GB2188900A - Fusogenic phospholipid vesicles - Google Patents

Abstract

Phospholipid vesicles (liposomes) containing a quaternary ammonium hydroxide salt in the membranes of the vesicles are described. The phospholipid vesicles containing the quaternary ammonium hydroxide salt in the vesicle membranes, in addition to their ability to attach to animal and plant cells, will fuse with a cell membrane and induce cell-to-cell fusion, providing a convenient way of transferring a material such as a drug into a cell.

Description

SPECIFICATION Fusogenic liposomes and method of making same Field ofinvention This invention relates to liposomes. More particularly, this invention relates to liposomes containing a quaternary 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.

Background 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 introducing drugs or other regulatory substances into an animal or plant cell to modify cellular physiology. Since the realization 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 molecule which is to be transferred such as described in "Procedure for Preparation of Liposomes with Large Internal Aqueous Space and High Capture by Reverse-Phase Evaporation," by Szoka and Papahadjopoulos, 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 High Yield Drug Entrapment in Liposomes," by Kirby and Gregoriadis, Bio/Technology, November 1984, pup.979984. U.S.

Patent No. 4,235,871 discloses a method of encapsulating numerous biologically active materials in synthetic, oligolamellar lipid vesicles by providing a mixture of lipid in an organic solvent and an aqueous mixture of the material for encapsulating, emulsifying the provided mixture, removing the organic soivent, and suspending the resultant gel in water. Further, a variety of applications for liposomes as a transfer means have been suggested in numerous patents. Thus, the above-noted '871 patent discloses numerous active compounds and compositions encapsulated in the liposome for incorporation into cells. U.S. Patent No. 4,394,448 is directed specifically to the insertion of deoxyribonucieic 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 aminocontaining surfactant which can be a quaternary ammonium halide salt. U.S. Patent No. 4,483,929 discloses an immunoreactant 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 animals, 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-iike processes.Accordingly, only a small percentage of the molecules which are entrapped within the liposomes 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 concentration, phospholipid liposomes are inert and not immunogenic. Only at relatively high concentrations are liposomes immunogenic and toxic. These liposomes of the prior 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.

It has also been established that envelopes of certain animal viruses, such as those obtained from Sendai virus, are fusogenic 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 Highly Fusogenic Sendai Virus Envelopes," by Vainstein et al, Biochimica et Biophysica Acta, 773 (1984), pp. 181-188. The fusogenic activity of these virus is believed to be due to the presence of specific viral glycoproteins in the virus envelopes. It appears, however, that the presence of a protein in the fusogenic viral envelopes makes these vesicles immunogenic and, therefore, impractical for in vivo 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.

The reported literature estabiishes, 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 limitations of prior art liposomes this technique has experienced only limited success.

Primary objects and general description ofinvention Accordingly, it is a primary object of the present invention to provide liposomes which are fusogenic and which will fuse with a cell membrane and induce cell-to-cell fusion.

It is another primary object of the present invention to provide a method of producing liposomes which will fuse to a cell membrane and induce cell-to-cell fusion.

The objects of the present invention are accomplished by incorporating a quaternary ammonium hydroxide salt into the membrane of a liposome either during the preparation of the liposome 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 nonfusogenic. 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 into cells through an endocytic-like process. The fusogenic characteristics of the liposomes of the present invention, on the other hand, permit a convenient means of incorporating any cell-modifying substance which can be entrapped into a phospholipid vesicle directly into a plant or animal cell in substantially quantitative amounts.

The quaternary ammonium hydroxide salt for incorporation into the wall or membrane of the phospholipid vesicle has the formula

wherein R1 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-membered heterocyciic radical such as, for example, pyrolle or pyridine.Particularly preferred compounds are the surfactants cetyl benzyldimethyl ammonium hydroxide, hexadecyitrimethyl 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), N-alkyl (Ca2,C14,Cl6) dimethylbenzyl ammonium hydroxide, and octylcresoxy ethoxyethyl dimethyl benzyl ammonium hydroxide.It is essential that the quaternary ammonium 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 > 9 /1 mg phospholipid and preferably from about 50 to 250 ffi 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-containing lipid having one fatty acid chain of from 12 to 20 carbon atoms and one fatty acid chain of at least 8 carbon atoms exemplified by dimyristoylphosphatidylcholine, dioleoylphosphatidylcholine, dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, phosphatidylcholine, and sphingomyeiin; as well as cholesterol and the like. The liposomes can be prepared by any of the well known published methods such as sonication of phospholipid suspensions, reverse evaporation, or by dehydration of dried layers of phsopholipid molecules.Suitable techniques are described in "Procedure for Preparation of Liposomes with Large Internal Aqueous Space and High Capture by Reverse-Phase Evaporation," by Szoka et al, previously noted; "Dehydration-Rehydration Vesicles: A Simple 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 Papahadjopoulos, Annu. Rev. Biophys Bioeng., 9(1980), pp.467480). Additionally, the cell-modifying substance which can be entrapped 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 previously suggested.It has been found that substances which can be encapsulated in the liposome and microinjected into a cell in accordance with the present invention include DNA and DNA fragments; pharmaceutically active compounds and compositions thereof such as carbohydrates, nucleotides, polynucleotides, 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 Having described the invention in general terms, the following two examples will illustrate presently preferred preparations of the fusogenic liposomes of the present invention.

EXAMPLE 1 Incorporation of quaternary ammonium hydroxyide salt into liposome membranes during liposome preparation 2 mg of an uncharged phospholipid, phosphotidyl choline (PC), and 1 mg cholesterol were dried from their chloroformic solution. The dry layer obtained from the PC and cholesterol is solubilized with 1 ml of diethylether, and the 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 7.0. The suspension obtained is then vigoroulsyvortexed and briefly sonicated in a bath sonicator. Reverse evaporated, large unilamellar liposomes 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 Szoka et al, supra.

Excess, non-incorporated O-salt is removed by the addition of SM-2 Bio-beads utilizing 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-described phospholipids and Q-salt for 40-60 minutes at room temperature, with gently 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 phospholipid bilayer (300-400 yg Q-salt/l mg PC). For preparation of liposomes loaded with any component of interest, the desired components, such as drugs, enzymes, RNA or DNA, are added to the buffer in which the phospholipids are suspended, as described in "Procedure for Preparation of Liposomes with Large Internal Aqueous Space and High Capture by Reverse-Phase Evaporation," by Szoka et al, supra.

The method for the preparation of fusogenic liposomes as set forth in Example 1 is not recommended when charged molecules are trapped within the liposomes. Under certain conditions and at a certain pH, any negatively charged component will be complexed to the quaternary ammonium hydroxide salt and precipitated with it. Since the quaternary ammonium hydroxide salt is positively charged, it will electrostatically 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 of such complexes between the positively charged quaternary ammonium hydroxide salt and the negatively charged molecules decreases the trapping efficiency (number of molecules trapped within each liposome) and the fusogenic activity of the liposomes.

Accordingly, in Example 2, a second technique for preparation of fusogenic liposomes is set forth in which direct contact between the quaternary ammonium hydroxide salt and the trapped molecules is avoided.

EXAMPLE 2 Insertion of O-salt into already prepared, loaded liposomes Loaded liposomes are prepared as described in Example 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 liposomes but added after the preparation of the loaded liposomes to a suspension containing loaded, resealed liposomes. Incubation of O-salt with a suspension of resealed liposomes results in the incorporation of the hydrophobic part of the O-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 400 iil of acetate buffer, is added to a tube containing 3 mg of Q-salt in 20 iil of acetate buffer. The mixture obtained is vigorously vortexed, after which it is incubated for 10-15 minutes at 37"C with gentle shaking. Excess, free non-incorporated O-salt is removed by adsorption to SM-2 Bio-beads, as previously described.A quantitative estimation again revealed that about 30% of the added Q-salt was incorporated into the phospholipid bilayen This method of Example 2 can be advantageous for certain preparations over the method of Example 1 in that molecules 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 addition, the fusogenic properties of liposomes bearing the Q-salt prepared by the method of Example 2 are superiorto those of liposomes prepared by the technique of Example 1.

The ability of liposomes composed of phosphatidylcholine and cholesterol (PC/cholesterol liposomes) as described 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 wh ich 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 2.In these experiments, either free Q-salt (5-10 ,lug) or free C-salt (5-10 019) or free B-salt (S10 pwg) or liposomes bearing these ammonium quaternary molecules (20 ,ag of PC) were incubated with a cell suspension (106 cells) for 15 minutes at 370C. Agglutination of cells and cell-cell fusion were followed by observation in a phase microscope. The results are tabulated in Table I.

TABLE I Characterization of liposomes bearing fusogenic and non-fusogenic quaternary ammonium salts Agglutination Cell-cell fusion Human erythrocytes Incubated with: PC/Cholesterol Liposomes Free Q-salt (OH- Salt) + + PC/Cholesterol Liposomes Bearing O-salt (OH- Salt) + Free C-Salt (CISalt) + PC/Cholesterol C-Salt (Cl-Salt) + Free B-Salt + PC/Cholesterol Liposomes Bearing B-Salt + HTC Incubated with: PC/Cholesterol Liposomes Free O-Salt (OH- Salt) + + 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-Salt + 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 ammonium quaternary molecules used (0-salt, C-salt and B-salt), either in their free form or incorporated into liposomes, were able to induce cell-cell agglutination. This is believed due to attachment of the positively charged quaternary ammonium salt molecules to the negatively charged cell membranes.

(3) Only the Q-salt, i.e., the hydroxide salt, either in free form or incorporated into liposomes, was able to induce cell-cell fusion. Induction of cell-cell fusion by liposomes bearing O-salt was much higher than by free Q-salt. The quaternary 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 agglutination.

Fusion of liposomes bearing a quaternary ammonium hydroxide salt with cells is also shown from their ability to microinject their content into the cytoplasm of animal and plant cells. Three systems have been used to ascertain the fusion of liposomes bearing O-salt with, 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 protein synthesis, and consequentiy 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 outside the cell, it does not have any lethal effect.

(2) The SV40-DNA, namely DNA extracted from the virus SV40, was enclosed within the liposomes.

Liposomes loaded with SV40-DNA were incubated with cultured HTC. Microinjection of the SV40 DNA into the HTC cells was observed by the appearance of a specific protein, the SV40-T-antigen. Synthesis of SV40-Tantigen is induced only when SV40-DNA is introduced into the cell-cytoplasm from which it is transferred to the cell nucleus.

It is also noted that (a) appearance of SV40-T-antigen occurred only in living cells, as it required active protein synthesis; and (b) free SV40-DNA molecules, when incubated with living cells, are unable to enter into the cells and, consequently, these free molecules do not induce synthesis of the T-antigen.

(3) Fluorescently labeled protein molecules, namelyfluorescently 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 intracellularfluorescence.

(a) Fusion-mediated micro injection of ricin A chain by fusogenic liposomes 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 substantially no liposome-cell fusion.

TABLE II Ability of fusogenic liposomes loaded with ricin A to inhibitprotetin synthesis and to kill HTC Inhibition of Protein HTC Killed HTC Incubated with Synthesis (%) (%) PC/Cholesterol Liposomes O 0 Free Ricin A Chain 5 4 PC/Cholesterol Liposomes Loaded With Ricin A 6 7 PC/Cholesterol Liposomes Bearing Q-Salt (OH- Salt) 5 8 PC/Cholesterol Liposomes Loaded With Ricin A Chain and Bearing Q-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 llg of liposomes were incubated with 106 cells for 30 minutes at 37"C, a period during which the liposomes interacted with the cells. At the end of the incubation periods, the cells were washed and further incubated in a growth medium for another twelve hours, following which protein synthesis (by 3H-leucine inporporation) and viability of cells (by staining with Trypan blue) were estimated.

(b) Fusion-mediated microinjection of SV40-DNA The results in Table III demonstrate that fusogenic liposomes can be used for the microinjection of active genes (DNA molecules) into cells in culture such as HTC. All experimental conditions and incubation with HTC were as described in Table II for ricin A. SV40-DNA was entrapped in liposomes as described in "Procedure for Preparation of Liposomes with Large Internal Aqueous Space and High Capture by Reverse-Phase Evaporation," by Szoka et al, supra. Appearance of SV40-T-antigen was estimated as above described with the use of specific, fluorescently labeled anti-SV40-T-antigen antibody.

TABLE Ill Ability ofloaded fusogenic liposomes (liposomes bearing O-salt) to micro-inject SV40-DNA HTC Incubated with Liposomes SV40-T-Antigen Positive Loaded with SV40-DNA Cells (% of total) PC/Cholesterol Liposomes 0 PC/Cholesterol Bearing O-Salt (OH- Salt) 10-15 PC/Cholesterol Bearing C-Salt (Cl- Salt) O The specific T-antigen appeared in the nucleus of 1 O1 5% of cells incubated with liposomes loaded with SV40-DNA and bearing O-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).

(c) Fusion-mediated injection of Fluorescent BSA into Petunia hybrida plant protoplasts and plant cell suspension Protoplasts of cells from Petunia hybrida were prepared by conventional methods as described in the literature. Fluorescent BSA was enclosed within liposomes as described for ricin A chain, and then Q-salt was incorporated into the membrane of the loaded liposomes as described above. In the experiment, 10-20 ssLg of loaded liposomes were incubated with 5x 105 protoplasts or plant cells. After 30-60 minutes of incubation at 28"C, the appearance of intracellular fluorescence was followed by the use of fluorescence microscopy.

TABLE IV Fusion-mediated injection oPfluorescent of fluorescent bovine serum albumin into petunia hybrida plant protoplasts and plant cell suspension Appearance or Intracellular Fluorescence (% of total cells) Petunia Pro to plasts Incubated with Fluorescent BSA-Loaded Liposomes PC/Cholesterol 0 PC/Cholesterol Bearing Q-Sait (OH- Salt) 80 Petunia Cell Suspension Incubated with Fluorescent BSA-Loaded liposomes PC/Cholesterol o PC/Cholesterol Bearing O-salt (OH- 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 fluorescence can result only from fusion of the loaded liposomes with the plant protoplasts. Any other process would lead to another form appearance of fluorescence. The results in Table IV show that, in addition to fusion (or microinjection) to plant protoplasts, loaded liposomes were able to fuse and microinject their content into plant cells that are surrounded by cell wall. Due to the presence of the highly charged Q-salt, the liposomes appear to be able to cross the cell wall and fuse with the cell membrane.

Applications of the liposome bearing A quaternary ammonium hydroxide salt Having illustrated the preparation of liposomes containing quaternary ammonium hydroxide salts and having demonstrated 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 (OH-) as a reagent to induce cell-cell fusion Animal cell-cell fusion as demonstrated for the presently disclosed liposomes is of paramount importance for gene expression studies. Further, plant protoplastfusion has many commercial applications which can lead to the development of new plant species of improved properties.

(2) Use of fusogenic liposomes for microinjection 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 molecules which can be enclosed within the liposomes. Microinjection of DNA, RNA, or organelles into cells, especially into plant protoplasts or plant cells, has substantial commercial application as does the entire field of genetic engineering of plant and animal cells. Furthermore, based on experiments 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, namely tissue or cells of whole animals Fusogenic liposomes can be used, after injection into laboratory animals or human beings, as a vehicle for the delivery 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.

(4) The use of fusogenic liposomes for the development of a new, non-isotopic, sensitive assay system for diagnostic purposes Liposomes bearing quaternary ammonium hydroxide salts will fuse with other liposomes. Fusion between liposomes leads to the leakage of their content. This leakage is specifically due to the interaction and fusion between the liposomes. Due to the presence 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. However, any two molecules capable of interaction with each other with high affinity will force liposomes bearing the quaternary ammonium hydroxide salt to fuse with each other by overcoming the repulsive forces of the positive charge. Accordingly, if a specific antigen is incorporated into the liposome membranes bearing the quaternary ammonium hydroxide salt, an antibody prepared against such antigen will force the liposomes to interact. The interaction between liposomes bearing quaternary ammonium hydroxide salt will lead to fusion and release of the liposomal content any quantitative estimation of the release process (release of fluorescent dye or any other reagent) will be a measure of the antigen-antibody interaction. Thus, fusion between liposomes bearing a quaternary 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 modifications can be made within the scope of the aforesaid description. Such modifications being within the ability of one skilled in the art form a part of the present invention and are embraced by the appended claims.

Claims (11)

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 hydrox ide, 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 octylcre soxy 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, dipalmitoylphosphatidy Icholine, distearoylphosphatidylcholine, sphingomyelin,-cholesterol, and mixtures thereof.

5. The phospholipid vesicle of claim 1 having encapsulated therein DNA or a DNA fragment.

6. The phospholipid vesicle of claim 1 having encapsulated therein a pharmaceutically active compound or composition.

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, hexadecyltrimethyi ammonium hydroxide, cetyl trimethyl 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 ethoxy ethyl dimethyl benzyl ammonium hydroxide.

9. The method of claim 7 wherein said quaternary hydroxide salt is oxtylcresoxy 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.