CA1262863A

CA1262863A - Fusogenic liposomes and method of making same

Info

Publication number: CA1262863A
Application number: CA000505702A
Authority: CA
Inventors: Abraham Loyter; Adolfo G. Gitman
Original assignee: International Genetic Sciences Partnership
Current assignee: International Genetic Sciences Partnership
Priority date: 1985-04-04
Filing date: 1986-04-02
Publication date: 1989-11-14
Also published as: DE3610873A1; ES8802401A1; BR8601554A; GB8608468D0; SE8601408L; GB2188900B; IL78348A; IT8683340A1; IT1189877B; AU5562286A; SE464448B; JPS61274739A; FR2579891B1; IT8683340A0; GB2188900A; NL8600794A; IL78348A0; BE904536A; ZA862466B; NZ215696A

Abstract

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ABSTRACT OF THE DISCLOSURE
Phospholipid vesicles (liposomes) containing a qua-ternary ammonium hydroxide salt in the membranes of the vesicles are described. The phospholipid vesicles containing the quaternary ammonium hydroxide salt in the vesicle mem-branes, in addition to their ability to attach to cells, will fuse with a cell membrane and induce cell to cell fusion, pro-viding a convenient way of transferring a material such as a drug into a cell.

Description

~Z~i28~3 I

FUSOGL~NIC LIPOSOM~i'S AND METI~OD OF MAKIN~ SAMÆ

_~ r. D OF JNV~N~'[ON
This invention relates to liposomes. More particu-larly, this invention relates to liposomes containing a qua-ternary ammonium hydroxide salt in the membranes thereof, permitting the liposomes to fuse with animal and plant cells, and microinject their content into the animal and plant cells.
_ACKGROUND AND PRIOR ART
Liposomes generally, and as the term is used herein, are vesicles made of phospholipid molecules. It has become commonly recognized that liposomes provide a means of intro-ducing drugs or other regulatory substances into an animal or plant cell to modify cellular physiology. Since the realiza-tion that liposomes can have an important role in introducing drugs or a material which will modify cellular physiology into a cell, various methods have been proposed for creating or preparing liposomes including liposomes with a large internal aqueous space for entrapping a drug or other modifying mole-cule which is to be transferred such as described in "Proce-dure for Preparation of Liposomes with Large Internal AqueousSpace and High Capture by Reverse-Phase Evaporation," by Szoka and Papahadjopoulosj Proc. Natl. Acad. Sci., USA 75, 1978, pp.
4194-4198. Other methods are directed primarily to improved procedures for entrapping a drug or the like in the liposome, such as described in "Dehydration-Rehydration Vesicles: A
Simple Method for Hlgh Yield Drug Entrapment in Liposomes," by Kirby and Gregoriadls, Bio/re_hnology, November 1984, pp. 979-984. U.S. Patent No. 4,235,871 discloses a rnethod of encap-sulating nulnerous hiologically active materials in synthetic, oligolamellar lipid vesicles by providing a rnixture of lipid in an organic so]vent and an aqueous mixture o~ the rnaterial for encapsulating, emulsifying the provided mixture, rernoving the organic solvent, and suspending the resultant gel in water. Further, a variety o~ applications for liposoines as a transEer means have been suggested in nurnerous patents.
Thus, the above-noted '871 patent discloses numerous active compounds and compositions encapsulated in the liposorne for incorporation into cells. U.S. Patent No. 4,394,448 is directed specifically to the insertion of deoxyribonucleic acid (DNA) or fragments thereof into a living cell whereby the DNA or fragment is encapsulated in a lipid vesicle and the vesicle brought into contact with a cell whereby insertion occurs. U.S. Patent Nos. 4,199,565; 4,201,767; 4,261,975, and 4,235,877 disclose the incorporation of a viral or bacterial antigen into liposomes which contain a positively charged amino-containing surfactant which can be a quaternary ammonium halide salt. U.S. Patent No. 4,483,929 discloses an immuno-reactant liposome reagent for use in the determination of a chemical compound capable of entering into an immunospecific reaction with a known antibody. In all applications known to applicant, although it has been demonstrated that phospholipid liposomes are able to introduce their content into cultured cells as well as into cells of specific tissues of whole ani-mals, these liposomes have been shown to be relatively poor carriers. It is believed that these prior art liposomes introduce the carried substances, i.e., the drug, into cells by endocytic-like processes. Accordingly, only a small per-centage of the molecules which are entrapped within the lipo-somes reach the cytoplasm of the recipient cells. Moreover, when liposomes are injected into animals, as well as into humans, it has been established that up to a certain concen-tration, phospholipid liposomes are inert and not irnmunogenic.Only at relatively high concentrations are liposomes immuno-genic and toxic. These liposornes of the pr;or art, while capable of agglutinating cells, are not fusogenic and do not undergo and are unable to fuse with a cell membrane or induce cell~cell fusion.
Et has also ~een estahlished that envelopes of cer-tain animal viruses, such as those obtained from Sendai virus, are Eusogenic and have been shown to serve as an efficient carrier for the introduction of molecules into cultured cells as reported in "A New Method for Reconsititution of ~ighly Fusogenic Sendai Virus Envelopes," by Vainstein et al, Bio-chimica et Biophysica ~cta, 773 (198~), pp~ 181-188. The fusogenic activity of these virus is believed to be due to the presence of specific viral glycoproteins in the virus enve-lopes. It appears, however, that the presence of a protein in the fusogenic viral envelopes makes these vesicles immunogenic and, therefore, impractical for in _ iVO use. Injection of viral envelopes into animals induces the formation of specific antiviral antibodies, a fact which limits their use as a biological carrier in vivo.
rrhe reported literature establishes, therefore, that although phospholipid vesicles have been recognized as a potentially important tool for incorporating drugs and other cell-modifying materials into cells~ because of the limita~
tions of prior art liposomes this technique has experienced only limited success.

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Z8~3 . 4 ~'R:tM~I~Y OBJE:C'I'S Al`ID
GE:NI:R~I. Dl:SCR:[ f'T [ON Ol;` .lNVr,N'l'ION

Accorc]ingly, in one aspect the present invention seeks to provide liposomes which are fusogenic and which will fuse with a cell membrane and induce cell-to-cell fusion, and more par-ticularly provides phospholipid vesicles containing a quaternary ammonium hydroxide salt in the membrane of the vesicle, the salt being present in an amount sufficient to render the vesicles fusogenic.
In another and further aspect, the present inven-tion seeks to provide a method of producing liposomes which will E~se to a cell membrane and induce cell-to-cell Eus:ion and more particularly provides the method of rendering a phospholipid vesicle fusogenic comprising incorporating into the rnembrane of the vesicle a quaternary ammonium hydroxide salt.
More particularly, the present invention provides for incorporating a quaternary ammonium hydroxide salt into the membrane of a liposome either during the preparation of the liposorne or after the liposome is prepared. It has been found that the presence of the quaternary ammonium hydroxide salt in the phospholipid vesicle membrane permits the vesicle to fuse with a cell membrane and induce cell-to-cell fusion. If the vesicle is loaded with a drug or other cell-modifying substance, the content of the vesicle will be microinjected into the animal or plant cell. The ability of the quaternary ammonium hydroxide salt to convert the phospholipid vesicles from non-fusogenic to fusogenic vesicles is particularly surprising in that quaternary ammonium salts other than the hydroxide salts provides vesicles which are non-fusogenic. Thus, it has been found -that phospholipid vesicles containing quaternary ammonium halide salts, quaternary ammonium acetate salts and the like will not fuse with a cell and, accordingly, will not microinject the content of the vesicles into a cell. These vesicles react similarly to the known non-fusogenic vesicles, and apparently incorporate the content of vesicles in-to cells through an endocytic-like process. l'he fusogenic characteristics of the liposomes of the present invention, on the othex hand, permit a convenient means of incorpora-ting any cell-modifying substance which can be entrapped into a phospholipi~ vesicle directly into a plant or anima:L cell in substan-tially guantitative amounts.

,~

~Z~ 3 The quaternary ammonium hydroxide salt or incorpor-at;on into the wall or membrane of the phospholipld vesicle has the formula -L ~1 - N - R3 ~ -OH

wherein Rl is a large alkyl group or a combination of an alkyl and aryl radical so as to impart surfactant characteristics to the salt, and R2, R3, and R4 are branched chain alkyl radicals of from 1 to 20 carbon atoms, or an aryl radical, or R2 and R3 can together be a 5~membered or 6-rnembered heterocyclic radi-cal such as, for example, pyrrole or pyridine. Particularlypreferred compounds are the surfactants cetyl ~enzyldimethyl ammonium hydroxide, hexadecyltrimethyl ammonium hydroxide, cetyltrimethyl ammonium hydroxide, di-isobutyl cresoxy ethoxy ;~ ethyl dimethylbenzyl ammonium hydroxide, di-isobutyl phenoxy ethoxy ethyl dimethylbenzyl ammonium hydroxide, methyl dode-cylbenzyl trimethyl ammonium hydroxide, methyl dodecyl xylene bis(trimethyl ammonium hydroxide), N-alkyl (C12~C14~C16) dimethylbenzyl amrnonium hydroxide, and octylcresoxy ethoxy-ethyl dimethyl benzyl ammonium hydroxide. It is essential that the quaternary amrnonium hydroxide salt have surfactant characteristics and contain the hydroxy radical so as to impart fusogenic characteristics to the phospholipid vesicle.
The amount of the quaternary ammonium hydroxide salt contained in the vesicles will normally range from about 5 to 750 ~ug/l mg phospholipid and preferably from about 50 to 250 ~g quaternary ammonium hydroxide salt per 1 mg phospholipid.
The liposomes useful in accordance with the present invention can be any of the prior art liposomes. Illustrative liposomes include the natural and synthetic phosphocholine~
~90 containing lipid having one fatty acid chain of from 12 to 20 carbon ato~s and one fatty acid chain oE at least 8 carbon atoms exemp]ified by dimyristoylphosphatidylcholine, dioleoyl~
phosphatidylcholine, dipalrnitoylphosphatidylcholine, distear-oylphosphatidylcholine, phosphatidylcholine, and sphingomyelin;
as well as cholesterol and the like. The liposomes can be prepared by any of the well known published rnethods such as sonication of phospholipid E;uspensions, reverse evaporation, or by dehyclration of dried layers oE phospholipid molecules.
Suitable techniques are described in "Procedure for Prepara-tion of Liposomes with Large Internal Aqueous Space and MiyhCapture by Reverse~Phase Evaporation," by Szoka et al, pre-viously noted; "Dehyclration-Rehydration Vesicles: A S;mple Method for High Yield Drug Entrapment in Liposomes," by Kirby and Gregoriadis, previously noted, as well as other known techniques such as use of detergents (see Szoka and Papahadjo-poulos, Annu. Rev. Biophys. Bioeng., 9 (1980~, pp. ~67 480).
Additionally, the cell-modifying substance which can be en-trapped within the liposome and which can be mircroinjected ; into animal or plant cells through fusion with the liposomes of the present invention~can be any of the substances pre-viously suggested. It has been found that substances which can be~encapsulated in the liposome and microinjected înto a cell in accordance with the present invention include DNA and DNA fragments; pharmaceutically active compounds and com-positions thereof such as carbohydrates, nucleotides~ poly-nucleotides, both naturally occurring and synthetic; influenza vaccines and antigens, as well as other substances which can affect the physiology of animal and plant cells.
PRESENTLY PREFERRED EMBODIMENTS OF INVENTION
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Having described the-invention in general terms, the following two examples will illustrate presently preferred preparations of the fusogenic liposomes of the present inven-tion.

~ample 1 ____ .
Incorporation of Quaternary Ammoniurn ~ydroxide Salt into Liposome Membranes During_LIposome P paration. _ _

2 mg of an uncharged phospholipid, phosphotidyl cho-line (PC), and 1 mg cholesterol were dried Erorn their chloro-formic solution. The dry layer obtained from the PC and cholesterol is solubillzed with 1 ml of diethylether, and thé
solution obtained is added to a suspension containing 2 mg of octylcresoxy ethoxyethyl dimethyl benzyl ammonium hydroxide (Q--salt) in acetate buffer, at a pH of 7Ø The suspension obtained is then vigorously vortexed and briefly sonicated in a bath sonicator. Reverse evaporated, large unilamellar lipo-somes are then prepared according to the method described in "Procedure for Preparation of Liposomes with Large Internal Aqueous Space and High Capture by Reverse-Phase Evaporation,"
by S~oka et al, supra.
~ xcess, non-incorporated Q-salt is removed by the addition of SM-2 Bio-beads utiliæing the technique described in "A New Method for Reconsititution of Highly Fusogenic Sendai Virus Envelopes," by Vainstein et al, supra. Thus, about 50-80 mg of SM~2 Bio-beads are incubated with the above-dcscribed phospholipids and Q-salt for 40-60 minutes at room temperature, with gentle shaking, in a final volume of 1 ml of acetate buffer. A quantitative estimation revealed that about 30-40% of the added Q-salt is incorporated into the phospholi-pid bilayer (300-400 ~ug Q-salt/l mg PC). For preparation of liposomes loaded with any component of interest, the desired components, ~uch as drugs, enzymes, RNA or DNA, are added to the buffer in which the phospholipids are suspended, as de~
scr;bed in "Procedure for Preparation of Liposomes with Large Internal Aqueous Space and High Capture by Reverse-Phase Evaporation," by Szoka et al, supra.

~L2~;2J51~3 The method for the preparation of fusogenic l;posomes as set Eorth in Example 1 is not recornrnended when charged molecules are trapped within the liposomes. Under certain conditions and at a certain pH, any negatively charged cornpo-nent will be complexed to the quaternary ammonium hydroxide salt and precipi~ated with it. Since the quaternary ammonium hydroxide salt is positively charged, it will electrostati~
cally interact with any negatively charged molecule, such as the nucleic acids exemplified by DNA or RNA which are strongly negatively charged at neutral pH. The formation o such corn-plexes between the positively charged quaternary ammonium hydroxide salt and the negatively charged rnolecules decreases the trapping efficiency (number of molecules trapped within each liposome) and the fusogenic activity of the liposornes.
Accordingly, in Example 2, a second technique ~or preparation of Eusogenic liposomes is set forth in which direct contact between the quaternary ammonium hydroxide salt and the trapped molecules is avoided.
Example 2 Insertion of Q-Salt Into Already Prepared ! Loaded Liposomes.
Loaded liposomes are prepared as described in Exarnple 1 or in accordance with previously published methods described above. However, as opposed to the method described in Example 1, the Q-salt is not added during the preparation of the lipo-somes but added after the preparation of the loaded liposomes to a suspension containing loaded, resealed liposomes. Incu-bation of Q-salt with a suspension of resealed liposomes re-sults in the incorporation of the hydrophobic part of the Q-salt molecule into the liposome phospholipid bilayer.
The procedure is performed as follows: A suspension containing phospholipid vesicles ~liposomes) prepared from 2 mg of PC and 1 mg of cholesterol, suspended in ~00 pl of ~6~286~

acetate buffer, is added to a tube containing 3 mg of Q-salt in 20 ~1 of acetate buffer. The mixture obtained is vigor-o~sly vortexed, after which it is incubated for 10-15 minutes at 37C with gentle shaking. Excess, free non-incorporated Q-salt is removed by adsorption to SM-2 Bio-beads, as previously described. A quantitative estimation again revealed that about 30~ of the ac3ded Q-salt was incorporated into the phos-pholipid bilayer.
Ihis method of Example 2 can be advantageous for cer-tain preparations over the method of Example 1 in that mole-cules which are enclosed within the liposomes are trapped inside the liposome before the addition of the Q-salt; and therefore no interaction can take place between the enclosed material and the added surfactant. In additiorl, the fusogenic properties of liposomes bearing the Q-salt prepared by the method of Example 2 are superior to those of liposomes pre-pared by the technique of Example l.
.
The ability of liposomes composed of phosphatidyl-choline and cholesterol (PC/cholesterol liposomes) as de-scribed in Example 2 to induce cell-cell fusion was studied by incubation with human erythrocytes and with hepatoma tissue cultured cells (HTC) in comparison with controls. Q-salt which is octylcresoxy ethoxyethyl dimethyl benzyl ammonium hydroxide; C-salt which is octylcresoxy ethoxyethyl dimethyl benzyl ammonium chloride, and B-salt which is benzyldimethyl `~ hexadecyl ammonium bromide were incorporated into already -~ formed liposomes, as described in Example 2O In these experi-ments, either free Q-salt ~5-10 ~g) or free C-salt (5-10 ~g) or free B-salt (5-10 ~g) or liposomes bearing these ammonium quaternary molecules (20 ~g of PC) were incubated with a cell suspension (106 cells) for 15 minutes at 37C. Agglutination of cells and cell-~cell fusion were followed by observation in a phase microscope. The results are tabulated io Table I.

~lZ~i2863 T A B L E
Characterization o~ Liposomes Bearing Fusogenic and Non-Fusoqenic Quaternary Ammonium Salts _, _ __ ~gglutina ion Cell-Cell Fu _ on Eluman Erythrocytes Incubated with __ PC/Cholesterol Liposomes........
Free Q-Salt (OH- Salt)..... ,....... + -~
PC/Cholesterol Liposomes 10 Bearing Q-Salt ~OH- Salt)......... ... -~ +~+
Free C-Salt (Cl- Salt).......... ... +
PC/Cholesterol C-Salt (Cl- Salt)...................... ... +
Free B-Salt................ ~....... +
PC/Cholesterol Liposomes Bearing B-Salt..................... +

HTC Incubated with:
PC/Cholesterol Liposomes~.......... - -Free Q-Salt (OH- Salt)............. + -~
20 PC/Cholesterol Liposomes Bearing Q-Salt (OH- Salt).......... + +~-+
Free C-Salt (Cl- Salt)............. ~ -PC/Cholesterol Liposomes Bearing C-Salt (Cl- Salt).......... ~ -B-Salt............................. +
~; PC/Cholesterol Liposomes Bearing B-SaltO.................... +

The results summarized in Table I establish:
(1) Liposomes composed of PC/cholesterol but not bearing any of the ammonium quaternary molecules did not have any effect on the agglutination or cell~cell fusion of the human erythrocytes or the HTC cells.
(2) All ammonlum quaternary molecules used (Q-salt, ~::: : : :
: -:

~ ~Z~i3 C-salt and s-salt), either in thelr free form or incor-porated into liposomes, were able to induce cell-cell agglutination. This is believed due to attachment of the positively char~ed quaternary ammonium salt molecules to the ne~atively charged cell membranes.

(3) Only -the ~-salt, i~e., the hydroxide salt, either in ~ree form or incorporated into liposomes, was able to induce cell-cell fusion. Induction o~ cell-cell fusion by linosomes bearing Q-salt was much higher than hy ~ree Q-salt. The ~uafernary ammonium chloride and bromide salts did not induce cell cell fusion despite the fact that they are able to bind to cell membranes, as can be inferred from their ability to induce cell-cell agglutina-tion.
Pusion of liposomes bearing a quaternary ammonium hydroxide salt with cells is also shown from their ability to microinject their content into the cytoplasm oE animal and plant cells. Three systems have been used to ascertain the fusion of liposomes bearing ~-salt withj and microinjection of their content into living cells, as follows~
~-~ (1) The toxin ricin A-chain was enclosed within the liposomes, and these loaded liposomes were incubated with cells in culture such as HTC. Ricin A inhibits cell pro-tein synthesis, and consequently kills cells only when present inside the cell. As opposed to the whole molecule of ricin (ricin containing both A and B subunits), the ricin A chain cannot, by itself, enter cells. Thus, when present outs;de the cell, it does not have any lethal effect.
(2) The SV40-DNA, namely DNA extracted from the virus SV~o, was enclosed within the liposomes. Liposomes loaded with SV40-DNA were ;ncubated w;th cultured HTC.
Microinjection of the SV40-DNA into the HTC cells was o~served by the appearance of a specific protein, the ~2~ i3 SV40-T-antigen. Synthesis of SV40-T--antigen is incluced only when SV~o~DNA is introduced int:o the cell-cytoplasm from which it is transferred to the cell nucleus.
It i5 also noted that (a) appearance of SV40-r-antigen occurred only in living cells, as it required active protein synthesis; and (b) free SV~o~DNA molecules, when incubated with living cells, are unable to enter into the cells and, consequently, these free rnolecule.s do not induce synthesis oE the T-antigen.
(3) Fluorescently labeled protein molecules, namely ~luorescently labeled bovine serum albumin, BSA; were enclosed within the liposomes. Loaded liposomes were then incubated with either plant protoplasts or a suspension of plant cells. Fusion-mediated microinjection was observed by following the appearance of intracellular fluorescence.
(a) Fusion-Mediated MicroInjection of Ricin A Chain by Fusogenic ripOsOmes.
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The results summarized in Table II show that only liposomes loaded with ricin A chain and bearing the quaternary ammonium hydroxide salt (Q-salt) caused strong inhibition of protein synthesis and a high degree of killing in HTC. No killing was observed either with unloaded liposomes or with free ricin A chain. As can be seen, very little killing above the controls was caused by liposomes bearing the quaternary ammonium chloride salt (C-salt), indicating no, or substan-tially no liposome-cell fusion.

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1~ ~ 13 ~ [
~bility of Fusogenic ripo<;omes roaded With Ricin A*to In11ihit Protein S~nthesis and to Kill HTC
Inhibition of I'rotein HrC Killed 1-3TC Incubated with _ _ _ _ ~SynthcSlS (~
PC/Cholesterol IJ;posomes........... 0 0 Free Ricin A Chain.................. 5 4 PC/Cholesterol Liposomes l0 Loaded With Ricin*A................... 6 7 PC/Cholesterol Liposomes Bearing Q-Salt (OH~ Salt~........... S
PC/Cholesterol Li~osomes Loaded With Ricin A Chain and Bearing ~-Salt (OH- Salt)........... 90 95 PC/Cholesterol Liposomes Loaded With Ricin*A Chain and Bearing C-Salt (Cl- Salt)........... 17 14 Ricin*A chain was first trapped within PC/cholesterol liposomes using the technique described in "Procedure for Preparation of Liposomes with Large Internal Aqueous Space and High Capture by Reverse-Phase Evaporation," by Szoka et al, supra, after which quaternary ammonium salt (either the OH- or the Cl- salts) was incorporated into the liposome bilayer, as above described. In the experiments, 10-20 /ug of liposomes were incubated with 106 cells for 30 minutes at 37C, a period during which the liposomes interacted with the cells. At the end of the incubation periods, the cells were washed and fur-ther incubated in a growth medium for another twelve hours, following which protein synthesis (by 3H-leucine incorpora-tion? and viability of cells (by staining with Trypan hlue) were e~stimated.
* Trade mark .~
~' ~Zi~363 (b) Fusion-Mediated MicroInjection of SV40-DNA.
The ~csults in Tal~le III demonstrate that ~usoyenic liposomes can be used for the microinjection of active yenes (DNA molecules) into cells in culture such as IITC. All ex~
perimental conditiolls and incubation with EITC were ~s de-scribed in Table II Eor ricin A. SV40-DNA was cntrapped in liposomes as described in "Procedure for Preparation of Liposomes with Large Internal Aqueous Space and lligh Capture by Reverse-Phase Ævaporation," by Szoka et al, su~ra. Appear ance of SV40 T-antigen was estimated as above described with the use of specific, fluorescently labeled anti~SV40-T~antigen antibody.

T A B L E III
Ability of Loaded Fusogenic Liposomes (Liposomes Bearing Q-Salt) to Micro-Inject SV40-DNA
-- - HTC Incubated with L;posomes SV40-T-Antigen Positive Loaded with SV40-DNACells (% of total PC/Cholesterol Liposomes.................... 0 ~; PC/Cholesterol Bearing Q-Salt (OH- Salt)....... ,....................... 10 - 15 PC/Cholesterol Bearing C-Salt (Cl- Salt)................~ ............. 0 :: ~

.
The specific T-antigen appeared in the nucleus of 10-15% of celIs incubated with liposomes loaded with SV~o-DNA
and bearing Q-salt (OH- salt). No T antigen appeared in cells incubated with the PC/cholesterol loaded with SV40-DNA but bearing the C-salt (Cl- salt).

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~2G28~i3 (c) Fusion--Mediated Injection of Fluorescent BSA into Pet_nia _ybrida Plant Protoplast~
and Plant Cell Suspension.
Protoplasts oE cells from Petun a ~brida were pre-pared by conventional methods as described in the literature.
Fluorescent BSA was enclosed within liposomes as described ~or ricin A chain, and t:hen Q-salt was incorporated into the membrane of the loaded liposomes as descrihed above., In the experiment, 10~~0 ~g of loaded liposomes were incubated with 5 x 10 protoplasts or plant cells. AEter 30-60 minutes of incubation at 2~C, the appearance of intracellular fluores-cence was followed by the use of ~luorescence microscopy.

T A B L E IV_ Fusion~Mediated Injection o~ Fluorescent Bovine Serum Albumin into Petunia Hybrida Plant Protoplasts and Plant Cell Suspension Appearance of Intracellular Fluorescence t% of total cells) 20 Petunia Protoplasts Incubated with ' `,~ Fluorescent BSA-Loaded Liposomes PC/Cholesterol.........O......................... 0 PC/Cholesterol Bearing Q-salt (OH- Salt)........ 80 Petunia Cell Suspension Incubated with Fluorescent BSA~Loaded liposomes PC/Cholesterol.................................. 0 PC/Cholesterol Bearing p,,-salt (~H- Salt)...... 60 From the results in Table IV, it is apparent that liposomes bearing Q-salt (OH~ salt) can also microinject their content into plant protoplasts. The appearance of fluores-~LZ~ 8~i3 cence can rcsult only frorn Eusion of the loaded liposomes with the plant protop]asts. Any other process would lead to another form appearance of fluorescence. The results in Table IV show that, in addition to fusion lor microinjection) to plant protoplasts, loaded liposomes were able to Euse and microinject their content into plant cells that are surrounded by cell wall. Due to the presence of the highly charged ~-salt, the liposomes appear to be able to cross the cell wall and fuse with the cell membrane.
Applications of the liposomes Bearing A ~uaterna~Ammonium Hvdroxide Salt.
Having illustrated the preparation of liposomes con-taining quaternary ammonium ilydroxide salts and having demon-strated the ability of the liposomes containing the quaternary ammonium hydroxide salt in the vesicle membranes to fuse with cells and to induce cell-to-cell fusion, microinjecting the content of the vesicles into the cell, various applications of the liposomes disclosed in this invention are set forth.
(1) Use of the Free Q-Salt (OH-) Or Liposomes Bearing Q-Salt IOH-) As A Rea~ent to Induce Cell-Cell Fusion.
Animal cell-cell fusion as demonstrated for the pre-sently disclosed liposomes is of paramount importance for gene expression studies. Further, plant protoplast fusion has many commercial applications which can lead to the development of new plant species of improved properties.
(2) Use of Fusogenic Liposomes for Micro-Injection of Various Components Into Animal and Plant Cells Grown in Culture.
Loaded, fusogenic liposomes can be used for the microinjection of DNA, RNA proteins (enzymes, hormones), and drugs into cells in culture. In addition, they can be used for microinjection of organelles or any other components or 6~

molecules wh;ch can be enclosed within the liposornes. Micro-injection o~ l)NA, RNA, or organelles into cells, especially into plant protoplasts or plant cells, has substantial colt,rner-cial application as does the entire field of genetic engineer-;n~ of plant and animal cells. Furthermore, based on expari-ments with plant and animal cells, liposomes bearing a quaternary ammonium hydroxide salt will be able to fuse and microinject their content into any cells with which they can be incubated. Accordingly, these liposomes will be abLe to fuse with bacteria, yeast, and algae protoplasts.
(3) Use of Fusogenic Liposomes for the Delivery of Various Components into Cells in vivo, Narnel~ Tissue or Cells of Whole Animals .. . . .. ~
Fusogenic liposomes can be used, after injection into laboratory animals or human beings, as a vehicle for the deli-very of drugs, hormones, or proteins such as enzymes and DNA.
This method may be used for drug, enzyme and for gene therapy.
Non-fusogenic, loaded liposomes are currently being used as carriers of drugs for therapy. However, fusogenic liposomes can be far more efficient than non-fusogenic liposomes as biological carriers, since they inject their content directly into cell cytoplasm~
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(4) The Use of Fusogenic Liposomes for the Development of a New, Non--Isotopic, Sensitive Assay SYstam for Diaanostic Purposes Liposomes bearing quaternary ammonium hydroxide salts will fuse with other liposomes. Fusion between liposomes leads to the laakage of their content. This leakage is speci-fically due to the interaction and fusion between the liposo-mes. Due to the prasence of the positively-charged quaternary ammonium hydroxide salt molecules, liposomes bearing these ammonium quaternary molecules do not interact and repulse each other. As noted above, they can interact and fuse only with liposomes leaking the quaternary ammonium hydroxide salt.
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~Z~i2~3 Ilowever, any two rnolecules capable of interacting with each other with high affinity will force liposomes bearing the quaternary ammonium hydroxide salt to fuse with each other by overcoming the repu]sive forces oE the positive charge. Ac-cordingly, iE a specific antigen is incorporated into the liposome membranes bearing the quaternary arnmonium hydroxide salt, an antibody prepared against such antigen will force the liposomes to interact. The interaction betwecrl liposomes bearing quaternary ammonium hydroxide salt will lead to fusion and re]ease of the liposomal content. Any quantitative esti-mation of the re]ease process (release of fluorescent dye or any other reagent~ will be a measure of the antigen-antibody interaction. Thus, fusion between liposomes bearing a quater-nary ammonium hydroxide salt can be used to quantitatively estimate the interaction between antibody and its antigen or hormone and its appropriate receptor. This system, therefore, can be used to estimate any small quantities of antigen or hormone present in any biological fluid.
As will be apparent to one skilled in the art, various modi~ications can be made within the scope of the aforesaid description. Such modlfications being within the ability of one skilled in the art form a part of the present invention and are embraced by the appended claims.

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Claims

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. Phospholipid vesicles containing a quaternary ammonium hydroxide salt in the membrane of said vesicle, said salt being present in an amount sufficient to render said vesicles fusogenic.

2. The phospholipid vesicle of claim 1 wherein said quaternary ammonium hydroxide salt is a member of the group consisting of cetyl benzyldimethyl ammonium hydroxide, hexadecyltrimethyl ammonium hydroxide, cetyltrimethyl ammonium hydroxide, di-isobutyl cresoxy ethoxy ethyl dimethylbenzyl ammonium hydroxide, di-isobutyl phenoxy ethoxy ethyl dimethyl-benzyl ammonium hydroxide, methyl dodecylbenzyl trimethyl ammonium hydroxide, methyl dodecyl xylene bis(trimethyl ammo-nium hydroxide), and octylcresoxy ethoxyethyl dimethyl benzyl ammonium hydroxide.

3. The phospholipid vesicles of claim 1 wherein said quaternary ammonium hydroxide salt is octylcresoxy ethoxyethyl dimethyl benzyl ammonium hydroxide.

4. The phospholipid vesicles of claim 1 wherein said phospholipid is a member of the group consisting of phosphatidylcholine, dimyristoylphosphatidylcholine, dioleoylphosphatidylcholine, dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, sphingomyelin, cholesterol, and mixtures thereof.

5. The phospholipid vesicle of claim 1 having encap-sulated therein DNA or a DNA fragment.

6. The phospholipid vesicle of claim 1 having encap-sulated therein a pharmaceutically active compound or com-position.

7. The method of rendering a phospholipid vesicle fusogenic comprising incorporating into the membrane of said vesicle a quaternary ammonium hydroxide salt.

8. The method of claim 7 wherein said quaternary ammonium hydroxide salt is a member of the group consisting of cetyl benzyldimethyl ammonium hydroxide, hexadecyltrimethyl ammonium hydroxide, cetyltrimethyl ammonium hydroxide, di-isobutyl cresoxy ethoxy ethyl dimethylbenzyl ammonium hydroxide, di-isobutyl phenoxy ethoxy ethyl dimethylbenzyl ammonium hydroxide, methyl dodecylbenzyl trimethyl ammonium hydroxide, methyl dodecyl xylene bis(trimethyl ammonium hydroxide), and octylcresoxy ethoxyethyl dimethyl benzyl ammonium hydroxide.

9. The method of claim 7 wherein said quaternary ammonium hydroxide salt is octylcresoxy ethoxyethyl dimethyl benzyl ammonium hydroxide.

10. The method of claim 7 wherein said quaternary ammonium hydroxide salt is incorporated during the formation of said phospholipid.

11. The method of claim 7 wherein said quaternary ammonium hydroxide salt is incorporated into said vesicle membrane after the formation of the phospholipid.