NZ247547A - Process for preparing heterovesicular lipid vesicles or liposomes; vesicles containing a chloride and an active agent - Google Patents

Description

New Zealand Paient Spedficaiion for Paient Number £47547 24 7 5 4 ? Priority Data(s): Complete Specification Filed: 3ass: ..test S5J Publication Da!o:..2 6 jAN ]ggg ^ O. ,Journal No: ft F* jk « 5 $$ -Ww Uncksf t!>s provisions of Fu 33 (1) %s«:Wicfisaof» has bean to.,.. .J.1L..mag£& v&£ tL.... 1/ tritsms Divided from No. 237464 dated 18 March 1991 No.: Date: NEW ZEALAND PATENTS ACT, 1953 COMPLETE SPECIFICATION HETEROVESICULAR LIPOSOMES „VWe, RESEARCH DEVELOPMENT FOUNDATION, a non-profit corporation of the State of Nevada, USA, of 402 North Division Street, Carson City, Nevada 89703, USA, hereby declare the invention for which t?We pray that a patent may be granted to^We, and the method by which it is to be performed, to be particularly described in and by the following statement:- (Followed by page la) 24 75 4 7 la Field of the Invention The present invention and/or the invention of New Zealand Patent Specification 237464 relates to the synthetic heterovesicular lipid vesicles or liposomes, processes for their manufacture and encapsulation of various materials therein, and treatment of patients with them.

Background Art Multivesicular liposomes are one of the three main types of liposomes, first made by Kim, et al. (1983, Biochim, Biophys. Acta 782, 339-348), and are uniquely different from the unilamellar (Huang, 1969, Biochemistry 8,344-352; Kim, et al. 1981, Biochim. Biophys. Acta 646, 1-10) and multilamellar (Bangham, et al. 1965, J. Mol, Bio. 13,238-252) liposomes in that there are multiple non-concentric aqueous chambers within. Previously described techniques for producing liposomes relate to the production of non-multivesicular liposomes; for example, U.S. Patent Nos. 4,522,803 - Lenk, 4,310,506 - Baldeschwieler, 4,235,871 - Papahadjopoulos, 4,224,179 - 4,078,052 -Papahadjopoulos, 4,394,372 - Taylor, 4,308,166 - Marchetti, 4,485,054 - Mezei, and 4,508,703 - Redziniak. For a comprehensive review of various methods of liposome preparation, refer to Szoka, et al. 1980, Ann. Rev. Biophys. Bioeng. 9:467-508.

Heterovesicular liposomes are lipid vesicles or liposomes with multiple internal aqueous chambers where at least two substances of different compositions are each encapsulated in separate chambers within one liposomes. The lipid vesicles or liposomes with multiple internal aqueous 24 7 547 chambers include, but are not limited to, multilamellar liposomes, stable paucilamellar liposomes, and multivesicular liposomes. It is highly advantageous to provide a liposome delivery system in which two or more 5 different substances are each encapsulated in separate compartments of a single liposome rather than encapsulated together in each compartment of the liposome.

Summary of the Invention The composition of the present invention comprises 10 heterovesicular liposomes, i.e. lipid vesicles or liposomes with multiple internal aqueous chambers where two or more substances of different compositions are each encapsulated separately in different chambers within one liposome, and at least one, but not two,of said substances is biologically active.

Briefly, the method of the invention comprises 15 making a "water-in-lipid" emulsion by dissolving amphipathic lipids in one or more organic solvents for the first lipid component, adding an immiscible first aqueous component including a substance to be encapsulated, preferably in the presence of hydrochloric acid, and then emulsifying the 20 mixture mechanically. In the emulsion, the water droplets suspended in the organic solvent will form- the internal aqueous chambers, and the monolayer of amphipathic lipids lining the aqueous chambers will become one leaflet of the bilayer membrane in the final product. A second lipid 25 component is then formed by dissolving amphipathic lipids in a volatile organic solvent and adding an immiscible second aqueous component including a second substance to be encapsulated, preferably in the presence of hydrochloric acid. A second emulsion is then created. A chimeric 3 0 emulsion is then formed by combining the first and second emulsions. The chimeric emulsion consists of multiple water droplets suspended in organic solvent where the substances of two different compositions are each dissolved separately in different aqueous droplets. The chimeric emulsion is 3 5 then immersed in a third aqueous immiscible component preferably containing one or more nonionic osmotic agents RECFfvrn 247547 and acid-neutralizing agent of low ionic strength and then mechanically dividing it to form solvent spherules suspended in the third aqueous component. The solvent spherules contain multiple aqueous droplets where the substances of two different compositions are each dissolved separately in different aqueous droplets within a single solvent spherule. The volatile organic solvent is evaporated from the spherules preferably by passing a stream of gas over the suspension. When the solvent is completely evaporated, the spherules convert into heterovesicular liposomes with multiple internal aqueous chambers where two substances of different compositions are encapsulated separately in different chambers within one liposome.

The use of hydrochloric acid with a neutralizing agent, or other hydrochlorides which slow leakage rates is preferably for high encapsulation efficiency and for a slow leakage rate of encapsulated molecules in biological fluids and in vivo. It is also preferable to use neutralizing agent of low ionic strength to prevent solvent spherules from sticking to each other.

In a first aspect the present invention provides a heterovesicular lipid vesicle or liposome where different substances are encapsulated separately in different chambers of the vesicle or liposome.

In a further aspect the present invention provides a heterovesicular liposome containing a biologically active substance encapsulated in the presence of hydrochloric acid or other hydrochlorides.

In a further aspect the present invention provides a heterovesicular liposome containing a biologically active substance encapsulated in 1 » I > ► 24 75 4 the presence of hydrochloric acid or other hydrochlorides and a neutralizing agent.

In a still further aspect the present invention provides methods of producing such heterovesicular lipid vesicles or liposomes.

In a further aspect the present invention provides processes for producing such heterovesicular lipid vesicles or liposomes by providing a first lipid component dissolved in one or more organic solvents and adding to the lipid component an immiscible first aqueous component containing a first substance to be encapsulated, forming a first water in oil emulsion from the first two immiscible components, providing a second lipid component dissolved in one or more organic solvents and adding into the lipid component an immiscible second aqueous component containing a second substance to be encapsulated, forming a second water in oil emulsion from the second two immiscible components, forming a chimeric emulsion by combining the first water in oil emulsion and second water in oil emulsion, transferring and immersing the chimeric emulsion into a third immiscible aqueous component,, dispersing the chimeric emulsion to form solvent spherules containing multiple droplets of the first aqueous component containing the first substance and the second aqueous component containing the second substance, and evaporating the organic solvent from the solvent spherules to form the heterovesicular lipid vesicles or liposomes.

In a still further aspect the present invention provides such a process in which a variety of hydrophilic biologically active materials and can be encapsulated separately in chambers of the heterovesicular lipid vesicles or liposomes.

Other and further features and advantages of the invention appear throughout the specification and claims.

Brief Description of the Drawings Figures 1-8 are schematic diagrams illustrating preparation of a heterovesicular vesicle or liposome.

Description of Preferred Embodiments The term "multivesicular liposomes" as used throughout the specification and claims means man-made, microscopic lipid-vesicles consisting of lipid bilayer membranes, enclosing multiple non-concentric aqueous chambers which all contain the same component. In contrast, t' the term "heterovesicular liposomes as used throughout the specification and claims means man-made, microscopic liquid vesicles consisting of lipid bilayer membranes enclosing multiple, aqueous chambers wherein at least two of the chambers separately contain substances of different compositions. The microscopic lipid vesicles include but are not limited to multilamellar liposomes, stable paucilamellar liposomes, and multivesicular liposomes.

The term "chimeric emulsion" as used throughout the specification and claims means an emulsion that consists of multiple water droplets suspended in organic solvent where the substances of two different compositions are each dissolved separately in different aqueous droplets.

The term "solvent spherule" as used throughout the specification and claims means a microscopic spheroid droplet of organic solvent, within which is multiple smaller droplets of aqueous solution. The solvent spherules are suspended and totally immersed in a second aqueous solution.

The term "neutral lipid" means oil or fats that have no membrane-forming capability by themselves and lack a hydrophilic "head" group. 4 ^ ip ,7 u q 4 The term amphipathic lipids means those molecules that have a hydrophilic "head" group and hydrophobic "tail" group and have membrane-forming capability.

The composition of the present invention is a heterovesicular lipid vesicle or liposome having at least two substances of different compositions each encapsulated separately in different chambers of the vesicle or liposome.

Many and varied biological substances can be incorporated by encapsulation within the multivesicular 10 liposomes. These include drugs, and other kinds of materials, such as DNA, RNA, proteins of various types, protein hormones produced by recombinant DNA technology effective in humans, hematopoietic growth factors, monokines, lymphokines, tumor necrosis factor, inhibin, 15 tumor growth factor alpha and beta, mullerian inhibitory substance, nerve growth factor, fibroblast growth factor, platelet-derived growth factor, pituitary and hypophyseal hormones including LH and other releasing hormones, calcitonin, proteins that serve as immunogens for 2 0 vaccination, and DNA and RNA sequences.

The following Table 1 includes a list of representative biologically active substances which can be encapsulated in heterovesicular liposomes in the presence of a hydrochloride and which are effective in humans.

Antiasthma metaproterenol aminophylline theophylline terbutaline Tegretol ephedrine isoproterenol adrenalin norepinephrine TABLE 1 Antiarrhythmic propanolol atenolol verapamil captopril isosorbide Tranquilizers chlorpromaz ine benzodiazepine butyrophenones hydroxyzines meprobamate phenothiazines reserpine thioxanthines 24 7 5 4 T Cardiac glycosides digitalis digitoxin lanatoside C digoxin Hormones antidiuretic corticosteroids testosterone estrogen thyroid growth ACTH progesterone gonadotropin mineralocorticoid LH LHRH FSH calcitonin Steroids prednisone triamcinolone hydrocortisone dexamethasone betamethosone prednisolone Ant ihvpert ens ive s apresoline atenolol Antidiabetic Diabenese insulin Antihistamines pyribenzamine chlorpheniramine diphenhydramine 40 Antiparasitic praziquantel metronidazole pentamidine Antibiotic penicillin tetracycline erythromycin cephalothin imipenem cefofaxime carbenicillin Anticancer azathioprine bleomycin cyclophosphamide adriamycin daunorubicin vincristine methotrexate 6-TG 6-MP vinblastine VP-16 VM-26 cisplatine FU Immunootherapies interferon interleukin-2 monoclonal antibodies gammaglobulin Sedatives & Analgesic morphine dilaudid codeine codeine-like synthetics demerol oxymorphone phenobarbital barbiturates Vaccines influenza respiratory syncytial virus Hemophilus influenza vaccine 24 7 5 4 7 Antibiotic (continued^ vancomycin gentamycin tobramycin piperacillin moxalactam amoxicillin ampicillin cefazolin cefadroxil cefoxitin other aminoglycosides Antifungal amphotericin B myconazole muramyl dipeptide clotrimazole Antihypotension dopamine dextroamphetamine Antiviral acyclovir and derivatives Winthrop-51711 ribavirin rimantadine/amantadine azidothymidine & derivatives adenine arabinoside amidine-type protease inhibitors Proteins and Glycoproteins lymphokines interleukins - 1, 2, 3, 4, 5, and 6 cytokines GM-CSF M-CSF G-CSF tumor necrosis factor inhibin tumor growth factor Mullerian inhibitors substance nerve growth factor fibroblast growth factor platelet derived growth factor coagulation factors (e.g. VIII, IX, VII) insulin tissue plasminogen activator histocompatibility antigen oncogene products myelin basic protein collagen fibronectin laminin other proteins made by recombinant DNA technology Other cell surface receptor blockers Nucleic Acids & Analogs DNA RNA methylphosphonates and analogs A preferred method of making the heterovesicular vesicle or liposome is illustrated in the drawing to which reference is now made. In step 1 (Figure 1) a first aqueous substance of composition 10 to be encapsulated is added to a 247547 first lipid component 12 in the vial 14. The vial 14 is sealed and in step 2 (Figure 2) is mixed and shaken, such as being attached to the head of a vortex mixer to form the first water in oil emulsion 16 containing the first substance of composition 10 to be encapsulated. In step 3 (Figure 3), a second vial 14a, a second aqueous substance 10a to be encapsulated is added to a second lipid component 12a, and the vial 14a is sealed and in step 4 (Figure 4) is mixed, such as being attached to the head of a vortex mixer to form a second water-in-oil emulsion l6a containing the substance of composition 10a to be encapsulated.

In step 5 (Figure 5) the first 16 and second 16a water in oil emulsions are added together and mixed, such as by hand to make a "chimeric" emulsion.

In step 6 (Figure 6) a portion of the chimeric emulsion from step 5 is individually added to vials containing a third immiscible aqueous component 18a such as by squirting rapidly through a narrow tip pasteur pipette into two one-dram vials, here shown as one.

In step 7 (Figure 7) vials from step 6 are shaken, such as by a vortex mixer to form a chloroform spherical suspension, and in step 8 (Figure 8) the chloroform spherical suspension in each vial is transferred from step 7 and the chloroform is evaporated, such as by a stream of nitrogen gas, thereby providing the heterovesicular liposome that contains the first aqueous substance 10 in one or more internal aqueous chambers and the second aqueous substance 10a in the remaining internal aqueous chambers within a single liposome.

Preferably, each of the substances to be encapsulated are encapsulated in the presence of a hydrochloride, such as hydrochloric acid, which slows their leakage rate from the liposome or vesicle.

As previously mentioned, any biologically active substance, such as illustrated in Table 1, can be encapsulated separately in chambers of the vesicle or liposome. 4 7 5 The following examples set forth presently preferred methods of encapsulating two substances of different compositions in separate chambers of a vesicle or liposome.

Example 1 Preparation of Dideoxycvtidine/Glucose Heterovesicular Liposomes Step l: A first aqueous substance (one ml of 2 0 mg/ml dideoxycytidine solution in water with 0.1 N hydrochloric acid) was added into a one-dram vial containing the first lipid component (9.3 umoles of dioleoyl lecithin, 2.1 umoles of dipalmitoyl phosphatidylglycerol, 15 umoles of cholesterol, 1.8 umoles of triolein and one ml of chloroform).

Step 2: The first vial was sealed and attached to the head of a vortex mixer and shaken at maximum speed for 6 minutes to form the first water-in-oil emulsion.

Step 3: In second vial, the second aqueous substance (one ml of 3 0 mg/ml glucose solution in water with 0.1 N hydrochloric acid) was added into the second lipid component (which is identical to the first lipid component).

Step 4; The second vial was sealed and attached to the head of a vortex mixer and shaken at maximum speed for 6 minutes to form the second water-in-oil emulsion.

Step 5: 0.5 ml of the first emulsion was added to the second vial and mixed by hand to make a "chimeric" emulsion.

Step 6: Half of the "chimeric" emulsion was individually squirted rapidly through a narrow tip Pasteur pipette into one-dram vials, each containing a third immiscible aqueous component (2.5 ml water, 3 2 mg/ml glucose, 4 0 mM free-base lysine.

Step 7; The vials from step 6 were shaken on the vortex mixer for 3 seconds at "5" setting to form solvent spherules containing multiple droplets of the first and second aqueous substances within. 4 7S Step 8: The chloroform spherule suspensions in each vials were transferred into the bottom of a 2 L beaker containing 4.5 ml of water, 3 5 mg/ml glucose, and 22 mM free-base lysine. A stream of nitrogen gas at 7 L/min was flushed through the beaker to evaporate chloroform over 5 minutes at 15 deg. C.

The above example describes a method of making heterovesicular liposomes which separately contain glucose in approximately 5/6 of the internal aqueous chambers and separately contain dideoxycytidlne in the remaining 1/6 of the internal aqueous chambers within a single liposome. Heterovesicular liposomes containing dideoxycytidine solution as one aqueous substance and glucose as the second aqueous substance were markedly more stable than non-heterovesicular liposomes.

Example 2 / This example is for the synthesis of heterovesicular liposomes containing IL-2 (interleukin-2) and lysine hydrochloride: For each batch of liposomes prepared, one ml of water containing 10 mg/ml HSA (Human serum albumin), 1 ug of IL-2, 200 mM lysine HC1 pH 7.13 was added into a one-dram vial containing 9.3 umoles of dioleoyl lecithin, 2.1 umoles of dipalmitoyl phosphatidylglycerol, 15 umoles of cholesterol, and 1.8 umoles of triolein and one ml of chloroform (this is the first water-in-oil emulsion). For the second water-in-oil emulsion, 1 ml of lysine HC1 (without IL-2) was added into one-dram vial containing 9.3 umoles of dioleoyl lecithin, 2.1 umoles of dipalmitoyl phosphatidylglycerol, 15 umoles of cholesterol, and 1.87 umoles of triolein and one ml of chloroform. Each of the two vials were individually attached to the head of a vortex mixer and shaken sequentially at the maximum speed for 6 minutes. 0.5 ml of the first water-in-oil emulsion was added to the 2 ml of the second emulsion and mixed to make a "chimeric" water-in-oil emulsion. Half of the "chimeric" > I > ► 24 7 5 4 emulsion was individually squirted rapidly through a narrow tip Pasteur pipette into one-dram vials, each containing 2.5 ml of 4% glucose in water and 0.1 ml of lysine free base, 200 mM, and shaken at maximum speed for 3 seconds to form chloroform spherules. The chloroform spherule suspensions were transferred into 250 ml Erlenmeyer flask containing 5 ml of 4% glucose in water and 0.2 ml of lysine free base, 200 mM. A stream of nitrogen gas at 7 L/min was flushed through the flask to evaporate chloroform over 5 minutes at 3 7 degrees C.

Example 3 This example is for the synthesis of heterovesicular liposomes containing ara-C solution as the first aqueous substance and distilled water as the second aqueous substance. For each batch of liposomes prepared, one ml of water containing 100 mg/ml ara-C, pH 1.1 was added into a one-dram vial containing 9.3 umoles of dioleoyl lecithin, 2.1 umoles of dipalmitoyl phosphatidylglycerol, 15 umoles of cholesterol, and 1.8 umoles of triolein and one ml of chloroform, attached to the head of the vortex mixer and shaken at maximum speed for 6 minutes (this is the first water-in-oil emulsion). For the in situ generation of the second water-in-oil emulsion, 1/2 of the content was removed from the first water-in-oil emulsion, and then 1 ml of distilled water was added into the remaining first water-in-oil emulsion and the one-dram vial was shaken for 10 seconds at maximum speed. This resulted in a "chimeric" water-in-oil emulsion. Half of the "chimeric" emulsion was individually squired rapidly through a narrow tip Pasteur pipette into one-dram vials, each containing 2.0 ml of 4% glucose in water and 0.5 ml of lysine free base, 2 00 mM, and shaken at maximum speed for 3 seconds to form chloroform spherules. The chloroform spherule suspensions were transferred into 250 ml Erlenmeyer flask containing 4 ml of 4% glucose in water and 0.5 ml of lysine free base, 200 mM. A stream of nitrogen gas at 7 L/min was flushed through the 24 7 5 4 flask to evaporate chloroform over 5 minutes at 3 7 degrees C.

Example 4 Synthesis of Heterovesicular Liposomes Containing Granulocvte-Macrophase Colony Stimulating Factor (GM-CSF) Exactly the same procedure was used as in Example 2 except IL-2 was replaced with 1 ug of GM-CSF.

Example 5 Synthesis of Heterovesicular Liposomes of Various Lipid Composition, and Incorporation of Various Materials into Liposomes In place of using dioleoyl lecithin, dipalmtoyl phosphatidylglyerol, cholesterol, and triolein (TO), and other amphipathic lipids such as phosphatidyl cholines (PC), cardiolipin (CL), dimyristoyl phosphatidylglycerol (DMPG), phosphatidyl ethanolamines (PE), phosphatidyl serines (PS), dimyristoyl phosphatidic acid (DMPA), and other neutral lipids such as tricaprylin (TC) in various combination can be used with similar results. For example, PC/C/CL/TO in 4.5/4.5/1/1 molar ration; DOPC/C/PS/TO in 4.5/4.5/1/1 molar ratio; PC/C/DPPG/TC in 5/4/1/1 molar ratio; PC/C/PG/TC in 5/4/1/1 molar ratio; PE/C/CL/TO in 4.5/4.5/1/1 molar ratio; and PC/C/DMPA/TO in 4.5/4.5/1/1 molar ratio can all be used. To incorporate other water-soluble materials, such as glucose, sucrose, methotrexate, Ponceau S, simply substitute the desired materials for IL-2 in Example 2. Also, other biologically active substances, such as set forth in Table 1, in suitable doses can be similarly substituted for IL-2 as in Example 2.

Example 6 In this example, the triolein in lipid components of above examples are substituted either singly or in combination by other triglycerides, vegetable oils, animal > > ► > 24 7 5 4 7 fats, tocopherols, tocopherol esters, cholesteryl esthers, or hydrocarbons with good results.

Example 7 To make liposomes smaller than that in the foregoing examples, and with reference to Examples 1 or 2, the mechanical strength or duration of shaking or homogenization in Step 4 of Example 1 or 2 was increased.

To make liposomes larger, the mechanical strength or duration of shaking or homogenization in Step 4 of Example 1 or 2 was decreased.

The heterovesicular liposomes can be administered to the patients in the normal manner when it is desirable to provide two separate biologically active compounds to the patient for the particular purpose of treatment desired.

The dosage range appropriate for human use includes the range of 1-6000 mg/m to body surface area. The reason that this range is so large is that for some applications, such as subcutaneous administration, the dose required may be quite small, but for other applications, such as intraperitoneal administration, the dose desired to be used may be absolutely enormous. While doses outside the foregoing dose range may be given, this range encompasses the breadth of use for practically all the biologically active substances.

The multivesicular liposomes may be administered by any desired route; for example, intrathecal, intraperitoneal, subcutaneous, intravenous, intralymphatic, oral and submucosal, under many different kinds of epithelia including the bronchialar epithelia, the gastrointestinal epithelia, the urogenital epithelia, and various mucous membranes of the body, and intramuscular.

When encapsulating more than two substances separately in chambers of a liposome, a third (or fourth) aqueous component containing the third or fourth biologically active substance is formed, mixed to form a third or fourth water in oil emulsion, and then combined with the first and second emulsions and mixed to form a "chimeric" emulsion containing the three or more biologically active substances. The remainder of the process is the same as described when encapsulating two biologically active compounds or substances.

The present invention, therefore, obtains the objects and ends and has the advantages mentioned as well as others inherent therein.

While examples of the invention have been given for the purpose of disclosure, changes can be made therein which are within the scope of the invention as defined by the appended claims. 247547 16

Claims (33)

WHAT WE CLAIM IS:

1. Heterovesicular lipid vesicles or liposomes wherein two different substances are encapsulated separately in different chambers of one liposome and at least one, but not both, of said substances is biologically active.

2. A process for producing heterovesicular lipid vesicles or liposomes comprising the steps of: (a) providing a first lipid component dissolved in one or more organic solvents and adding into the said lipid component an immiscible first aqueous component; (b) forming a first water-in-oil emulsion from the first two immiscible components; (c) providing a second lipid component dissolved in one or more organic solvents and adding into the said second lipid component an immiscible second aqueous component; (d) forming a second water-in-oil emulsion from the second two immiscible components; (e) forming a chimeric emulsion by combining the first water-in-oil emulsion and the second water-in-oil emulsion; (f) transferring and immersing the chimeric emulsion into a third immiscible aqueous component; (g) dispersing the chimeric emulsion to form solvent spherules containing multiple droplets of the first and second aqueous components within and (h) evaporating the organic solvents from the solvent spherules to form heterovesicular liposomes having multiple aqueous chambers.

3* The process according to claim 2 wherein the first or secorvd-lipld component is a phospholipid or an admixture of sever ^ ! >rvd-lii 1 - MOV 1995 phospholipids.

4. The process according to claim 2 wherein tHree—or more water-in-oil emulsions each containing an immiscrbi-e—-aqueous component are combined to form the chimeric emulsion. 247547

5. The process according to claim 2 wherein the lipid components are identical.

6. The process according to claim 3 wherein the phospholipids are selected from the group consisting of phosphatidylcholine, cardiolipin, phosphatidylethanolamine, sphingomyelin, lysophosphatidylcholine, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, and phosphatidic acid.

7. The process according to claim 3 wherein at least one of the phospholipids is selected from a group with a net negative charge or charges.

8. The process according to claim 3 wherein the phospholipid is provided in admixture with cholesterol.

9. The process according to claim 3 wherein the phospholipid is provided in admixture with stearylamine.

10. The process according to claim 3 wherein a lipophilic biologically active material is provided in admixture with the first or second lipid component.

11. The process according to claim 2 wherein at least one of the first and second lipid components is a neutral lipid either singly or in combination with a substance selected from the group consisting of triglycerides, vegetable oils, animal fats, tocopherols, tocopherol esters, cholesteryl esters, and hydrocarbons.

12. The process according to claim 2 wherein the organic solvent is chosen singly,, or in combination from the group consisting of diethyl ether, isopropyl ether, chloroform, tetrahydrofuran, ethers, hydrocarbons, halogenated hydrocarbons, halogenated ethers, and esters.

13. The process according to claim 2 wherein the first aqueous component and the second aqueous component each contain a hydrochloride.

14. The process according to claim 13 wherein the hydrochloride is selected either singly or in combination from the group consisting of hydrochloric acid, lysine hydrochloride, histidine hydrochloride.

15. The process according to claim 2 wherein a 247547 hydrophilic biologically active material is encapsulated within the liposomes.

16. The process according to claim 15 wherein the hydrophilic biologically active material is selected from the group consisting of interleukin-2, ara-C cytosine arabinoside, methotrexate, 5-fluorouracil, cisplatin, floxuridine, melphalan, mercaptopurine, thiguanine, thiotepa, vincristine, vinblastine, streptozocin, leuprolide, interferon, calcitonin, doxorubicin, daunorubicin, mitoxanthrone, amsacrine, factinomycin, and bleomycin.

17. The process according to claim 2 wherein the emulsification of the two lipid components is carried out using methods selected from the group consisting of mechanical agitation, ultrasonic energy, and nozzle atomization.

18. The process according to claim 4 wherein each of the three or more water-in-oil emulsions contain a hydrochloride.

19. The process according to claim 18 wherein each of the three or more water-in-oil emulsions contain at least one acid neutralising agent.

20. The process according to claim 19 wherein the neutralizing agent is selected either singly or in combination from the group consisting of free-base lysine and free-base histidine.

21. The process according to claim 4 wheirein the second aqueous component of one of the three or more water-in-oil emulsions is of low ionic strength.

22. The process according to claim 19 wherein the aqueous component of one of the three or more water-in-oil emulsions is an aqueous solution further containing solutes selected from the group consisting of carbohydrates and amino acids.

23. The process according to claim 19 wherein the aqueous component of one of the three or more water-in-oil emulsions is an aqueous solution further containing solutes selected either singly or in combination from the group consisting of glucose, ucrose, lactose, free-base lysine and free-base histidine.

24. The process according to claim 2 wherein the average ize and number of the aqueous chambers within liposomes is 247547 determined by the type, intensity, and/or duration of the emulsification used.

25. The process according to claim 2 wherein the formation of solvent spherules is carried out using methods selected from the group consisting of mechanical agitation, ultrasonic energy, and/or nozzle atomization.

26. The process according to claim 25 wherein the average size of the liposomes is determined by the type, intensity, and duration of the emulsification used.

27. The process according to claim 2 wherein the evaporation of the organic solvents from the solvent spherules in step (g) is provided by passing nitrogen gas thereover.

28. The process of claim 3 where, the substance to be encapsulated is selected from the group consisting of the substances of Table 1 and Examples 1, 2, 3, 4 and 5.

29. Heterovesicular liposomes made according to the method of claim 3.

30. A heterovesicular liposome containing a biologically active substance encapsulated in the presence of hydrochloric acid or other hydrochlorides and a neutralizing agent.

31. The heterovesicular liposome of claim 30 where, the biologically active substance is selected from the group consisting of the substances of Table 1 and Examples 1, 2, 3, 4 and 5.

32. A heterovesicular lipid vesicle or liposome according to any one of claims 1 and 29 to 31 " substantially as herein described with reference to any example thereof

33. A process for producing a heterovesiciAar^liplildJ l.! vesicle or liposome according to any one of clai|ms 2^o-2|j|Qy jggg received 247547 20 substemtially as herein described with reference to any example thereof. _ JMW wttwytoed Agents AJ.RNRK&30N.