Compositions and methods for maintaining reservoirs of bioactive agents by sequestering the reservoir in a gel matrix are described. In particular, liposomes containing an entrapped bioactive agent are sequestered in a gel matrix. The resulting liposome-gel compositions may be used in vivo or in vitro to provide for sustained release of the bioactive agent. The gel matrix inhibits the dispersion and clearance of the sequestered liposomes without interfering with the ability of the liposomes to release the entrapped bioactive agent. Furthermore, the rate of release of the bioactive agent from the liposome-gel compositions may be varied by altering the composition of the liposomes and/or gels.

TABLE OF CONTENTS 
1. Field of the Invention 
2. Background of the Invention 
2.1. Liposomes 
2.2. Polymer Matrices and Gels 
3. Summary of the Invention 
4. Detailed Description of the Invention 
4.1. Preparation of Liposomes 
4.2. Gel Matrices 
4.3. Bioactive Agents 
4.4. Release of Bioactive Agent 
4.5. Use of the Liposome-gel Preparation in Living Systems 
5. Example: Liposomes in Methylcellulose 
5.1. Preparation of SPLVs and Methylcellulose Gel 
5.2. Subcutaneous Administration of the SPLV-Methylcellulose Preparation 
5.3. Intramuscular Administration of the SPLV-Methylcellulose Preparation 
6. Example: Liposomes in Agarose 
6.1. Preparation of SPLVs and Agarose Gel 
6.2. Intraperitoneal Administration of the SPLV Agarose Preparation 
6.3. Intramuscular Administration of the SPLV-Agarose Preparation 
7. Example: Liposomes in Collagen 
7.1. Preparation of SPLVs and Collagen Gel 
7.2. Intramuscular Administration of the SPLV-Collagen Preparation 
7.3. Release of SPLV-entrapped Agent from the Site of Inoculation 
7.4. Subcutaneous Administration and Release of SPLV-entrapped Agent from 
the Site of Inoculation 
1. FIELD OF THE INVENTION 
The invention describes compositions and methods for maintaining and 
immobilizing a reservoir of a biologically active agent which provides for 
the sustained release of the biologically active agent in living systems. 
According to the present invention, a biologically active agent is 
entrapped in liposomes which are sequestered in a gel matrix. When used in 
living systems the liposomes sequestered in the gel matrix provide for 
prolonged release of liposome entrapped agents and the gel matrix provides 
for immobilization of the liposomes. 
According to one embodiment of the present invention, the liposome-gel 
compositions of the present invention can be implanted in vivo to provide 
for the prolonged release of the entrapped bioactive agent to the host 
organism. When administered in vivo, the gel matrix provides for 
protection of the liposomes from rapid clearance without interfering with 
release of the liposome entrapped agent. In another embodiment of the 
present invention, the liposome-gel composition may be used as a support 
or overlay for cells grown in culture and thus provide for the prolonged 
release of the entrapped bioactive agent into the culture medium. 
2. BACKGROUND OF THE INVENTION 
2.1. Liposomes 
Liposomes are lipid vesicles which can entrap a variety of pharmaceutical 
agents and can be used for delivery of these agents to cells and tissues 
in vivo. A multitude of liposomes can be constructed from one or more 
lipids such that they are small unilamellar vesicles (SUV), large 
unilamellar vesicles (LUV), oligolamellar vesicles prepared by reverse 
phase evaporation (REV), or multilamellar vesicles (MLV). See review by 
Deamer and Uster, 1983, "Liposome Preparation: Methods and Mechanisms," 
1983, in Liposomes, Ostro, ed., Marcel Dekker, Inc., New York, pp. 27-51. 
Much has been written regarding the possibilities of using liposomes for 
drug delivery systems. See for example, the disclosures in U.S. Pat. No. 
3,993,754, U.S. Pat. No. 4,145,410. In liposome delivery systems the 
medicament is entrapped in the liposome which is administered to the 
patient to be treated. See U.S. Pat. No. 4,224,179 and U.S. Pat. No. 
4,235,871. 
Aqueous suspensions of liposomes may be inoculated in any desired way 
(e.g., intravenously, intramuscularly, intraperitoneally, etc.). However, 
after their inoculation, most of the liposomes are dispersed from the site 
of inoculation, and either degraded or endocytosed by phagocytic cells 
such as polymorphonuclear and mononuclear leucocytes, and macrophages 
(Poste, 1983, Biol. Cell, 47: 19-38). Thus, the release of entrapped drug 
from liposomes is limited to the period of time between inoculation and 
degradation or clearance of liposomes from body fluids. 
Sustained drug release characteristics can be ascribed to other types of 
drug microcarriers such as lipid microvesicles (microreservoirs) described 
by Sears, U.S. Pat. No. 4,298,594. 
2.2. Polymer Matrices and Gels 
Polymer matrices and gels have been used to localize delivery or retard 
dispersion of drugs from the site of administration in vivo. Harris et 
al., 1980, J. Pharm. Sci., 69: 1271-1273, used cross-linked starch gel for 
localized delivery of prostaglandin E2. Cotes et al., 1980, incorporated 
human growth hormone into a 16% partially hydrolysed gelatin solution 
which was subcutaneously injected into animals in an attempt to extend the 
period of elevated plasma hormone concentration (J. Endocrinol. 
87:303-312). More recently, Morimoto et al., 1983, demonstrated enhanced 
absorption of insulin when the peptide was incorporated into polyacrylic 
acid aqueous gel bases containing various long chain fatty acids 
(Internatl. J. Pharm. 14:149-157). 
A variety of other polymeric compounds have been utilized to provide 
sustained-release drug delivery systems, including: silicone elastomers of 
two types, i.e., the matrix type wherein a powdered drug is dispersed 
uniformly in a solid phase elastomer, and a membrane type wherein a 
reservoir of drug is enclosed within a layer of silicone elastomer 
(Wadsworth and Ratnasooriya, 1981, J. Pharmacol. Methods 6:313-320; see 
also Cheesman et al., 1982, Fertil. and Sterl. 38:475-481); 
polymethacrylate or silastic polymers impregnated with progesterone 
(Ainsworth and Wolynetz, 1982, J. Am. Sci. 54:1120-1127); co-polymers of 
lactic acid and glycolic acids which provide controlled release of 
levonorgestrel for six months to one year (Pitt et al., 1981, Natl. Inst. 
Drug Abuse Res. Monogr. Ser., 28:232-253; Wise et al., 1980, J. Pharm. 
Pharmacol. 32: 399-403); a fibrin excipient that enables controlled 
release of biochemical agents (Brown et al. in U.S. Pat. No. 4,393,041); 
anti-inflammatory and analgesic gel compositions (Noda et al. in U.S Pat. 
No. 4,393,076; and protective gel compositions for wounds (Mason et al. in 
U.S. Pat. No. 4,393,048). 
3. SUMMARY OF THE INVENTION 
This invention describes compositions and methods for maintaining 
reservoirs of bioactive agents by sequestering the reservoir in a gel 
matrix. More particularly, liposomes containing bioactive agents are 
sequestered in a gel matrix which is administered in vivo or in vitro. The 
gel matrix inhibits both dispersion of the liposomes in vivo or in vitro 
and clearance of the liposomes in vivo without blocking (1) the diffusion 
into the gel of body fluids or culture media which interact with the 
liposome bilayer; (2) the ability of liposomes to release the entrapped 
agent; or (3) the diffusion of the released agent through the gel to the 
surrounding environment. 
Although incorporation of a bioactive agent directly into a gel matrix may 
provide for a certain degree of sustained-release, entrapment of a 
bioactive agent in liposomes can provide for a more prolonged release of 
the agent because the liposome membrane can be prepared or modified to 
further retard the leak of the entrapped agent. However, because liposomes 
themselves are degraded or cleared when administered in vivo, it is 
difficult to achieve prolonged release of a liposome-entrapped agent in 
vivo. The present invention is based upon the discovery that sequestering 
a liposome preparation in a gel matrix, as described herein, protects the 
liposomes from clearance but does not impair the ability of the liposomes 
to release their contents slowly. 
4. DETAILED DESCRIPTION OF THE INVENTION 
According to one embodiment of the present invention, a suspension of 
liposomes which entrap a biologically active agent is mixed with a 
suspension of the gel material. The resulting mixture can be administered 
in vivo to form a gel at the site of administration; alternatively, the 
preparation can be allowed to gel before administration. Either method of 
administration results in sequestering the liposomes in the gel matrix at 
the site of injection; the resistance of the liposomes to clearance or 
degradation; and the release over a period of time of the 
liposome-entrapped agent at the site of administration. 
In another embodiment of the present invention the liposome-gel preparation 
may be used in cell or tissue culture systems to provide for the prolonged 
release of the bioactive agent into the culture medium. The liposome-gel 
preparation may serve as a support for cell adhesion and growth; 
alternatively the liposome-gel preparation may be applied to the cell 
culture as an overlay. 
The rate of release of the entrapped bioactive agent is dependent on the 
type of liposomes used and the composition of the liposome membranes. In 
fact populations of different liposomes may be sequestered in the gel 
matrix. 
Any type of bioactive agent that can be entrapped in a liposome may be used 
according to the present invention. Examples of these are listed infra. In 
fact, two or more bioactive agents entrapped in the same or different 
populations of liposomes may be sequestered in a gel matrix for use 
according to the method of the present invention. Finally, one bioactive 
agent may be entrapped in the liposomes, and the same or a different 
bioactive agent may be contained in the gel matrix. When this liposome-gel 
preparation is administered, the bioactive agent contained in the gel 
matrix is released quickly whereas the bioactive agent entrapped in the 
sequestered liposomes is released slowly. Thus, when one bioactive agent 
is entrapped in both the sequestered liposomes and in the gel matrix one 
dose may provide for both the initial dose of the agent and for its 
sustained release, thereby avoiding the necessity of administering 
maintenance doses. Alternatively, when one bioactive agent is entrapped in 
the sequestered liposomes and another bioactive agent is entrapped within 
the gel matrix, concurrent therapy using any mixture of bioactive agents 
is possible. The subsections below are illustrative of the types of 
liposomes, gels and bioactive agents which may be used in the practice of 
the present invention. 
4.1. Preparation of Liposomes 
Liposomes used in the present invention can be prepared by a number of 
methods, including but not limited to: the original methods of Bangham et 
al. (1965, J. Mol. Biol. 13:238-252) which yield MLVs; SUVs as described 
by Papahadjopoulos and Miller (1967, Biochem. Biophys. Acta. 135:624-638); 
REVs as described by Papahadjopoulos in U.S. Pat. No. 4,235,871; and LUVs 
as described by Szoka and Papahadjopoulos in 1980, Ann. Rev. Biophys. 
Bioeng, 9:467-508; as well as methods described in U.S. patent application 
Ser. No. 476,496 by Lenk et al., filed Mar. 24, 1983 which issued as U.S. 
Pat. No. 4,522,803 yield stable plurilamellar vesicles (hereinafter 
referred to as SPLVs); and methods described in U.S. patent application 
Ser. No. 521,176 by Fountain et al. filed Aug. 8, 1983 which yield 
monophasic vesicles (hereinafter referred to as MPVs). The procedures for 
the preparation of SPLVs and MPVs are described below. 
SPLVs are prepared as follows: an amphipathic lipid or mixture of lipids is 
dissolved in an organic solvent. Many organic solvents are suitable, but 
diethyl ether, fluorinated hydrocarbons and mixtures of fluorinated 
hydrocarbons and ether are preferred. To this solution are added an 
aqueous phase and the active ingredient to be entrapped. This biphasic 
mixture is converted to SPLVs by emulsifying the aqueous material within 
the solvent and evaporating the solvent. Evaporation can be accomplished 
during or after sonication by any evaporative technique, e.g., evaporation 
by passing a stream of inert gas over the mixture, by heating, or by 
vacuum. The volume of solvent used must exceed the aqueous volume by a 
sufficient amount so that the aqueous material can be completely 
emulsified in the mixture. 
In practice, a minimum of about 3 volumes of solvent to about 1 volume of 
aqueous phase may be used. In fact, the ratio of solvent to aqueous phase 
can vary up to 100 or more volumes of solvent to 1 volume aqueous phase. 
The amount of lipid must be sufficient so as to exceed that amount needed 
to coat the emulsion droplets (about 40 mg of lipid per ml of aqueous 
phase). The upper boundary is limited only by the practicality of 
cost-effectiveness, but SPLVs can be made with 15 gm of lipid per ml of 
aqueous phase. 
Most amphipathic lipids may be constituents of SPLVs. Suitable hydrophilic 
groups include but are not limited to: phosphato, carboxylic, sulphato and 
amino groups. Suitable hydrophobic groups include but are not limited to: 
saturated and unsaturated aliphatic hydro-carbon groups and aliphatic 
hydrocarbon groups substituted by at least one aromatic and/or 
cycloaliphatic group. The preferred amphipathic compounds are 
phospholipids and closely related chemical structures. Examples of these 
include but are not limited to: lecithin, phosphatidyl-ethanolamine, 
lysolecithin, lysophatidylethanolamine, phosphatidylserine, 
phosphatidylinositol, sphingomyelin, cardiolipin, phosphatidic acid and 
the cerebrosides. Specific examples of suitable lipids useful in the 
production of SPLVs are phospholipids which include the natural lecithins 
(e.g., egg lecithin or soybean lecithin) and synthetic lecithins, such as 
saturated synthetic lecithins (e.g., dimyristoylphosphatidylcholine, or 
dipalmitoylphosphatidylcholine or distearoyl-phosphatidylcholine) and 
unsaturated synthetic lecithins (e.g., dioloylphosphatidylcholine or 
dilinoloyl-phosphatidylcholine). The SPLV bilayers can contain a steroid 
component such as cholesterol, coprostanol, cholestanol, cholestane and 
the like. When using compounds with acidic hydrophilic groups (phosphato, 
sulfato, etc.) the obtained SPLVs will be anionic; with basic groups such 
as amino, cationic liposomes will be obtained; and with polyethylenoxy or 
glycol groups neutral liposomes will be obtained. The size of the SPLVs 
varies widely. The range extends from about 100 nm to about 10,000 nm (10 
microns) and usually about 100 nm to about 1,500 nm. The SPLVs are 
characterized by a few to over 100 lipid bilayers enclosing aqueous 
compartments. 
The following is an example of the proportions that may be used in SPLV 
synthesis: SPLVs may be formed by adding 50 micromoles of phospholipid to 
5 ml of diethyl ether containing 5 micrograms of butylatedhydroxytoluene 
(BHT) and then adding 0.3 ml of aqueous phase containing the active 
substance to be encapsulated. The resultant mixture which comprises the 
material to be entrapped and the entrapping lipid is sonicated while 
streaming an inert gas over the mixture thus removing most of the solvent. 
Another suitable liposome preparation which may be used is lipid vesicles 
prepared in a monophasic solvent system, hereinafter referred to as 
monophasic vesicles or MPVs. MPVs are particularly stable and have a high 
entrapment efficiency. MPVs are prepared by a unique process as follows: a 
lipid or a mixture of lipids and an aqueous component are added to an 
organic solvent or a combination of organic solvents in amounts sufficient 
to form a monophase. The solvent or solvents are evaporated until a film 
forms. Then an appropriate amount of aqueous component is added, and the 
film is resuspended and agitated in order to form the MPVs. 
The organic solvent or combination of solvents used in the process must be 
(1) miscible with water and (2) once mixed with water should solubilize 
the lipids used to make the MPVs. 
For example, an organic solvent or mixture of solvents which satifies the 
following criteria may be used in the process: (1) 5 ml of the organic 
solvent forms a monophase with 0.2 ml of aqueous component and (2) the 
lipid or mixture of lipids is soluble in the monophase. 
Solvents which may be used include but are not limited to ethanol, acetone, 
2-propanol, methanol, tetrahydrofuran, glyme, dioxane, pyridine, diglyme, 
1-methyl-2-pyrrolidone, butanol-2, butanol-1, isoamyl alcohol, 
isopropanol, 2-methoxyethanol, or a combination of chlorform methanol 
(e.g., in a 1:1 ratio). 
The evaporation should be accomplished at suitable temperatures and 
pressures which maintain the monophase and facilitate the evaporation of 
the solvents. In fact, the temperatures and pressures chosen are not 
dependent upon the phase-transition temperature of the lipid used to form 
the MPVs. The advantage of this latter point is that heat labile products 
which have desirable properties can be incorporated in MPVs prepared from 
phospholipids such as distearoylphosphatidylcholine, which can be formed 
into conventional liposomes only at temperatures above the 
phase-transition temperature of the phospholipids. The process usually 
allows more than 30-40% of the available water-soluble material to be 
entrapped during evaporation and 2-15% of the available water-soluble 
material to be entrapped during the resuspension; and up to 70-80% of the 
available lipid-soluble material can be entrapped if the lipid:drug ratio 
is increased significantly. With MLVs the entrapment of aqueous phase, 
which only occurs during the rehydration step since no aqueous phase is 
present during the drying step, usually does not exceed 10%. 
Most lipids may be constituents of MPVs. Suitable hydrophilic groups 
include but are not limited to: phosphato, carboxylic, sulphato and amino 
groups. Suitable hydrophobic groups include but are not limited to: 
saturated and unsaturated aliphatic hydrocarbon groups and aliphatic 
hydrocarbon groups substituted by at least one aromatic and/or 
cycloaliphatic group. The preferred amphipathic compounds are 
phospholipids and closely related chemical structures. 
Specific examples of suitable lipids useful in the production of MPVs are 
phospholipids which include but are not limited to the natural lecithins 
or phosphatidylcholines (e.g., egg lecithin or soybean lecithin) and 
synthetic lecithins, such as saturated synthetic lecithins (e.g., 
dimyristoylphosphatidylcholine or dipalmitoylphosphatidylcholine or 
distearoylphosphatidylcholine) and unsaturated synthetic lecithins (e.g., 
dioleoylphosphatidylcholine or dilinoleoylphosphatidylcholine). Other 
phospholipids include but are not limited to phosphatidylethonolamine, 
lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, 
phosphatidylinositol, sphingomyelin, cardiolipin, phosphatidic acid, 
ceramides and the cerebrosides. The MPV bilayers can contain a steroid 
component such as cholesterol, coprostanol, cholestanol, cholestane and 
the like. When using compounds with acidic hydrophilic groups (phosphato, 
sulfato, etc.) the obtained MPVs will be anionic; with basic groups such 
as amino, cationic liposomes will be obtained. 
MPVs may advantageously be used in delivery systems wherein a bioactive 
agent is entrapped within the MPV ("entrapped" is defined as entrapment 
within the aqueous compartment or within the membrane bilayer). In order 
to entrap one or more agents in MPVs, the agent or agents may be added to 
the monophase prior to evaporation and formation of the film. 
Alternatively, the agent or agents may be added with the aqueous component 
used to resuspend the film and form the MPVs. In fact, to obtain a high 
entrapment efficiency, the agent or agents may be added to both the 
monophase and to the aqueous component used to resuspend the film. Two or 
more agents can also be entrapped in one MPV preparation by adding one 
agent to the monophase and the other to the aqueous component used to 
resuspend the film. 
4.2. Gel Matrices 
Any type of gel matrix may be used in the present invention. The only 
constraints are (1) the gel matrix must be capable of seqestering the 
liposomes; i.e., the pores of the gel matrix must be of the appropriate 
size relative to the size of the liposomes in order to sequester the 
liposomes in the gel; (2) when used in vivo the gel matrix must be 
compatible with the recipient organism (i.e., the level of toxicity should 
be kept to a minimum so as not to outweigh the beneficial effects of 
administering the bioactive agent in the liposome-gel preparation); (3) 
the gel must be capable of forming a gel or of remaining gelled at the 
temperatures and conditions of the environment in which it is administered 
or applied. For example the gel must remain gelled in the body fluids and 
at the temperatures to which it is exposed in vivo. Similarly, when used 
in cell or tissue culture the gel must remain gelled in the culture media 
and at the incubation temperatures used. Those skilled in the art can 
appreciate that the gel will degrade with the passage of time, especially 
when applied in vivo; however, once the sustained delivery of bioactive 
agent has substantially been accomplished, degradation and metabolism of 
the gel matrix by the host organism is a desirable result. 
Any gel can be used in the practice of the present invention. The materials 
which can be used to form such gels include but are not limited to: 
carbohydrates such as cellulosics, methylcellulose, starch and modified 
starch, agarose, gum arabic, ghatti, karay, tragacanth, guar, locust bean 
gum, tamarind, carageenan, alginate, xanthan, chickle, collagen, 
polyacrylamide, polysiloxanes (polyanhydrides, e.g., malic anhydride 
copolymers, polyacrylates, e.g., hydroxyethylpolymethycrylate 
polymethylmethacrylate, polyethylethacrylate polymethacrylate, 
ethylenevinylacetate copolymers, ethylenevinylalcohol copolymers, 
polyorthoesters, .epsilon.-caprolactones, amino acid polymers such as 
gelled albumin, amino acid polymers and copolymers and gelatins, and other 
organic or inorganic polymers which can be mixed with liposomes in vitro. 
After the mixture forms a gel the resulting liposome-gel matrix can be 
implanted in tissues. In a particularly useful embodiment of the present 
invention soft gel matrices such as agarose, collagen and the like 
containing sequestered liposomes may be injected in vivo. 
Alternatively, gels such as methylcellulose can be formed in the tissues 
after inoculation of liposomes in a suspension containing the gel 
material. After inoculation the suspension forms a gel and the liposomes 
remain sequestered in the gel matrix rather than dispersed and cleared. 
Regardless of the method used for preparing and implanting the gel matrix, 
the release of a liposome entrapped bioactive agent is prolonged and the 
relative concentration of the agent at the site of inoculation is 
increased. 
4.3. Bioactive Agents 
Virtually any bioactive agent can be entrapped within the liposomes for use 
according to the present invention. Such agents include but are not 
limited to antibacterial compounds, antiviral compounds, antifungal 
compounds, anti-parasitic compounds, tumoricidal compounds, proteins, 
toxins, enzymes, hormones, neurotransmitters, lipoproteins, glycoproteins, 
immunoglobulins, immunomodulators, dyes, radiolabels, radio-opaque 
compounds, fluorescent compounds, polysaccharides, cell receptor binding 
molecules, anti-inflammatories, antiglaucomic agents, mydriatic compounds, 
anesthetics, nucleic acids, polynucleotides, etc. 
In fact, if concurrent therapy is desired, two or more bioactive agents may 
be entrapped in one liposome population which is sequestered in the gel 
matrix. Alternatively, two or more liposome populations (of the same or 
different types of liposomes, e.g. mixtures of SPLVs, MPVs, SUVs, LUVs, 
REVs, etc.) which each entrap the same or different bioactive agents may 
be sequestered in the gel matrix. 
In yet another embodiment of the present invention the gel can be used as a 
vehicle for the same or different bioactive agents than those entrapped by 
liposomes. 
In certain therapeutic applications it may be desired to deliver a 
relatively high dose of a drug compound (i.e., compound A) followed by a 
sustained dose of the same or another compound (i.e., compound B). 
According to the present invention, this is readily accomplished by 
entrapping compound B in liposomes, sequestering the liposomes in a gel 
matrix containing compound A, and administering the same in vivo in a 
single inoculation. Thus, rapid delivery of compound A by diffusion from 
the gel, and slow sustained delivery of compound B by release from the 
liposomes is effected 
4.4. Release of Bioactive Agent 
The release of the bioactive agents may be controlled by the type of 
liposomes used and the membrane composition of the liposome bilayers as 
well as by the type and porosity of the gels used. The rate of release is 
also dependent upon the size and composition of the bioactive agent 
itself. 
The liposome itself is the first rate limiting factor in the release of 
entrapped bioactive agents. The rate of release may depend upon the number 
of bilayers, the size of the liposomes and most importantly the bilayer 
composition. 
A number of researchers add "stabilizers" such as sterols, cholesterols and 
the like to the phospholipid bilayers in order to alter the permeability 
of the liposome (Papahadjopoulos, D., Kimilberg, H. K., 1974, in Progress 
in Surface Science, ed. S. G. Davison, pp. 141-232, Oxford: Pergamon; 
Demel, R. A., Bruckdorf, K. R., Van Deenan, L. L., 1972, Biochem. Biophys. 
Acta, 255:331-347). For the present invention it is important that the 
stable liposomes will release their contents upon contact with body fluids 
or culture media. For a particularly useful liposome preparation, see U.S. 
patent application Ser. No. 516,268, by Fountain, filed July 22, 1983, 
which describes liposome preparations containing a titratable agent 
incorporated into the membrane which allows for a controlled release of 
the liposome entrapped agent. Thus the rate of release may be controlled 
by modifying liposome membranes accordingly. 
The gel matrix may be the second rate limiting factor in the release of the 
bioactive agent. In general, for low molecular weight bioactive agents 
(e.g., approximately 2,000 Daltons or less in molecular weight) the 
porosity of the gel matrix will not be relevant to the rate of release of 
bioactive agent because in most cases these agents of low molecular weight 
will freely diffuse through any gel. For example, most antibiotic 
compounds will freely diffuse through gel matrices of the present 
invention. In this situation the composition of the liposome membrane will 
be more important in determining the rate of release of entrapped agent. 
On the other hand, pore size of the gel may become a rate limiting factor 
in diffusion of the bioactive agent which is released from the sequestered 
liposomes when the bioactive agent is of a greater molecular weight. 
Generally, polyacrylamide gels exclude molecules of 10.sup.6 Daltons or 
larger in molecular weight. The pore size of a polyacrylamide gel depends 
upon the concentration of acrylamide used to make the gel (generally 4 to 
20% acrylamide is used to prepare these gels). The pore size can be varied 
further by the extent of crosslinking of the gel. If the molecular weight 
of the liposome-entrapped bioactive agent is known, one skilled in the art 
could prepare a gel to obtain the approximate diffusion rate desired by 
controlling the acrylamide concentration and crosslinking of the gel. 
Soft gels such as methylcellulose, collagen and agarose can be used to 
control the diffusion of larger molecules that are excluded by 
polyacrylamide gel (e.g., molecules greater than or equal to 10.sup.6 
Daltons in molecular weight) and thus may be used to control the release 
of bioactive agents of still larger molecular weight. As previously 
explained, the upper limit on pore size of the gel must be determined with 
a view to sequestering the liposomes. It should be noted that the use of 
the soft gels is not limited to the release of agents of larger moleculer 
weight. Soft gels could also be made to control the diffusion of smaller 
molecules as well. The porosity of soft gels is also controlled by its 
concentration as well as other factors. 
In addition to the parameters such as size of the bioactive agent and 
porosity of the gel which may be used to control the rate of diffusion of 
bioactive agents released from the sequestered liposomes, the nature of 
the bioactive agent itself and the gel will further affect the rate of 
diffusion. Thus, if the bioactive agent has any affinity for the gel 
matrix (e.g., affinity based upon charge, hydrogen bonding, van der Waals 
forces, etc.) diffusion through the gel of the bioactive agent released 
from the sequestered liposomes will be slowed. 
Finally, no matter what gel matrix is used to sequester liposomes 
containing an entrapped bioactive agent, the matrix will be freely 
permeable to fluids to which it is exposed, e.g., to tissue or body fluids 
or culture media except the molecules which have molecular weights higher 
than the permeability limit of the gel. Thus, the liposomes within the gel 
matrix will interact only with molecules which are able to diffuse through 
the gel matrix. This is important since the interaction of body fluids or 
culture media with the liposome membrane may be a significant factor in 
altering the permeability of the liposome membrane. 
4.5. Use of the Liposome-Gel Preparation in Living Systems 
The liposome-gel compositions of the present invention may be used for 
sustained delivery of a bioactive agent to cells and/or fluids in vivo and 
in vitro. A number of embodiments are discussed below. 
When used in vivo, the liposome-gel compositions of the present invention 
may be administered before or after gel formation. Routes of 
administration include but are not limited to: inoculation or injection, 
(e.g., intraperitoneal, intramuscular, subcutaneous, intra-aural, 
intra-articular, intra-mammary, etc.), topical application (e.g., on 
areas, such as eyes, ears, skin or on afflictions such as wounds, burns, 
etc.), and by absorption through epithelial or mucocutaneous linings (e.g. 
vaginal and other epithelial linings, gastrointestinal mucosa, etc.). 
For example the liposome-gel preparations of the present invention may be 
inoculated in vivo to provide for the sustained systemic release of the 
bioactive agent. Such applications may be particularly useful for the 
systemic release of drugs such as hormones (e.g., to control growth, 
fertility, sugar metabolism, etc.) or antimicrobials to control and treat 
infections, etc. 
In an alternative example, the liposome-gel preparation may be applied 
topically. Topical application may be particularly useful for the 
treatment of wounds (either surgical or non-surgical wounds) where the 
sustained release of antimicrobials and/or blood clotting factors may be 
helpful in the healing process. Similarly, the liposome-gel preparation 
may be topically applied to burns for the sustained release of 
antimicrobials and/or cell growth factors. The liposome-gel preparation 
may also be applied in the ear to treat infections by providing sustained 
release of antimicrobials; this would reduce the necessity of repeated 
applications of the bioactive agent in the form of ear drops. 
In another alternative embodiment, the liposome-gel preparation may be 
administered orally for sustained release. Such application may be useful 
for sustained release to oral epithelium and other oral tissues and for 
sustained release to epithelia of the alimentary tract. 
The liposome-gel preparations of the present invention may also be used in 
vitro to provide for sustained release of a bioactive agent into the cell 
or tissue culture medium. Such bioactive agents may include but are not 
limited to nutrients, drugs, hormones, growth factors, etc. The 
liposome-gel preparation may be used as a support for cell adhesion and 
growth; for instance, a liposome-collagen gel may be especially useful for 
culturing muscle cells, nerve cell, or liver cells. When the liposome-gel 
preparation is applied as an overlay, a liposome-agarose gel may be 
particularly useful.

The specific embodiments described above and below are given by way of 
example only and the invention is limited only by the appended claims. 
5. EXAMPLE: LIPOSOMES IN METHYLCELLULOSE 
5.1. Preparation of SPLVs and Methylcellulose Gel 
SPLVs containing radiolabeled gentamicin sulfate (.sup.125 I-GS/SPLVs) were 
prepared as described in Section 4.1: 100 mg egg phosphatidylcholine (egg 
PC) was added to 5 ml diethylether to which 0.3 ml phosphate buffered 
saline (PBS) containing .sup.125 I-p-hydroxyphenyl propionic acid 
derivatized gentamicin sulfate (.sup.125 I-GS) was added. The SPLVs were 
formed by sonicating the resultant mixture while evaporating the 
diethylether under a stream of nitrogen. The SPLVs were resuspended in 1 
ml PBS. 
A 2% solution of methylcellulose usually 400-2000 centipoise in PBS pH 7.2 
was prepared by mixing at 4.degree. C. until homogeneous, autoclaving at 
120.degree. C. and cooling for 24 hours at 4.degree. C. 
5.2. Subcutaneous Administration of the SPLV-Methylcellulose Preparation 
An aliquot of the 2% methylcellulose solution was mixed with an equal 
aliquot of the .sup.125 I-GS/SPLV suspension at room temperature. Aliquots 
(0.1 ml) of this mixture (the .sup.125 I-GS/SPLV-methylcellulose 
preparation) were inoculated subcutaneously in the abdominal region of 
adult Swiss Webster mice. Two groups of control mice were treated as 
follows: one group was inoculated subcutaneously in the abdominal region 
with 0.1 ml 1% methylcellulose (prepared as described in section 5.1) 
containing .sup.125 I-GS (i.e., the .sup.125 I-GS was not entrapped in 
SPLVs); the second group was inoculated subcutaneously in the abdominal 
region with 0.1 ml of the .sup.125 I-GS/SPLVs suspended in PBS (as 
prepared in section 5.1). Since methylcellulose forms a gel at 37.degree. 
C., shortly after subcutaneous inoculation of the methylcellulose 
preparations (within seconds) a semi-solid "bump" formed under the skin. 
This bump was visible or palpable for at least 24 hours. In contrast, the 
inocula of SPLVs suspended in PBS which were inoculated subcutaneously in 
the control group of mice did not result in the formation of any visible 
or palpable sign that lasted more than a few minutes. 
The subcutaneous immobilization of .sup.125 I-GS was verified by measuring 
the levels of .sup.125 I-GS in the area of inoculation. To this end, skin 
and underlying tissues around the site of inoculation (approx. 1.5 
cm.sup.2 of abdominal ventral wall) were excised 24 hours after 
inoculation and radioactivity was determined using a gamma counter. The 
results (Table I) indicated that indeed liposomes were immobilized at the 
site of inoculation by the methylcellulose gel matrix. 
TABLE 1 
______________________________________ 
RETENTION OF RADIOLABELED GENTAMICIN 
SULFATE AT THE SITE OF SUBCUTANEOUS 
INOCULATION IN MICE 
% Radiolabel 
Inoculum Remaining after 24 hours 
______________________________________ 
.sup.125 I-GS/SPLV-methylcellulose 
83.7 
.sup.125 I-GS/SPLVs in PBS 
15.0 
.sup.125 I-GS in methylcellulose 
3.5 
______________________________________ 
5.3. Intramuscular Administration of the SPLV-Methylcellulose Preparation 
In another similar experiment, mice were inoculated intramuscularly in the 
lower femural region of the leg with 0.05 ml of the .sup.125 
I-GS/SPLVs-methylcellulose preparation. The control mice were inoculated 
with an equivalent amount of the .sup.125 I-GS/SPLVs suspended in PBS. At 
intervals post inoculation the mice were sacrificed, the entire leg 
dissected and the radioactivity in the limb determined. The results shown 
in Table 2 demonstrate that the radioactivity decreased more rapidly in 
the legs of control mice than in the legs of mice which received the 
liposome-gel inoculation. 
TABLE 2 
______________________________________ 
RETENTION OF RADIOLABELED GENTAMICIN 
SULFATE AT THE SITE OF INTRAMUSCULAR 
INOCULATION IN MICE 
% Radiolabel 
Inoculum Remaining after 88 hours 
______________________________________ 
.sup.125 I-GS/SPLV-methylcellulose 
96.0 
.sup.125 I-GS/SPLV 46.8 
______________________________________ 
Thus, the clearance of .sup.125 I-GS is attenuated when liposomes 
containing this agent are sequestered in the methylcellulose gel matrix 
and administered intramuscularly. 
6. EXAMPLE: LIPOSOMES IN AGAROSE 
6.1. Preparation of SPLVS and Agarose Gel 
SPLVs were prepared as described in Section 5.1 using 100 mg egg PC and 0.3 
ml PBS containing .sup.125 I-GS or 0.3 ml HEPES buffer containing .sup.125 
I-human growth hormone (.sup.125 I-HGH, New England Nuclear). 
Solutions of 0.5%-2% agarose (Bio-rad standard low Mr) in water or buffer 
were prepared by melting the polymer powder at 100.degree. C., then 
sterilizing the solution at 20.degree. C. by autoclaving. After cooling, 
the resulting gel was melted at 60.degree. C. The temperature was then 
decreased to 42.degree. C. 
In order to sequester the liposomes and to inoculate the resulting 
gel-liposome preparation, the following procedure was used: 
One volume of agarose solution was mixed with one volume of SPLVs suspended 
in PBS buffer (pH 7.2). This suspension was immediately aspirated into a 
needle having a large internal diameter (1.5 mm) using a syringe adapted 
to the needle. The syringe was placed horizontally at 4.degree. C. to 
permit the agarose to gel. The gel containing liposomes is a cylinder (its 
volume depending on the internal diameter of the needle and the amount of 
aspirated solution) which can be easily extruded and inoculated. 
6.2. Intraperitoneal Administration of the SPLV Agarose Preparation 
Adult Swiss-Webster mice were inoculated intraperitoneally with .sup.125 
I-GS/SPLVs sequestered in 1% agarose gel (one gel cylinder/mouse). After 
24 hr, the mice were sacrificed, the gels were recovered and their 
radioactivity determined. The results showed that 95% of the initial 
radioactivity was associated with the recovered gels indicating that the 
liposomes were efficiently sequestered and maintained at the site of 
inoculation. 
6.3. Intramuscular Administration of the SPLV Agarose Preparation 
.sup.125 I-HGH/ SPLVs were sequestered in 0.5-2% agarose gels. Adult 
Swiss-Webster mice were inoculated intramuscularly in the leg with 0.1 ml 
of the .sup.125 I-GS/SPLV-agarose gel preparation. Groups of control mice 
were inoculated intramuscularly in the leg with 0.1 ml of .sup.125 
I-GS/SPLVs suspended in HEPES buffer, pH 7.2; agarose gels containing 
.sup.125 I-HGH (i.e., the .sup.125 I-HGH was not entrapped in SPLVs); or 
.sup.125 I-HGH in HEPES buffer. At intervals post-inoculation, the mice 
were sacrificed, the inoculated leg dissected and the residual 
radioactivity determined. The results are presented in Table 3. 
The results in Table 3 indicate that the in vivo retention of .sup.125 
I-HGH is prolonged when the hormone is entrapped in the SPLVs or when the 
hormone is sequestered in the agarose gel; however, the hormone is 
optimally retained at the site of inoculation when the liposomes 
containing the hormone are sequestered in a gel matrix. 
TABLE 3 
______________________________________ 
RETENTION OF GROWTH HORMONE AT THE 
INTRAMUSCULAR SITE OF INOCULATION 
% of .sup.125 I-Growth Hormone Remaining in Leg 
Hours Days 
Inoculum.sup.1 
3-5 24 2 3 7 14 28 
______________________________________ 
.sup.125 I-HGH 
5.0 0.7 ND 0.2 0.3 0.1 ND 
.sup.125 I-HGH/ 
81.0 57.0 ND 16.0 0.7 0.4 ND 
SPLVs 
.sup.125 I-HGH/ 
Agarose 
0.5% 26.0 20.0 14.0 16.0 14.0 14.0 8.0 
1.0% 36.0 38.0 30.0 29.0 26.0 19.0 9.0 
.sup.125 I-HGH/ 
SPLV 
Agarose 
0.5% 59.0 55.0 47.0 45.0 26.0 23.0 29.0 
1.0% 74.0 63.0 54.0 37.0 24.0 20.0 17.0 
2.0% 100.0 100.0 ND 100.0 53.0 38.0 ND 
______________________________________ 
.sup.1 Percentages indicate the final concentration of agarose in the 
liposomegel preparation. 
7. EXAMPLE: LIPSOMES IN COLLAGEN 
7.1. Preparation of SPLVs and Collagen Gel 
.sup.125 I-HGH/SPLVs were prepared as described in Section 5.1 using 100 mg 
egg PC and 0.3 ml HEPES buffer containing .sup.125 I-HGH. 
A gel of acid soluble rat tail collagen was prepared by a modification of 
the method described by Michalopoulous and Pitot, 1975, Exper. Cell Res. 
94:70-78. Briefly, 1-3 g of rat tail collagen fibers, dissected from 2 rat 
tails, was suspended in 300 ml of dilute solution of glacial acetic acid 
in water (1:1000), and stirred at 4.degree. C. for 48 hours. After 
allowing the mixture to settle for 24 hours, the clear solution of 
solubilized rat tail collagen was decanted from sedimented collagen 
fibers. Stock concentrations of acid-solubilized collagen of 0.3% or 0.9% 
(weight of fibers used per volume of acid solution) were stored cold until 
use. 
The liposomes were sequestered in the gel as follows: 
1.7 ml of acetic acid solubilized rat tail collagen (0.3-1%) was mixed with 
0.4 ml of a 2 to 1 mixture of 10.times.concentrated HEPES buffer (50 mM 
HEPES, 0.75M NaCl, 0.75M KCl) and 0.28M NaOH. Immediately after addition 
of buffer, an aliquot of .sup.125 I-HGH/SPLVs (0.1 ml to 0.2 ml) suspended 
in HEPES buffer was added to the solution which was mixed to ensure 
uniform distribution. This suspension was allowed to gel at 37.degree. C. 
for 1 hour. 
An experiment was done to determine the relationship between the amount of 
lipids (as liposomes) in gels and the sequestration ability of the gel. 
Accordingly a 0.2 ml aliquot of various liposome dilutions was added to 
the rat tail collagen in HEPES buffer as described above and allowed to 
gel at 37.degree. C. for 1 hour. Liposomes which were not sequestered in 
the gel were removed by filtration under vacuum through a nylon mesh 
filter (81.2 .mu. pore size; McMaster-Carr Supply Co., Dayton, NJ). 
Radioactivity in the gel collected on the filter was determined. Table 4 
illustrates that approximately 54-74% of the .sup.125 I-HGH/SPLVs could be 
sequestered in a 0.3% collagen gel. 
Table 4 also demonstrates that the quantity of liposomes which could be 
sequestered in the collagen gel might be increased by increasing the 
concentration of collagen in the gel to 0.9%. 
Another liposome preparation (Table 4) contained fibronectin covalently 
crosslinked to lipids. The glycoprotein fibronectin which has high 
affinity for collagen was covalently cross-linked to the liposome bilayer 
by an enzyme catalyzed method described in U.S. patent application Ser. 
No. 533,583 by Weiner et al., filed Sept. 19, 1983. Briefly, .sup.125 
I-HGH/SPLVs were prepared as described Section 5.1 using egg PC and 
phosphatidylethanolamine (8:2 mole %) and Tris (tris 
(hydroxymethyl)aminomethane) saline buffer containing .sup.125 I-HGH. In 
order to covalently link the fibronectin to the liposomes the .sup.125 
I-HGH/SPLV suspension was incubated for 2 hours at 37.degree. C. with 1 mg 
fibronectin (Seragen Inc., Boston, MA; or Collaborative Research Inc., 
Lexington, MA) in 1 ml Tris saline buffer with 20 mM CaCl.sub.2, 100 .mu.g 
Factor XIII (trans-glutaminase, Alpha Therapeutic Corp., Los Angeles, CA); 
1 unit thrombin (Sigma, St. Louis, MO). (One unit of thrombin will clot a 
25 mg % fibronectin solutions in 15 seconds at 37.degree. C.). After 
incubation the fibronectin modified-SPLVs (.sup. 125 I-HGH/FN-SPLVs) were 
pelleted at 10,000.times.g for 10 minutes, and washed 3 times. 
TABLE 4 
______________________________________ 
SEQUESTRATION OF LIPOSOMES IN A COLLAGEN 
GEL MATRIX 
% Radiolabel 
Sequestered 
Lipid Added to Gel 
% Collagen Within Gel 
______________________________________ 
.sup.125 I-HGH/SPLVs (0.2 ml) 
100.0 mg 0.3 54 
33.3 mg 0.3 57 
12.3 mg 0.3 74 
7.9 mg 0.3 72 
5.0 mg 0.3 66 
3.8 mg 0.3 71 
10.0 mg 0.9 81 
.sup.125 I-HGH/FN-SPLVs (0.2 ml) 
8.3 mg 0.3 86 
______________________________________ 
As illustrated in Table 4, when fibronectin-modified liposomes were added 
to the collagen gel, a significantly enhanced sequestration of liposomes 
was achieved. 
7.2. Intramuscular Administration of the SPLV-Collagen Preparation 
In order to determine the effect of liposome sequestration on the retention 
of growth hormone in tissues, adult Swiss Webster mice were inoculated 
intramuscularly in the leg with the .sup.125 I-HGH/SPLV or .sup.125 
I-HGH/FN-SPLV sequestered in 0.3% collagen gels (prepared as described 
above in Section 7.1). Control groups were inoculated with .sup.125 
I-HGH/SPLVs suspended in buffer, or .sup.125 I-HGH in buffer. At intervals 
post-inoculation, mice were sacrificed, the inoculated legs dissected and 
the residual radioactivity determined. 
The results (Table 5) show that the hormone is optimally retained at the 
site of inoculation when the liposomes containing the hormone are 
sequestered in the collagen gel matrix. Modification of the liposome 
membrane by the attachment of fibronectin, despite a greater sequestration 
of liposomes in collagen gel (see Table 4) did not significantly enhance 
retention of bioactive agent at the site of inoculation. However a more 
linear rate of release of .sup.125 I-growth hormone in this group was 
observed. 
TABLE 5 
______________________________________ 
RETENTION OF GROWTH HORMONE AT THE 
INTRAMUSCULAR SITE OF INOCULATION 
% of .sup.125 I Human Growth Hormone 
Remaining in leg.sup.a 
Hours Days 
Inoculum 3-5 24 3 7 14 
______________________________________ 
.sup.125 I-HGH 
5.0 1.0 0.2 0.3 0.1 
.sup.125 I-HGH/SPLVs 
81.0 57.0 16.0 1.0 0.4 
.sup.125 I-HGH/SPLVs- 
78.0 57.0 55.0 27.0 19.0 
Collagen 
.sup.125 I-HGH/FN-SPLVs- 
82.0 77.0 54.0 40.0 7.0 
Collagen 
______________________________________ 
.sup.a Mean values of three mice/group. 
In another experiment, sustained release of .sup.125 I-HGH from liposomes 
sequestered in collagen gel prepared using Vitrogen, a commercially 
available pepsin and acid digested bovine dermal collagen, was compared 
with that observed when the gel was prepared using the acid-solubilized 
rat collagen. 
Vitrogen, a product of the Collagen Corp., was obtained from Flow 
Laboratories (McLean, VA). A gel of Vitrogen (0.3%) was prepared according 
to manufacturer's instructions. The liposomes containing entrapped 
.sup.125 I-HGH were sequestered in the gel as decribed in Section 7.1. 
Adult Swiss Webster mice were inoculated intramuscularly in the leg with 
.sup.125 I-HGH/SPLV or .sup.125 I-HGH/FN-SPLV sequestered in 0.3% collagen 
gels prepared using either rat tail acid solubilized collagen or bovine 
(Vitrogen) collagen. Control groups were inoculated with .sup.125 
I-HGH/SPLV suspended in buffer or free .sup.125 I-HGH suspended in rat 
tail acid solubilized collagen gel. At intervals post-inoculation, mice 
were sacrificed, the inoculated legs dissected, and the residual 
radioactivity determined. 
As illustrated in Table 6, although sequestration of liposomes in Vitrogen 
gel enhanced retention of hormone at the site of inoculation at 7 days 
post-inoculation, this gel was not as effective as the acid solubilized 
rat tail collagen. Modification of the sequestered liposomes with 
fibronectin significantly enhanced retention of hormone at the site of 
inoculation with both forms of the gel at 1 day post-inoculation, but not 
at 7 days post-inoculation for the Vitrogen gel. 
TABLE 6 
______________________________________ 
RETENTION OF GROWTH HORMONE AFTER 
INTRAMUSCULAR INOCULATION: EFFECT OF 
VARIOUS LIPOSOME COLLAGEN MATRICES 
% of .sup.125 I Human Growth 
Hormone Remaining in leg.sup.a 
Days 
Inoculum 1 7 
______________________________________ 
.sup.125 I-HGH/Rat Tail 
1.0 0.2 
Collagen 
.sup.125 I-HGH/SPLVs 
57.0 1.0 
.sup.125 I-HGH/SPLVs- 
57.0 27.0 
Rat Tail Collagen 
.sup.125 I-HGH/FN-SPLVs- 
77.0 40.0 
Rat Tail Collagen 
.sup.125 I-HGH/SPLVs Vitrogen 
42.6 14.3 
.sup.125 I-HGH/FN-SPLVs- 
75.8 15.4 
Vitrogen 
______________________________________ 
.sup.a Mean values of five mice/group. 
7.3. Release of SPLV Entrapped Agent from Site of Inoculation 
An additional experiment was done to verify whether the growth hormone 
released in vivo from the liposomes sequestered in a collagen gel is in an 
active (functional) form. Bovine growth hormone (BGH) was entrapped in 
SPLVs (BGH-SPLV) prepared essentially as described in Section 5.1 at a 
ratio of 1.75 mg BGN/100 mg egg PC. A 0.25 ml aliquot of the BGH-SPLVs 
(containing 1.75 mg BGH) were sequestered in a 0.8% collagen gel (as 
described in section 7.1) which was then inoculated intramuscularly in 
female 39 days old Sprague Dawley rats which were hypophysectomized at 25 
days of age (Charles River Inc.). Control animals were not treated after 
hypophysectomy. The growth of the rats was determined daily and expressed 
in grams by subtracting the body weight at the time of inoculation from 
the actual weight post inoculation. The results (Table 7) show that the 
group of animals treated with the BGH/SPLV-collagen gel preparation gained 
weight steadily, indicating that the growth hormone was released from the 
collagen-sequestered liposomes and retained its activity. No gain in 
weight was observed in the control group of rats. 
TABLE 7 
______________________________________ 
WEIGHT GAIN IN HYPOPHYSECTOMIZED RATS 
Change in grams.sup.a 
Days 
Treatment 1 2 3 4 5 6 7 8 
______________________________________ 
Untreated 0 0 -0.8 -0.8 -0.2 0.5 -0.4 -0.9 
BGH/SPLV- 1.2 3.9 4.7 7.3 8.7 9.4 9.6 9.9 
Collagen 
Single Injection 
______________________________________ 
.sup.a Mean values of 8 rats/group. 
7.4. Subcutaneous Administration and Release of SPLV-Entrapped Agent From 
the Site of Inoculation 
SPLVs containing insulin (insulin/SPLVs) were prepared as described in 
Section 4.1: 100 mg dipalmitoyl phosphatidylcholine was dissolved in 5 ml 
diethyl ether. To this was added 0.3 ml of aqueous buffer (either PBS or 
0.01M Tris) at pH 7.4. containing 15 mg bovine insulin (25 unit/mg) (Sigma 
Chemical CO., St. Louis, MO). In order to solubilized the insulin in the 
aqueous buffer, it was necessary first to partition the hormone powder 
into a solution (50 mg/ml) of sonicated small unilamellar vesicles 
composed of EPC. Following solubilization, the aqueous droplet was 
emulsified into the ether phase by sonicating under a stream of nitrogen 
until the ether was completely evaporated. The lipid/insulin paste was 
rehydrated to form insulin/SPLVs. The insulin/SPLVs were washed three 
times in buffer containing 10 mm CaCl.sub.2. The CaCl.sub.2 facilitated 
pelleting of the insulin/SPLVs. Entrapment of insulin as determined by 
.sup.14 C-insulin label was 20-30%. 
Sustained release of insulin from insulin/SPLVs and insulin/SPLVs 
sequestered in collagen gel was examined in a diabetic animal model. 
Diabetes was developed in Sprague-Dawley rats, supplied by either Charles 
River Laboratories (Wilmington, MA) or Hilltop Laboratory Animals 
(Scottsdale, PA) by intraperitoneal injection of streptozotocin, 50 mg/kg 
on two consecutive days. Two weeks post-injection, diabetes was assessed 
by measuring water consumption, urine volume and urine glucose. Urine 
glucose was determined using a glucose assay kit No. 15-UV (Sigma Chemical 
CO., St. Louis, MO). 
Insulin/SPLVs were sequestered in collagen gel (0.9% collagen) as described 
in Section 7.1. Experimental animals received a single subcutaneous 
injection of insulin/SPLVs in the collagen gel in the hind limb 
corresponding to 4 mg insulin/kg body weight. Control animals received a 
single subcutaneous injection of either free insulin in buffer, free 
insulin in collagen gel, or free insulin/SPLVs in an amount equivalent to 
experimental animals. A minimum of five animals was used for each group. 
As illustrated in Table 8, urine glucose values were depressed in diabetic 
rats which received a single subcutaneous injection of either free 
insulin/SPLVs or insulin/SPLVs sequestered in collagen gel. In animals 
treated with insulin/SPLVs only, however, the maximum glucose depression 
(hence, greatest insulin release) was seen at 8 hours post-treatment. 
Glucose in urine began to rise again in these animals after 24 hours. On 
the other hand, in animals treated with insulin/SPLVs sequestered in 
collagen gel, maximum glucose depression was seen at 2 days 
post-treatment. A statistically significant greduction in urine glucose 
was still evident one week post-treatment. This indicates that 
sequestration of the liposomes in collagen gel impedes the release of 
insulin to the systemic circulation because no difference would be 
expected if the release of insulin was solely a liposome dependent 
phenomenon. 
It should be noted that free insulin subcutaneously injected was rapidly 
cleared (within 4 hours) from the systemic circulation (data not shown). 
TABLE 8 
______________________________________ 
SYSTEMIC RELEASE OF INSULIN AFTER SUB- 
CATANEOUS A ADMINISTRATION 
% Change In Urine Glucose From Untreated Diabetic Animals.sup.a 
Hours Days 
Animal.sup.b 4 8 1 2 7 
______________________________________ 
Normal (non-diabetic).sup.c 
5.8 10.3 10.4 5.3 24 
Untreated Diabetic 
123.9 144.5 128.4 222.0 
-- 
Insulin/SPLVs Diabetic 
63.4 27.6 52.4 104.8 
69.6 
Insulin/SPLVs-Collagen 
69.9 51.8 55.7 9.3 38.9 
Diabetic 
______________________________________ 
.sup.a Represents % change from value of urine glucose output (mg/dl) at 
time = 0. 
.sup.b Mean values of five rats/group. Rats injected with a single 
injection of free insulin in buffer cleared the hormone in 4 hours (data 
not shown). 
.sup.c Normal denotes nontreated nondiabetic animals included for 
comparison.