Heterovesicular liposomes
The present invention is a continuation-in-part of application Ser. No. 196,590, filed May 30, 1988, which in turn is a continuation-in-part application of application Ser. No. 496,846, filed Mar. 21, 1990, all abandoned in favor of this application. 
    
    
     FIELD OF THE INVENTION 
     The invention 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. (Biochim, Biophys. Acta, 782:339-348, 1983), and are uniquely different from the unilamellar (Huang, Biochemistry, 8:344-352, 1969; Kim, et al. Biochim. Biophys. Acta, 646:1-10, 1981) and multilamellar (Bangham, et al., J. Mol, Bio., 13:238-252, 1965) 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. Pat. 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. (Ann. Rev. Biophys. Bioeng., 9:467-508, 1980). 
     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 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 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 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. 
     Briefly, the method of the invention comprises making a &#34;water-in-lipid&#34; 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 a halogenohydroacid and then emulsifying the mixture mechanically. As used herein, the term &#34;halogenohydroacid&#34; means an acid represented by the formula HX, wherein X is a halogen (e.g., Br, F, Cl, I). The halogenohydroacids are typically obtained by dissolving the corresponding hydrogen halides (which can also be expressed as HX) in water. The preferred halogenohydroacid is hydrochloric acid (HCl). 
     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 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 halogenohydroacid. A second emulsion is then created. A chimeric 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 then immersed in a third aqueous immiscible component preferably containing one or more nonionic osmotic agents 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 halogenohydroacid with a neutralizing agent, or other forms of halides or other small anions 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. 
     Accordingly, it is an object of the present invention to provide 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. 
     A further object of the present invention is the provision of a heterovesicular liposome containing at least two biologically active substances of different compositions each encapsulated separately in chambers of the liposome in the presence of halogenohydroacid or other forms of halides or other small anions which slow the leakage of them. 
     It is a further object of the present invention to provide a heterovesicular liposome containing at least two biologically active substances of different compositions each encapsulated separately in chambers of the liposome in the presence of halogenohydroacid or other forms of halides or other small anions and a neutralizing agent. 
     It is a further object of the present invention to provide methods of producing such heterovesicular lipid vesicles or liposomes. 
     It is a further object of the present invention to provide 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. 
     It is a further object to provide 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. 
     It is a further object of the present invention to provide a method for the treatment of a patient with at least two separate biologically active substances of different compositions by administering them to the patient encapsulated separately in chambers of a heterovesicular vesicle or liposome. 
     Other and further objects, features and advantages of the invention appear throughout the specification and claims. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     The drawing is a schematic diagram illustrating preparation of a heterovesicular vesicle or liposome. 
     FIG. 1 is a schematic representation of a first aqueous substance added to a first lipid component in a vial. 
     FIG. 2 is a schematic representation of a first water in oil emulsion containing the aqueous substance and lipid components of FIG. 1. 
     FIG. 3 is a schematic representation of a two phase system in a vial containing a second aqueous component and a second lipid component. 
     FIG. 4 is a schematic representation of a water in oil emulsion formed from the aqueous component and lipid component of FIG. 3 by shaking. 
     FIG. 5 is a schematic representation of a chimeric emulsion made by mixing together the water in oil emulsion of FIG. 2 and the water in oil emulsion of FIG. 4. 
     FIG. 6 is a schematic representation of a vial containing an immiscible aqueous component into which the chimeric emulsion of FIG. 5 is introduced. 
     FIG. 7 is a schematic representation of solvent spherules containing droplets of the first and second aqueous components. 
     FIG. 8 is a schematic representation of the heterovesicular liposome formed by evaporation of solvent from the solvent spherules of FIG. 7. 
    
    
     DESCRIPTION OF PREFERRED EMBODIMENT 
     The term &#34;multivesicular liposomes&#34; 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, the term &#34;heterovesicular liposomes&#34; as used throughout the specification and claims means man-made, microscopic liquid vesicles consisting of lipid bilayer membranes enclosing multiple, aqueous chamber 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 &#34;chimeric emulsion&#34; 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 &#34;solvent spherule&#34; 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 &#34;neutral lipid&#34; means oil or fats that have no membrane-forming capability by themselves and lack a hydrophilic &#34;head&#34; group. 
     The term amphipathic lipids means those molecules that have a hydrophilic &#34;head&#34; group and hydrophobic &#34;tail&#34; 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 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, 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 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 hydrohalide and which are effective in humans. 
     
                                           TABLE 1                                 
__________________________________________________________________________
Antiasthmatics                                                            
              Antiarrhythmics                                             
                         Tranquilizers                                    
melairoterenol                                                            
              propanolol chlorpromazine                                   
aminophylline atenolol   benzodiazepine                                   
theophylline  verapamil  butyrophenones                                   
terbutaline   Antianginas                                                 
                         hydroxyzines                                     
norepinephrine                                                            
              isosorbide dinitrate                                        
                         meprobamate                                      
ephedrine     Hormones   phenothiazines                                   
isoproterenol thyroxine  thioxanthenes                                    
adrenalin     corticosteroids                                             
                         Steroids                                         
Cardiac glycosides                                                        
              testosterone                                                
                         preunisone                                       
digitalis     estrogen   triamcinolone                                    
digitoxin     progesterone                                                
                         hydrocortisone                                   
lanatoside C  mineralocorticoid                                           
                         dexamethasone                                    
digoxin       Antidiabetics                                               
                         betamethasone                                    
Antihypertensives                                                         
              Diabenese  preunisolone                                     
apresoline    insulin    Antihistamines                                   
atenolol      Antineoplastics                                             
                         pyribenzamine                                    
captopril     azathioprine                                                
                         chlorpheniramine                                 
reserpine     bleomycin  diphenhydramine                                  
Antiparasitics                                                            
              cyclophosphamide                                            
                         Sedatives and Analgesics                         
praziquantel  vincristine                                                 
                         morphine                                         
metronidazole methotrexate                                                
                         dilaudid                                         
pentamidine   6-TG       codeine                                          
ivermectin    6-MP       codeine-like synthetics                          
Nucleic Acids and Analogs                                                 
              vinblastine                                                 
                         demerol                                          
DNA           VP-16      oxymorphone                                      
RNA           VM-26      phenobarbital                                    
methylphosphonates and                                                    
              cisplatin  barbiturates                                     
analogs       5-FU       fentanyl                                         
Antisense nucleic acids                                                   
              FUDR       ketorolac                                        
Antibiotics   fludarabine phosphate                                       
                         Vasopressors                                     
penicillin    Immunomedalators                                            
                         dopamine                                         
tetracycline  interferon dextroamphetamine                                
amikacin      interleukin-2                                               
                         Antivitals                                       
erythromycin  gammaglobulin                                               
                         acyclovir and derivatives                        
cephalothin   monoclonal antibodies                                       
                         Gancyclovir and phosphates                       
imipenem      Antifungals                                                 
                         Winthrop-51711                                   
cefotaxime    amphotericin B                                              
                         ribavirlin                                       
carbenicillin myconazole rimantadine/amantadine                           
ceftazidime   muramyl dipeptide                                           
                         azidothymidine &amp; derivates                       
kanamycin     clotrimazole                                                
                         adenine arabinoside                              
tobramycin    ketoconozole                                                
                         amidine-type protease                            
ampicillin    fluconazole                                                 
                         inhibitors                                       
gentamycin    itraconazole                                                
cefoxitin                                                                 
cefadroxil                                                                
cefazolin                                                                 
other aminoglycosides                                                     
amoxicillin                                                               
moxalactam                                                                
piperacillin                                                              
vancomycin                                                                
ciprofloxacin                                                             
other quinolones                                                          
Vaccines                                                                  
other recombinant, killed and live vaccines and antigenic material for    
use                                                                       
as vaccines.                                                              
antigenic material for the treatment of allergies                         
influenza                                                                 
respiratory syncytial virus                                               
HIV vaccine                                                               
Hemophilus influenza vaccines                                             
Hepatitis A,B,C vaccines                                                  
mumps                                                                     
rubella                                                                   
measles                                                                   
tetanus                                                                   
malaria vaccines                                                          
herpes                                                                    
cancer vaccines                                                           
Anti-leu-3a vaccine                                                       
Monoclonal Antibodies (human, mouse other species-derived and/or          
recombinant                                                               
and/or fusions and/or fragments thereof)                                  
OKT3                                                                      
OKT4                                                                      
HA-IA                                                                     
Anti-Carcino-Embryonic Antigen Antibodies                                 
Anti-Ganglioside Antibodies: Anti GD2, Anti GM2, Anti GD3, Anti GM3       
Urinary Tract-Associated Antigen-related antibodies                       
Anti-Il-2 Receptor                                                        
Chimeric Anti-Leu-2                                                       
Anti-IL-2 receptor                                                        
Anti-Leu-2                                                                
Chimeric Anti-Leu-3a                                                      
Chimeric L6                                                               
MAb-L6                                                                    
Radiolabeled L6                                                           
Centorex                                                                  
Centoxin                                                                  
Panorex                                                                   
Anti-LPS                                                                  
Immunotoxin                                                               
Anti-tumor necrosis factor                                                
Anti-pseudomonas                                                          
Anti-tumor necrosis factor                                                
OncoRad 103                                                               
OncoScint CR103                                                           
OncoScint OV103                                                           
OncoScint PR356                                                           
OncoTher 130                                                              
KS 1/4-DAVLB                                                              
ADCC agent                                                                
Murine monoclonal antibodies to human B-cell lymphomas (anti-idiotypes)   
Murine monoclonal antibody (1Melpg1) (anti-idiotype) against murine       
monoclonal                                                                
antibody to melanoma-associated antigen                                   
Anti-B4-blocked ricin                                                     
Anti-My9-blocked ricin                                                    
ImmuRaid-CEA                                                              
MAb against colorectal, ovarian, and lung cancers                         
rhenium-186 MAb                                                           
Orthoclone OKT ®                                                      
E5 ™                                                                   
LYM-1                                                                     
TNT                                                                       
791aZyme ®                                                            
CD5 Plus ®                                                            
CD7 Plus ®                                                            
MelaZyme ®                                                            
 Herbicides                                                               
Triazine                                                                  
chloroacetamide                                                           
cyanazine                                                                 
bentazone                                                                 
Roundup                                                                   
Rodeo                                                                     
butachlor                                                                 
CNP                                                                       
chlomethoxynil                                                            
simetryne                                                                 
Atrazine                                                                  
Alachlor                                                                  
Cyanazine                                                                 
metolachlor                                                               
metribuzin                                                                
phenoxy herbicides: 2,4-D [(2,4-dichlorophenoxy)acetic acid],             
2,4-D amine (2,4-dichlorophenoxyacetic acid dimethylamine),               
2,4-D isooctyl (2,4-dichlorophenoxyacetic acid isooctyl ester),           
2,4,5-T amine (2,4,5-trichlorophenoxyacetic acid trimethylamine)          
other triazine herbicides                                                 
other chloroacetamide herbicides                                          
other phenoxyacid herbicides                                              
Pesticides                                                                
Abamectin                                                                 
other avermectins                                                         
atrazine                                                                  
lindane                                                                   
dichlorvos                                                                
dimethoate                                                                
warfarin                                                                  
p,p&#39;-DDD                                                                  
p,p&#39;-DDE                                                                  
HCH                                                                       
DMDT                                                                      
aldrin                                                                    
dieldrin                                                                  
Aldicarb                                                                  
EDB                                                                       
DCP                                                                       
DBCP                                                                      
simazine                                                                  
cyanazine                                                                 
Bacillus thuringiensis toxin                                              
Bacillus thuringiensis var. kurstaki                                      
bis(tri-n-butyltin)oxide (TBTO)                                           
other organochlorine pesticides                                           
Proteins and Glycoproteins                                                
lymphokines                                                               
interleukins - 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11.                         
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                         
erythropoietin                                                            
IL-3/GM-CSF fusion proteins                                               
Monoclonal antibodies                                                     
Polyclonal antibodies                                                     
antibody-toxin fusion proteins                                            
antibody radionuclide conjugate                                           
Interferons                                                               
Fragments and peptide analogs, and analogs of fragment of proteins,       
peptides                                                                  
and glycoproteins.                                                        
Epidermal growth factor                                                   
CD4 receptor and other recombinant receptors                              
other proteins isolated from nature                                       
Antidiuretic hormone                                                      
oxytocin                                                                  
adrenocorticotropin Hormone                                               
calcitonin                                                                
follicle stimulating hormone                                              
luteinizing hormone releasing hormone                                     
luteinizing hormone                                                       
gonadotrophin                                                             
transforming growth factors                                               
streptokinase                                                             
Human Growth Hormone,                                                     
Somatotropins for other species, including, but not limited to:           
1. Porcine,                                                               
2. Bovine,                                                                
3. Chicken,                                                               
4. Sheep,                                                                 
5. Fish,                                                                  
Growth Hormone releasing hormones for humans and various animal species,  
Glucagon,                                                                 
Desmopressin,                                                             
Thyroid Releasing Hormone,                                                
Thyroid Hormone,                                                          
Secretin,                                                                 
Magainins,                                                                
Integrins,                                                                
Adhesion Peptides, including, but not limited to, those having the        
Arginine-Glutamine-Aspartic Acid sequence,                                
Super Oxide Dismutase,                                                    
Defensins,                                                                
T-Cell Receptors,                                                         
Bradykinin antagonists,                                                   
Pentigetide,                                                              
Peptide T,                                                                
Antinflammins,                                                            
Major Histocompatibility (MHC) complex components and peptides            
targeted to the MHC,                                                      
Protease inhibitors,                                                      
Lypressin,                                                                
Buserelin,                                                                
Leuprolide,                                                               
Nafarelin,                                                                
Deslorelin,                                                               
Goserelin,                                                                
Historelin,                                                               
Triptorelin,                                                              
LHRH antagonists,                                                         
HOE-2013,                                                                 
Detirelix,                                                                
Org-30850,                                                                
ORF-21243,                                                                
Angiotensin Converting Enzyme inhibitor Peptide,                          
Renin inhibitory peptides,                                                
Ebiratide (HOE-427),                                                      
DGAVP,                                                                    
Opiate receptor agonists and antagonists, including, but not limited to:  
1. Enkephalins,                                                           
2. Endorphins,                                                            
E-2078,                                                                   
DPDPE,                                                                    
Vasoactive intestinal peptide,                                            
Atrial Natriuretic Peptide,                                               
Brain Natriuretic Peptide,                                                
Atrial Peptide clearance inhibitors,                                      
Hirudin,                                                                  
Oncogene Inhibitors,                                                      
Other Colony Stimulating Factors,                                         
Neurotransmitters                                                         
              Radionuclides                                               
                         Radio contrasts                                  
Dopamine      Technetium Gadolinium chelates                              
Epinephrine   Indium     Iohexol                                          
Norepinephrine                                                            
              Yttrium    Ethiodol                                         
acetylcholine Gallium    Iodexinol                                        
Gammaamino butyric acid                                                   
Others                                                                    
amino acids                                                               
vitamins                                                                  
cell surface receptor blockers                                            
__________________________________________________________________________
 
    
     As used herein, the term &#34;biologically active&#34;, when used to describe substances present in the chambers of the heterovesicular liposome, includes substances which possess biological activity in the form as presented in the vesicle as well as substances which become active after release from the vesicle chamber (i.e., possess &#34;quiescent&#34; biological activity). For example, in the latter instance, a first vesicle chamber could contain an enzyme and a second vesicle chamber could contain a prodrug which is converted upon interaction with the enzyme into an active moiety with therapeutic activity. Alternatively, the invention embraces a first vesicle chamber containing a substance possessing quiescent biological activity and a second vesicle chamber containing a substance possessing a different quiescent biological activity; where, upon release from their respective chambers, the substances interact with each other or components of the in vivo biological melieu such that both substances become biologically active in vivo. For example, a first vesicle chamber could contain the inactive compound 5-fluorocytosine (5-FC) and a second vesicle chamber could contain the enzyme cytosine deaminase. When cytosine deaminase is release from its vesicle and interacts with 5-FC released from its vesicle, the biologically active antitumor drug 5-fluorouracil (5-FU) is produced. 
     In addition, biologically active substances which can be incorporated include substances which act indirectly. For example, various excipients and stabilizers may be present. Such substances may act, for example, to increase the shelf life or bioavailability of a particular drug. Alternatively, many substances commonly classified as excipients may actually possess direct biological activity from very slight to quite significant. For example, the common excipient mannitol can also act biologically as a diuretic and even water may act biologically to affect dehydration. Such indirectly active substances include aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solutions are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic-aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer&#39;s dextrose, dextrose, and sodium chloride, lactated Ringer&#39;s, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer&#39;s dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like (see, Remingtons Pharmaceutical Sciences, 16th Ed., A. Oslo, ed., Mack, Easton, PA. 1980). Those of ordinary skill in the art can readily ascertain and envision various combinations of compounds which can be utilized in the vesicles of the invention without resorting to undue experimentation. 
     A preferred method of making the heterovesicular vesicle or liposome is illustrated in the drawing to which reference is now made. As shown in FIG. 1, in step 1 a first aqueous substance of composition 10 to be encapsulated is added to a first lipid component 12 in the vial 14. The vial 14 is sealed and in step 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 shown in FIG. 2 containing the first substance of composition 10 to be encapsulated. In a second vial 14a, a second aqueous 10a to be encapsulated is added to a second lipid component 12a, as shown in FIG. 3, and the vial 14a is sealed and in step 4 is mixed, such as being attached to the head of a vortex mixer to form a second water-in-oil emulsion 16a containing the substance of composition 10a to be encapsulated. 
     In step 5 the first 16 and second 16a water in oil emulsions are added together and mixed, such as by hand to make a &#34;chimeric&#34; emulsion as shown in FIG. 5. 
     In step 6, as shown in FIG. 6 a portion of the chimeric emulsion from step 5 is individually added to vials 20 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 vials from step 6 are shaken, such as by a vortex mixer, and in step 8 the chloroform spherule suspension 21 (as shown in FIG. 7), 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 22 (as shown in FIG. 8), that contains a first substance in one or more internal aqueous chambers and a second substance 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 halogenohydroacid, such as hydrochloric acid, which slows their leakage rate from the liposome or vesicle. Most preferably, the halogenohydroacid is HCl, HI, or HBr. Other hydrochlorides which are satisfactory include, but not limited to, guanine hydrochloride, glucosamine hydrochloride, lysine hydrochloride, histidine hydrochloride, arginine hydrochloride, and combinations thereof, which can be neutral, acidic, or basic. 
     As previously mentioned, any biologically active substance, such as illustrated in Table 1, can be encapsulated separately in chambers of the vesicle or liposome. 
     The heterovesicular 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. 
     The heterovesicular liposomes may be modified in order to impart organ or cell target specificity. Such modifications may be particularly relevant for using the vesicles of the invention to administer drugs which are highly toxic or capable of inducing severe side effects, such as taxol or other anti-neoplastic agents which might otherwise not be utilized in view of their deleterious effects on normal tissues when systemically disseminated. 
     The targeting of liposome has been classified based on anatomical and mechanistic factors. Anatomical classification is based on the level of selectivity, for example, organ-specific, cell-specific, and organelle-specific. Mechanistic targeting can be distinguished based upon whether the targeting is passive or active. Passive targeting utilizes the natural tendency of liposomes to distribute to cells of the reticulo-endothelial system (RES) in organs which contain sinusoidal capillaries. Active targeting, on the other hand, involves alteration of the liposome by coupling the liposome to a specific ligand such as a monoclonal antibody, sugar, glycolipid, or protein, or by changing the composition or size of the liposome in order to achieve targeting to organs and cell types other than the naturally occurring sites of localization (see, for example, Remington&#39;s Pharmaceutical Sciences, Gannaro, A. R., ed., Mack Publishing, 18 Edition, pp. 1691-1693, 1990, incorporated by reference). 
     The surface of the targeted delivery system may be modified in a variety of ways. In the case of a liposomal targeted delivery system, lipid groups can be incorporated into the lipid bilayer of the liposome in order to maintain the targeting ligand in stable association with the liposomal bilayer. Various linking groups can be used for joining the lipid chains to the targeting ligand (Mannino, et al., Bio Techniques, 6(7):682, 1988, incorporated by reference). The compounds bound to the surface of the targeted delivery system may vary from small haptens of from about 125-200 molecular weight to much larger antigens with molecular weights of at least about 6 KD, but generally of less than 10 6  KD. Proteinaceous ligand and receptors are of particular interest. 
     In general, surface membrane proteins which bind to specific effector molecules are referred to as receptors. In the present invention, antibodies are preferred receptors (U.S. Pat. Nos. 4,806,466 and 4,957,735; incorporated by reference). Antibodies can be used to target liposomes to specific cell-surface ligands. For example, certain antigens expressed specifically on tumor cells, referred to as tumor-associated antigens (TAAs), may be exploited for the purpose of targeting antibody-containing liposomes directly to malignant tumor. Since the composition incorporated in the liposome may be indiscriminate with respect to cell type in its action, a targeted delivery system offers a significant improvement over randomly injecting non-specific liposomes. A number of procedures can be used to covalently attach either polyclonal or monoclonal antibodies to a liposome bilayer. Antibody-targeted liposomes can include monoclonal or polyclonal antibodies or fragments thereof such as Fab, or F(ab&#39;) 2 , as long as they bind efficiently to the antigenic epitope on the target cells. Liposomes may also be targeted to cells expressing receptors for hormones or other serum factors (Malone, et al., Proc. Nat&#39;l. Acad. Sci, USA,,86:6077, 1989; Gregoriadis, Immunology Today, 11(3):89, 1990; both incorporated by reference). 
     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 Dideoxycytidine Glucose Heterovesicular Liposomes 
     Step 1: A first aqueous substance (one ml of 20 mg/ml dideoxycytidine solution in water with 0.1N 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 30 mg/ml glucose solution in water with 0.1N 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 &#34;chimeric&#34; emulsion. 
     Step 6: Half of the &#34;chimeric&#34; 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, 32 mg/ml glucose, 40 mM free-base lysine. 
     Step 7: The vials from step 6 were shaken on the vortex mixer for 3 seconds at &#34;5&#34; setting to form solvent spherules containing multiple droplets of the first and second aqueous substances within. 
     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, 35 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 dideoxycytidine 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 HCl 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 HCl (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 &#34;chimeric&#34; water-in-oil emulsion. Half of the &#34;chimeric&#34; 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 37 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 &#34;chimeric&#34; water-in-oil emulsion. Half of the &#34;chimeric&#34; 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, 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 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 flask to evaporate chloroform over 5 minutes at 37 degrees C. 
     EXAMPLE 4 
     Synthesis of Heterovesicular Liposomes Containing Granulocyte-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 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 &#34;chimeric&#34; 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 spirit of the invention as defined by the appended claims.