flask. A 
volume consisting of 5.0 ml of the clear dark red solution (10.1 mg AD) 
was combined with 10.7 mg of HSA/MS-Q (Example 18) in a screw cap test 
tube. The pH of the cloudy red mixture was adjusted from 2.99 to 5.83 by 
the addition of 0.1N NaOH and mixed with a rotary mixer at 4.degree. C. in 
the dark for 11 hrs. AD-HSA/MS-Q were removed from the free AD solution, 
washed, and dehydrated as described in Example 28. The wash was saved for 
analysis. The product was dark red powder, 7.1 mg, 54% yield. 
In Vitro Release 
The release of free AD from the AD-HSA/MS-Q (7.1 mg) produced in this 
procedure was measured and the results set forth in Table 12. 
TABLE 12 
______________________________________ 
AD Release from 18 wt % AD-HSA/MS-Q (26 .mu.m) 
7.08 mg AD-HSA/MS-Q (1.28 mg AD) 
Time (hrs) 
Drug Release 
2.0 4.0 6.0 8.0 10.0 12.0 14.0 
______________________________________ 
Wt AD (mg) 
0.68 0.21 0.12 0.06 0.03 0.03 0.01 
Cumulative 
0.68 0.89 1.01 1.07 1.10 1.13 1.14 
Wt AD (mg) 
% Released 
53 70 77 82 85 87 88 
______________________________________ 
EXAMPLE 30 
21 wt% Adriamycin-Polyglutamic Acid (11%)-Human Serum 
Albumin/Microspheres-Quenched 
A volume consisting of 5.0 ml of the stock AD solution (10.1 mg AD) 
prepared as above was combined with 11.0 mg of PGA (11%)-HSA/MS-Q 
(synthesized in Example 20) in a screw cap test tube. The pH of the cloudy 
red mixture was adjusted from 3.46 to 5.81 by the addition of 0.1N NaOH 
and mixed with a rotary mixer at 4.degree. C. in the dark for 11 hrs. 
AD-PGA (11%)-HSA/MS-Q were washed out of the free AD solution, dehydrated 
and concentration of the bound drug was determined as above. The product 
was a dark red powder, 10.88 mg, 82% yield. Concentration of the bound AD 
was 2.83 mg or 21 wt %. 
In vitro Release 
The release of the free AD from the AD-PGA(11%)-HSA/MS-Q (10.9 mg) was 
measured and the results set forth in Table 13. 
TABLE 13 
______________________________________ 
AD Release from 21 wt % AD-PGA (11%)-HSA/MS-Q (23 .mu.m) 
10.88 mg AD-PGA (11%)-HSA/MS-Q (2.23 mg AD) 
Time (hrs) 
Drug Release 
2.0 4.0 6.0 8.0 10.0 12.0 14.0 
______________________________________ 
Wt AD (mg) 
1.30 0.21 0.10 0.04 0.01 0.02 0.01 
Cumulative 
1.29 1.50 1.60 1.64 1.65 1.67 1.68 
Wt AD (mg) 
% Released 
58 67 72 73 74 75 75 
______________________________________ 
EXAMPLE 31 
25 wt% Adriamycin-Polyglutamic Acid (15%)-Human Serum 
Albumin/Microspheres-Quenched 
A volume consisting of 5.0 ml of the stock AD solution (10.1 mg AD) 
prepared as above was combined with 9.9 mg of PGA (15%)-HSA/MS-Q 
(synthesized in Example 20) in a screw cap test tube. The pH of the cloudy 
red mixture was adjusted from 3.59 to 5.84 by the addition of 0.1N NaOH 
and mixed with a rotary mixer at 4.degree. C. in the dark for 11 hrs. 
AD-PGA(15%)-HSA/MS-Q were washed out of the free AD solution, dehydrated 
and concentration of the bound drug was determined as described above. The 
product was dark red powder, 5.7 mg, 43% yield. Concentration of the bound 
AD was 3.33 mg or 25 wt %. 
In vitro Release 
The release of the free AD from the AD-PGA(15%)-HSA/MS-Q (5.7 mg) was 
measured and the results set forth in Table 14. 
TABLE 14 
______________________________________ 
AD Release from 25 wt % AD-PGA (15%)-HSA/MS-Q (28 .mu.m) 
5.70 mg AD-PGA (15%)-HSA/MS-Q (1.43 mg AD) 
Time (hrs) 
Drug Release 
2.0 4.0 6.0 8.0 10.0 12.0 14.0 
______________________________________ 
Wt AD (mg) 
0.77 0.08 0.02 0.02 0.02 0.02 0.01 
Cumulative 
0.77 0.85 0.87 0.89 0.91 0.93 0.94 
Wt AD (mg) 
% Released 
54 59 61 62 64 65 66 
______________________________________ 
EXAMPLE 32 
33 wt % Adriamycin-Polyglutamic Acid (19%)-Human Serum 
Albumin/Microspheres-Quenched 
A volume consisting of 5.0 ml of the stock adriamycin solution (10.1 mg AD) 
prepared as above was combined with 10.1 mg of PGA (19%)-HSA/MS-Q 
(synthesized in Example 20) in a screw cap test tube. The pH of the cloudy 
red mixture was adjusted from 3.71 to 5.84 by the addition of 0.1N NaOH 
and mixed with a rotary mixer at 4.degree. C. in the dark for 11 hrs. 
AD-PGA (19%)-HSA/MS-Q were washed out of the free AD solution, dehydrated 
and concentration of the bound drug was determined as described above. The 
product was a dark red powder, 7.9 mg, 53% yield. Concentration of the 
bound drug was 4.9 mg or 33 wt %. 
In vitro Release 
The release of the free AD from the AD-PGA (19%)-HSA/MS-Q (7.9 mg) was 
measured and the results set forth in Table 15. 
TABLE 15 
______________________________________ 
AD Release from 33 wt % AD-PGA (19%)-HSA/MS-Q (24 .mu.m) 
7.88 mg AD-PGA (19%)-HSA/MS-Q (2.58 mg AD) 
Time (hrs) 
Drug Release 
2.0 4.0 6.0 8.0 10.0 12.0 14.0 
______________________________________ 
Wt AD (mg) 
0.77 0.20 0.14 0.09 0.08 0.07 0.07 
Cumulative 
0.77 0.97 1.11 1.20 1.28 1.35 1.42 
Wt AD (mg) 
% Total AD 
30 38 43 47 50 52 55 
______________________________________ 
EXAMPLE 33 
Ion Exchange Release Properties of Adriamycin-Albumin/Microspheres-Quenched 
AD was bound to (1) HSA/MS-Q (quenched with 2-aminoethanol), (2) HSA-MS-Q 
(quenched with glycine) and (3) PGA (15%)-HSA/MS-Q according to the 
procedure described above. MS were washed out of the free drug solution, 
dehydrated and concentration of the bound AD was determined as described 
above. After dehydration, the AD-MS had a yield of (1) 8.28 mg, 75%, (2) 
7.98 mg, 77% and (3) 9.50 mg, 57%. Concentration of the bound drug for 
each sample was (1) 1.4 mg or 14%, (2) 1.6 mg or 20% and (3) 4.8 mg or 
28%. 
In vitro Release 
The AD-albumin/MS samples were separately loaded in the release column as 
described above. Water was used as the mobile phase for the first 5.0 hrs, 
then exchanged for physiological saline for the remainder of the 
experiment. Release data is set forth in Tables 16, 17 and 18. 
TABLE 16 
______________________________________ 
AD Release from 14 wt % AD-HSA/MS-Q (aminoethanol) 
(25 .mu.m) 8.28 mg AD-HSA/MS-Q (1.16 mg AD) 
Time (hrs) 
Drug Release 
2.0 4.0 6.0 8.0 10.0 12.0 14.0 
______________________________________ 
Wt AD (mg) 
0.16 0.12 0.17 0.30 0.05 0.02 0.0 
Cumulative 
0.16 0.28 0.45 0.75 0.80 0.82 0.82 
Wt AD (mg) 
% Released 
14 24 39 65 69 71 71 
______________________________________ 
TABLE 17 
______________________________________ 
AD Release from 20 wt % AD-HSA/MS-Q (glycine) (25 .mu.m) 
7.98 mg AD-HSA/MS-Q (1.60 mg AD) 
Time (hrs) 
Drug Release 
2.0 4.0 6.0 8.0 10.0 12.0 14.0 
______________________________________ 
Wt AD (mg) 
0.14 0.12 0.38 0.18 0.08 0.08 0.08 
Cumulative 
0.14 0.26 0.64 0.82 0.90 0.98 1.06 
Wt AD (mg) 
% Released 
9 16 40 52 56 61 66 
______________________________________ 
TABLE 18 
______________________________________ 
AD Release from 28 wt % AD-PGA (15%)-HSA/MS-Q (28 .mu.m) 
9.54 mg AD-PGA (15%)-HSA/MS-Q (2.67 mg AD) 
Time (hrs) 
Drug Release 
2.0 4.0 6.0 8.0 10.0 12.0 14.0 
______________________________________ 
Wt AD (mg) 
0.08 0.03 0.23 0.79 0.25 0.13 0.12 
Cumulative 
0.08 0.11 0.34 0.13 1.38 1.51 1.63 
Wt AD (mg) 
% Released 
3 4 13 42 52 57 61 
______________________________________ 
EXAMPLE 34 
In vitro Release of Adriamycin from Albumin/Microspheres in a Static System 
PGA(10%)-HSA/MS-Q containing AD, synthesized as described above were washed 
out of the free AD solution, dehydrated and concentration of the bound AD 
was determined as described above. The product was a dark red powder, 6.1 
mg, 75% yield. The concentration of the bound drug was 2.6 mg or 31.7 wt 
%. 
In vitro Release 
The AD-PGA(10%)-HSA/MS (6.1 mg) were combined with 2.0 ml of physiological 
saline in a screw cap test tube and placed in a 37.degree. C. shaker water 
bath. Every 30 mins, the cloudy red mixture was centrifuged and 50 .mu.l 
was removed from the clear red supernate, diluted in 5.0 ml of 
physiological saline. The concentration of the released AD was then 
determined as previously described above. This procedure was repeated 
until the amount of release drug remained constant (i.e., no more AD 
released). The AD-PGA(10%)-HSA/MS-Q were then washed (5X) with 10.0 ml 
volumes of water to remove all free AD. After the last water wash was 
decanted, the MS were re-suspended with 2.0 ml of saline and the process 
duplicated for the remainder of the experiment (15 hrs.) 
EXAMPLE 35 
In Vivo Studies 
Toxicity of Adriamycin-Polyglutamic Acid (15%)-Human Serum 
Albumin/Microspheres-Quenched in CD-1 Mice 
AD-PGA(15%)-HSA/MS-Q were synthesized according to the procedure described 
above. The product was a dark red powder with 27 wt % bound AD and total 
yield of 76%. MS had a size range of 20-40 .mu.m. 
CD-1 white female mice, 5-7 weeks of age, weighing 30-33 g were injected by 
the intraperitoneal (i.p.) route with AD at concentrations of 0.6 mg and 
1.5 mg dissolved in 0.5 ml of sterile saline. A second group of animals 
were injected with AD-PGA(15%)-HSA/MS-Q with 0.6, 1.5, and 2.5 mg of bound 
AD. For each concentration of the free and bound drug, 5 mice were used. 
PGA(15%)-HSA/MS-Q without AD was used for a control group and injected in 
equivalent weight amounts. Animals were then observed over a two-month 
period and fatalities were recorded for each group and tabulated. See 
Table 19. 
TABLE 19 
______________________________________ 
Toxicity of AD-PGA-HSA/MS-U in CD-1 Mice 
by i.p. Injection 
Preparation Survival from Time of Administration 
and dose 7 days 14 days 21 days 
28 days 
35 days 
______________________________________ 
AD (0.6 mg) 5/5 1/5 1/5 1/5 1/5 
AD (1.5 mg) 5/5 0/5 -- -- -- 
AD-PGA-HSA/MS 
5/5 5/5 5/5 5/5 5/5 
(AD 0.6 mg) 
(2.22 mg MS) 
AD-PGA-HSA/MS 
5/5 5/5 5/5 5/5 5/2 
(AD 1.5 mg) 
(4.44 mg MS) 
AD-PGA-HSA/MS 
5/5 4/5 1/5 0/5 -- 
(AD 2.5 mg) 
(9.26 mg MS) 
PGA-HSA/MS 5/5 5/5 5/5 5/5 5/5 
(2.22 mg MS) 
PGA-HSA/MS 5/5 5/5 5/5 5/5 5/5 
(4.44 mg MS) 
PGA-HSA/MS 5/5 5/5 5/5 5/5 5/5 
(9.26 mg MS) 
______________________________________ 
EXAMPLE 36 
HSA/MS were readily prepared as described above containing up to 18 wt % of 
the anti-tumor drug adriamycin (AD). The binding of AD to HSA/MS involved 
two mechanisms; (1) the covalent attachment of the primary amine 
associated with AD to free reactive mono-dialdehydes on HS/MS-unquenched 
(U) and (2) physical binding of AD to HSA/MS-quenched (Q). 
Terry, R.N., M.S., Thesis, University of Florida (1980) demonstrated that 
PGA readily formed stoichiometric ionic salts with basic drugs such as AD. 
This is due to interactions between the anionic carboxyl groups associated 
with PGA and the cationic primary amine group located on the daunosamine 
ring of AD. Terry also found that by subjecting the PGA-AD complex to an 
appropriate electrolyte, the ionic salt would dissociate and release the 
AD. The addition of PGA to the HSA/MS made possible the preparation of 
AD-PGA-HSA/MS containing up to 45% of the drug through the formation of 
this AD-salt complex. This is demonstrated in the comparison of the 
binding data for AD in Table 20 for (HSA/MS-U and PGA-HSA/MS-U and Table 
21 for (HSA/MS-Q and PGA-HSA/MS-Q). 
TABLE 20 
______________________________________ 
Amount of Bound AD for Unquenched 
HSA/MS and PGA-HSA/MS 
Wt % of Added PGA 
Wt % of Bound AD 
______________________________________ 
0 18 
12 33 
16 39 
22 46 
______________________________________ 
TABLE 21 
______________________________________ 
Amount of Bound AD for Quenched 
HSA/MS and PGA-HSA/MS 
Wt % of Added PGA 
Wt % of Bound AD 
______________________________________ 
0 18 
11 21 
15 25 
19 33 
______________________________________ 
The data shows that as the concentration of PGA increases in the MS, the wt 
% of bound AD also increases. This data also shows that the amount of 
bound AD is higher for the MS-U than the MS-Q at comparable amounts of PGA 
and is attributable to the increased amount of AD that can be covalently 
bound to have HSA/MS-U than the physically bound AD to HSA/MS-Q. 
The ionic AD-salt complex is sensitive to pH. Adjusting the pH directly 
affected the amount of AD bound to the PGA-HSA/MS-Q. The optimum pH for 
binding was found to be 6.0 and corresponded to 32 wt % complexed drug. 
AD-HSA/MS were mounted in epoxy and serial section with a ultramicrotome, 
the sections were mounted on TEM grids and examined with an optical 
microscope. The red chromophor of AD could be seen throughout the slices 
and indicated that the drug had penetrated into the matrix of the HSA/MS 
as well as on the surface. 
It is a feature of the invention that the hydrophilic nature of HSA/MS 
allows for the incorporation of therapeutic agents to the albumin/MS after 
their synthesis. This is inherently different from prior art procedures 
which add the drugs to the aqueous phase before the dispersion process and 
the formation of the microspheres. The advantage of drug addition after MS 
synthesis are: (1) higher drug loadings and (2) ability to bind chemically 
sensitive drugs that otherwise may be affected during the formation of the 
MS. 
The in vitro AD release rates for the AD-albumin/MS were found to be 
readily controlled and due to the three distinct binding mechanisms of AD 
to albumin/MS; (1) slow-hydrolytic degradation of covalent bonds, (2) 
medium-dissociation of the drug salt complex, and (3) fast-release of 
physically adsorbed drug. 
For the unquenched MS (HSA/MS-U and PGA-HSA/MS-U) the amount of AD released 
in 15 hrs varied from 23% to 50%. Increasing the concentration of PGA in 
HSA/MS increased the amount of released AD. Since the PGA-AD salt complex 
dissociates faster than hydrolytic degradation of covalently bound AD, 
increasing the amount of incorporated PGA increases the rate of AD 
release. For AD-HSA/MS-Q and AD-PGA/MS-Q, the amount of AD released in 15 
hrs varied from 55% to over 80%. As the amount of PGA increases, the 
percent of the total amount of AD that is released decreases. Since the MS 
are quenched, the number of reactive aldehydes that are available to 
covalently bind AD are reduced. The predominate drug binding mechanism 
would then be by physical association and drug-salt formation. The 
physically adsorbed AD is released faster than salt dissociation. 
Therefore, as the amount of the PGA is increased in the MS, a higher 
percentage of the bound AD is associated with the salt complex. This 
causes a larger % of the released AD to be by salt dissociation which 
reduces the release rate. With respect to quenching, glycine would 
incorporate a higher amount of terminal carboxyl groups than the amino 
alcohol due to basic structural differences between the two molecules. 
Higher amounts of free carboxyl groups would allow higher concentrations 
of AD bound by salt formation. Because the salt complex is stable in 
water, the MS with the highest concentration of carboxyl groups 
(AD-PGA-HSA/MS-Q, COOH) would release the lowest amount of AD when water 
was used as the mobile phase through the dynamic flow column. AD-HSA/MS-Q 
(OH), would contain the least amount of carboxyl groups (albumin itself 
contains some of these groups) and therefore would release the highest 
amount of AD during the water phase. When the mobile phase is exchanged 
with saline, the salt dissociates releasing AD. Dynamic flow in vitro 
release rates do not accurately represent kinetic behavior in vivo, only 
animal models can determine that type of information. It does, however, 
allow comparisons to be made between drug carriers so that adequate 
evaluations can be made before expensive laboratory animal models are 
used. Furthermore, the dynamic column system is, at best, a representation 
of controlled drug delivery in the blood circulatory system due to the 
fast continuous flow. In actuality, MS implanted inside a solid tumor 
would not be subject to such a rapid turnover of fluid. The slow fluid 
turnover in the tumor can be better represented by a semi-static in vitro 
release model. In a static system, drug release from AD-HSA/MS would be 
regulated by the mechanisms described as well as the concentration of 
released AD in the AD-HSA/MS environment. As the drug is released into the 
closed system (i.e., tumor mass), a concentration level would be reached 
where further drug release would be inhibited. Drug seepage out of the 
tumor area would then reduce the concentration gradient to a point where 
more drug would then be released from AD-HSA/MS. 
In the toxicity study reported above, a dose level of 600 .mu.g of free AD 
killed over 80% of the animals tested. The same amount of AD now bound to 
PGA-HSA/MS resulted in no deaths. When AD-PGA-HSA/MS with 1500 g of bound 
AD were administered i.p., 60% of the mice survived. Another hazard with 
AD is the severe necrotic lesions that develop at the site of injection 
when there is drug leakage around the needle. These ulcers can take months 
to heal. All animals treated with free AD developed these ulcers. No ulcer 
development was seen with animals injected with the AD containing MS. 
Control groups consisting of PGA-HSA/MS did not demonstrate any noticeable 
toxic effects. 
An important characteristic of a successful drug implant is the ability of 
the drug-carrier to biodegrade once implanted and release of the 
therapeutic agent. Albumin/MS synthesized in this study were examined for 
these properties. It was found that highly cross-linked FTIC-BSA/MS that 
were injected into CD-1 mouse muscle tissue started to degrade by the 
fourth week and remained immobilized at the site of injection. By varying 
the cross-linking density, variations in the rate of degradation in vivo 
may be achieved. 
AD-HSA/MS were also implanted into CD-1 muscle tissue and examined after 
tissue removal. The AD molecule has natural fluorescent abilities and 
could be easily observed with the optical microscope under fluorescent 
light. Tissue samples observed after four weeks from day of injection 
still showed the red chromophor of AD in the surrounding tissue next to 
the immobilized AD-HSA/MS. 
This study demonstrates three important characteristics of the albumin/MS 
produced according to the invention. These are: (1) to remain localized at 
site of injection, (2) biodegradation after implantation and (3) drug 
releasing properties after injection. 
Preparation of Bleomycin-Albumin/Microspheres 
EXAMPLE 37 
Bleomycin Sulfate (BLM) was supplied in 10.0 ml sealed glass vials 
containing between 8 and 9 mg of a lyophilized white powder that was 
amorphous in texture. To measure the exact concentration of the BLM, the 
glass vials were opened in the exhaust hood and the drug dissolved by the 
addition of known volumes of water. The concentration was then determined 
with the UV/VIS at an adsorbance of 294 nm and using an extinction 
coefficient of 12.15 ml/mg (Windholz, M., et al, The Merck Index, Merck & 
Co., Inc. p. 171 (1976)). 
EXAMPLE 38 
30 wt % Bleomycin-Human Serum Albumin/Microspheres-Unquenched 
A stock solution of BLM was prepared containing 66.7 mg BLM in 25.0 ml of 
water. An aliquot consisting of 5.0 ml of the clear solution (13.3 mg BLM) 
was combined with 11.3 mg HSA/MS-U (synthesized as described in Example 1) 
in a screw cap test tube. The pH of the Brown cloudy mixture was adjusted 
from 4.23 to 5.97 by the addition of 0.1N NaOH and mixed with a rotary 
mixer in the dark at 4.degree. C. for 7 hrs. BLM-HSA/MS-U were washed free 
of the unbound BLM and supernate saved for analysis as described above. 
After the last wash, the MS were dehydrated with acetone and allowed to 
air dry. The MS product was a brown powder, 11.7 mg, 73% yield containing 
30 wt % drug. 
In vitro Release 
The release of free BLM from BLM/HSA/MS-U (11.7 mg) was performed using the 
in vitro dynamic flow column described above. See Table 22. 
TABLE 22 
______________________________________ 
BLM Release from 29 wt % BLM-HSA/MS-U (29 .mu.m) 
11.67 mg BLM-HSA/MS-U (3.50 mg BLM) 
Time (hrs) 
Drug Release 
2.0 4.0 6.0 8.0 10.0 12.0 14.0 
______________________________________ 
Wt BLM (mg) 
1.29 0.06 0.04 0.03 0.02 0.02 0.01 
Cumulative 1.29 1.35 1.39 1.42 1.44 1.46 1.47 
Wt BLM (mg) 
% Released 37 39 40 41 41 42 42 
______________________________________ 
EXAMPLE 39 
31 wt % Bleomycin-Polyglutamic Acid (9%)-Human Serum 
Albumin/Microspheres-Unquenched 
An aliquot consisting of 5.0 ml of the BLM stock solution (13.34 mg BLM) 
prepared as described above was combined with 12.7 mg of PGA (9%)-HSA/MS-U 
(synthesized as described above) in a screw cap test tube. The pH of the 
mixture was adjusted from 4.71 to 6.00 by the addition of 0.1N NaOH and 
mixed at 4.degree. C. in the dark for 7 hrs. The BLM-PGA(9%)-HSA/MS-U were 
removed from solution and concentration of the bound drug was determined 
as described above. The product was a brown powder, 13.15 mg, 72% yield. 
Concentration of the bound drug was 5.69 mg or 31 wt %. 
In vitro Release 
The in vitro release of free BLM from BLM-PGA-(9%)-HSA/MS-U (13.15 mg) was 
measured as described above. See Table 23. 
TABLE 23 
______________________________________ 
BLM Release from 31 wt % BLM-PGA (9%)-HSA/MS-U (27 .mu.m) 
13.15 mg BLM-PGA (9%)-HSA/MS-U (4.08 mg BLM) 
Time (hrs) 
Drug Release 
2.0 4.0 6.0 8.0 10.0 12.0 14.0 
______________________________________ 
Wt BLM (mg) 
2.32 0.09 0.06 0.03 0.04 0.02 0.0 
Cumulative 2.32 2.41 2.47 2.50 2.54 2.56 2.56 
Wt BLM (mg) 
% Released 57 59 61 61 62 63 63 
______________________________________ 
EXAMPLE 40 
30 wt % Bleomycin-Polyglutamic Acid (14%)-Human Serum 
Albumin/Microspheres-Unquenched 
An aliquot consisting of 5.0 ml of the stock BLM solution (13.34 mg BLM) 
prepared as above was combined with 12.8 mg of PGA(14%)-HSA/MS-U 
(synthesized as described above) in a screw cap test tube. The pH of the 
cloudy mixture was adjusted from 4.83 to 6.00 by the addition of 0.1N NaOH 
and mixed with a rotary mixer at 4.degree. C. in the dark for 7 hrs. 
BLM-PGA(14%)-HSA/MS-U were washed out of the free drug solution, 
dehydrated and concentration of the bound drug was determined as described 
in Example 38. The product was a brown powder, 13.8 mg, 75% yield. 
Concentration of the bound BLM was 5.56 mg or 30 wt %. 
In vitro Release 
The in vitro release of the free BLM from BLM-PGA(14%)-HSA/MS-U (13.8 mg) 
was measured as described above. See Table 24. 
TABLE 24 
______________________________________ 
BLM Release from 30 wt % BLM-PGA (14%)-HSA/MS-U (31 .mu.m) 
13.8 mg BLM-PGA (14%)-HSA/MS-U (4.14 mg BLM) 
Time (hrs) 
Drug Release 
2.0 4.0 6.0 8.0 10.0 12.0 14.0 
______________________________________ 
Wt BLM (mg) 
2.41 0.06 0.03 0.02 0.02 0.01 0.01 
Cumulative 2.41 2.47 2.50 2.52 2.54 2.55 2.56 
Wt BLM (mg) 
% Released 58 60 60 61 61 62 62 
______________________________________ 
EXAMPLE 41 
23 wt % Bleomycin-Human Serum Albumin/Microspheres-Quenched 
A stock solution of BLM was prepared containing 62.7 mg dissolved with 25.0 
ml of water in a volumetric flask. An aliquot consisting of 5.0 ml (12.54 
mg) was combined with 11.9 mg of HSA/MS-Q in a screw cap test tube. The pH 
of the mixture was adjusted from 3.95 to 5.95 by the addition of 1.0N NaOH 
and mixed with a rotary mixer at 4.degree. C. in the dark for 19 hrs. The 
BLM-HSA/MS-Q were removed from solution, dehydrated and concentration of 
bound drug was determined as described above. The product was a brown 
powder, 11.8 mg, 76% yield. Concentration of the bound drug was 3.58 or 23 
wt %. 
In vitro Release 
The in vitro release of the free BLM from BLM-HSA/MS-Q (11.81 mg) was 
measured and the results set forth in Table 25. 
TABLE 25 
______________________________________ 
BLM Release from 23 wt % BLM-HSA/MS-Q (29 .mu.m) 
11.81 mg BLM-HSA/MS-Q (2.71 mg) 
Time (hrs) 
Drug Release 
2.0 4.0 6.0 8.0 10.0 12.0 14.0 
______________________________________ 
Wt BLM (mg) 
1.16 0.04 0.02 0.02 0.01 0.0 0.0 
Cumulative 1.16 1.20 1.22 1.24 1.25 1.25 1.25 
Wt BLM (mg) 
% Released 43 44 45 46 46 46 46 
______________________________________ 
EXAMPLE 42 
20 wt % Bleomycin-Polyglutamic Acid(9%)-Human Serum 
Albumin/Microspheres-Quenched 
An aliquot consisting of 5.0 ml of the stock BLM solution (12.54 mg BLM) 
was combined with 13.9 mg of PGA(9%)-HSA/MS-Q in a screw cap test tube. 
The pH of the mixture was adjusted from 3.68 to 5.98 by the addition of 
0.1N NaOH and mixed with a rotary mixer at 4.degree. C. in the dark for 19 
hrs. The BLM-PGA(9%)-HSA/MS were washed out of the free BLM solution, 
dehydrated and concentrations of the bound drug was determined as 
described above. The product was a brown powder, 16.2 mg, 84% yield. 
Concentration of the bound drug was 5.51 mg or 28 wt %. 
In vitro Release 
The release of the free BLM from BLM-PGA(9%)-HSA/MS-Q (16.20 mg) was 
measured and the results are set forth in Table 26. 
TABLE 26 
______________________________________ 
BLM Release from 28 wt % BLM-PGA (9%)-HSA/MS-Q (27 .mu.m) 
16.20 mg BLM-PGA (9%)-HSA/MS-Q (4.54 mg BLM) 
Time (hrs) 
Drug Release 
2.0 4.0 6.0 8.0 10.0 12.0 14.0 
______________________________________ 
Wt BLM (mg) 
2.53 0.05 0.02 0.01 0.01 0.0 0.0 
Cumulative 2.53 2.58 2.60 2.61 2.62 2.62 2.62 
Wt BLM (mg) 
% Released 56 57 57 57 58 58 58 
______________________________________ 
EXAMPLE 43 
29 wt % Bleomycin-Polyglutamic Acid (14%)-Human Serum 
Albumin/Microspheres-Quenched 
An aliquot consisting of 5.0 ml of the stock BLM solution (12.54 mg BLM) 
was combined with 12.1 mg of PGA(14%)-HSA/MS-Q in a screw cap test tube. 
The pH of the mixture was adjusted from 3.65 to 5.97 by the addition of 
0.1N NaOH and mixed with a rotary mixer at 4.degree. C. in the dark for 19 
hrs. The BLM-PGA(14%)-HSA/MS-Q were washed out of the free BLM solution, 
dehydrated and concentration of the bound drug was determined as described 
bove. The product was a brown powder, 13.2 mg, 78% yield. Concentration of 
the bound drug was 4.98 or 29 wt %. 
In vitro Release 
The release of the free BLM from BLM-PGA(14%)-HSA/MS-Q (13.21 mg) was 
measured and release results are set forth in Table 27. 
TABLE 27 
______________________________________ 
BLM Release from 29 wt % BLM-PGA (14%)-HSA/MS-Q (31 .mu.m) 
13.21 mg BLM-PGA (14%)-HSA/MS-Q (3.83 mg BLM 
Time (hrs) 
Drug Release 
2.0 4.0 6.0 8.0 10.0 12.0 14.0 
______________________________________ 
Wt BLM (mg) 
2.15 0.04 0.02 0.01 0.0 0.0 0.0 
Cumulative 2.15 2.19 2.21 2.22 2.22 2.22 2.22 
Wt BLM (mg) 
% Released 56 57 58 58 58 58 58 
______________________________________ 
EXAMPLE 44 
12 wt % Gentamycin-Human Serum Albumin/Microspheres-Unquenched 
A solution of GMC (gentamycin sulfate) was prepared containing 257.9 mg 
dissolved with 25.0 ml of water in volumetric flask. An aliquot consisting 
of 5.0 ml of the clear solution (51.58 mg) was combined with 50.5 mg of 
the dehydrated HSA/MS-U (synthesized as described in Example 1 in a screw 
cap test tube. The pH of the brown cloudy mixture was adjusted from 5.18 
to 5.70 by the addition of 0.1N NaOH and mixed at 4.degree. C. in the dark 
for 12 hrs. GMC-HSA/MS-U were washed free of the unbound GMC and supernate 
saved for analysis as described above. After the last wash, the MS were 
dehydrated with acetone and allowed to air dry. The MS product was a brown 
powder, 52.4 mg, 92% yield containing 12 wt % drug. 
Gentamycin sulfate (GMC) has an aminoglycoside structure with no detectable 
absorbance in either the ultraviolet or visable range. In order to 
quantitate the concentration of GMC for the drug binding experiments, a 
modification of the Barends et al [A. J. Chromatography, Vol. 222, pg. 316 
(1981)] procedure was used. 
EXAMPLE 45 
16 wt % Gentamycin-Polyglutamic Acid(17%)-Human Serum 
Albumin/Microspheres-Unquenched 
A solution of GMC was prepared containing 252.02 mg dissolved in 25.0 ml of 
water in a volumetric flask. An aliquot consisting of 10.0 ml of the clear 
solution (100.81 mg) was combined with 119.06 mg of PGA(17%)-HSA/MS-U in a 
screw cap test tube. The pH of the brown cloudy mixture was adjusted from 
5.30 to 5.74 by the addition of 0.1N NaOH and mixed with a rotary mixer at 
4.degree. C. in the dark for 12 hrs. GMC-PGA(17%)-HSA/MS-U were removed 
from the free drug solution by centrifugation, washed and dehydrated as 
described above. The supernate was saved for analysis. The MS product was 
a brown powder, 113.6 mg, 81% yield. 
EXAMPLE 46 
15 wt % Gentamycin-Polyglutamic Acid(19%)-Bovine Serum 
Albumin/Microspheres-Unquenched 
A solution of GMC was prepared containing 252.94 mg dissolved in 25.0 ml of 
water in a volumetric flask. An aliquot consisting of 10.0 ml of the clear 
solution (101.18 mg GMC) was combined with 127.72 mg of the dehydrated 
PGA(19%)-BSA/MS-U in a screw cap test tube. The pH of the brown cloudy 
mixture was adjusted from 5.12 to 5.83 by the addition of 0.1N NaOH and 
mixed with a rotary mixer at 4.degree. C. in the dark for 12 hrs. 
GMC-PGA(19%)-BSA/MS-U were removed from the free drug solution by 
centrifugation washed and dehydrated as described above. 
In vitro Release 
The in vitro release of GMC from GMC-PGA(19%)-BSA/MS-U was performed as 
described above. Concentration of the free drug in the collected fractions 
was analyzed as described in Example 44. Release data is set forth in 
Table 28. 
TABLE 28 
______________________________________ 
GMC Release from 18 wt % GMC-PGA (19%)-BSA/MS-U (18 .mu.m) 
16.37 mg GMC-PGA (19%)-BSA/MS-U (246 gm GMC) 
Time (hrs) 
Drug Release 
2.0 4.0 6.0 8.0 10.0 12.0 14.0 
______________________________________ 
Wt GMC (mg) 
1.66 0.40 0.16 0.01 0.0 0.0 0.0 
Cumulative 1.66 2.15 2.31 2.32 2.32 2.32 2.32 
Wt GMC (mg) 
% Released 66 86 94 95 95 95 95 
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EXAMPLE 47 
18 wt % Streptomycin-Polyglutamic Acid(14%)-Bovine Serum 
Albumin/Microspheres-Unquenched 
STM (259.6 mg) was dissolved with 25.0 ml of water, 5.0 ml of the clear 
solution (51.92 mg STM) was combined with 48.3 mg of PGA(14%)-BSA/MS-U 
prepared as described in Example 24 in a screw cap test tube. The pH of 
the cloudy white mixture was adjusted from 4.23 to 5.79 by the addition of 
0.1N NaOH and mixed with a rotary mixer at 4.degree. C. in the dark for 18 
hrs. The mixture was centrifuged (200 RPM.times.2 mins) and the clear 
supernate was removed and saved for analysis. The STM-PGA(14%)-BSA/MS-U 
were removed from the free drug solution, washed and dehydrated. The MS 
product was a white powder, 27.5 mg, 47% yield. 
Streptomycin sulfate (STM) has no detectable absorbance in either the 
ultraviolet or visable range. As with GMC, STM has to be modified in order 
to quantitate the amounts of STM bound STM to the MS. The manitol 
procedure by Grove and Randall in Assay Methods of Antibiotics a 
Laboratory Manual, Welch, H, and Martin Ibaneze, F, eds. Medical 
Encyclopedia, Inc., New York, N.Y., p. 34 (1975) was used. 
EXAMPLE 48 
23 wt % Streptomycin-Polyglutamic Acid(14%)-Bovine Serum 
Albumin/Microspheres-Quenched 
A volume of 5.0 ml of the stock STM solution (51.92 mg STM) was combined 
with 38.8 mg of the PGA(14%)-BSA-PGA/MS-Q prepared as described in Example 
22 in a screw cap test tube. The pH of the cloudy white mixture was 
adjusted from 4.32 to 5.86 by the addition of 0.1N NaOH and mixed with a 
rotary mixer in the dark for 4.degree. C. for 18 hrs. The 
STM-PGA(14%)-BSA/MS-Q were removed from the free drug solution and the 
bound STM was determined as described above. The product was a white 
powder, 34.8 mg, 70% yield. Concentration of bound STM was 11.28 mg or 23 
wt %. 
EXAMPLE 49 
12 wt % Streptomycin-Polyglutamic Acid(14%)-Bovine Serum 
Albumin/Microspheres-Quenched and Lyophilized 
A volume of 5.0 ml of the stock STM solution (51.92 mg STM) was combined 
with 39.7 mg of lyophilized PGA(14%)-BSA/MS-Q in a screw cap test tube. 
The pH of the cloudy white mixture was adjusted from 4.47 to 5.80 by the 
addition of 0.1N NaOH and mixed with a rotary mixer in the dark at 
4.degree. C. for 18 hrs. STM-PGA(14%)-HSA/MS-Q were washed out of the free 
STM solution, and concentration of the bound drug was 5.63 mg or 12 wt %. 
EXAMPLE 50 
13 wt % Streptomycin-Polyglutamic Acid(14%)-Bovine Serum 
Albumin/Microspheres-Quenched (Wet) 
A volume of 5.0 ml of the stock STM solution (51.92 mg STM) was combined 
with 3.0 ml of PGA(14%)-BSA/MS-Q (9.7 mg/ml) or 29.4 mg total MS, 
suspended in 3.0 ml of water, in a screw cap test tube. The combination of 
the two solutions (STM and MS) produced a final volume of 8.0 ml. The pH 
of the cloudy white mixture was adjusted from 5.10 to 5.91 by the addition 
of 0.1N NaOH and mixed with a rotary mixer in the dark at 4.degree. C. for 
18 hrs. STM-PGA(14%)-BSA/MS-Q were washed out of the free drug solution, 
and concentration of the bound drug was 4.39 mg or 13 wt %. 
EXAMPLE 51 
23 wt % Streptomycin-Carboxymethyldextran(12%)-Bovine Serum 
Albumin/Microspheres-Quenched 
STM (255.2 mg) was dissolved in 25.0 ml of water, 5.0 ml of the clear 
solution (51.04 mg STM) was combined with 58.4 mg of CMD(12%)-BSA/MS-Q in 
a screw cap test tube. The pH of the cloudy white mixture was adjusted 
from 3.35 to 5.90 by the addition of 0.1N NaOH and mixed with a rotary 
mixer at 4.degree. C. in the dark for 18 hrs. MS were washed out of the 
free drug solution, dehydrated and concentration of the bound STM was 
determined. The product was a white powder, 60.3 mg, 85% yield. 
Concentration of the bound drug was 23 wt. %. 
The albumin protein and polypeptide microsphere compositions of this 
invention when loaded with biologically active agents are uniquely 
valuable for localization and controlled release of such agents, as for 
example, in localized cancer chemotherapy, local delivery of 
anti-inflammatory drugs, and for localized concentration of antibiotics. 
The microsphere compositions of this invention are also useful in adjuvant 
systems as carriers and active agents in stimulating the immune system. 
Currently used adjuvants (such as Freund's adjuvant) for immunostimulants 
and vaccines are often highly irritating, produce undesirable fever or 
highly inflammatory reactions and are short-lived in activity due to 
metabolic deterioration. Adjuvant compositions in which microspheres of 
this invention are utilized, for example by loading with specific antigens 
(i.e., tumor-specific antigens) or non-specific immune stimulants such as 
BCG constituents of MDP (myramyl dipeptide) afford the advantages of 
prolonged activity and greater stability as well as increased efficacy and 
mimimal toxic side effects. 
Macrophage are the active cellular agents of the immune defense system of 
animals and humans and digest or destroy foreign cells or substances and 
dead cellular material. In some diseases, macrophage and other cells are 
invaded but do not attack the viral or parasitic invaders. Suprisingly, 
the microspheres of this invention have been found to be readily ingested 
by macrophage by phagocytic uptake. Consequently they are uniquely 
suitable as carriers for bioactive agents which can attack the dormal 
viral or parasitic cells or can activate microphage. They are, therefore, 
especially useful as macrophage activation compositions and for treatment 
of diseases, particularly parasitic or viral, in which the disease agent 
remains hidden and dormant within host cells for prolonged periods. 
The microsphere compositions of this invention are also valuable for 
diagnostic medical and biochemical analyses. When modified appropriately 
with such substances as polypeptides, antibodies, antigens, enzymes, 
enzyme substrates, and radiolabeled or modified with fluorescent 
compounds, they may be readily designed to provide highly sensitive 
systems for radioimmune assays or fluorescence assays to detect a wide 
range of disease conditions. Important among such diagnostic tests are 
assays to detect cancer and venereal disease as well as parasitic, viral 
fungal and bacterial infections. The microporous, particulate, hydrophilic 
nature of the microspheres of this invention make them readily modifiable 
for such diagnostic applications by covalent attachment or physical 
association of specific-binding biological substances. Biospecific 
affinity chromatography, i.e., for separation of specific antibodies, 
antigens, enzymes and other metabolites, is similarly also an important 
application for the hydrophilic protein and polypeptide microspheres of 
this invention.