SE464448B - FUSOGEN Phospholipid Vesicle Containing A Quaternary Ammonium Hydroxide Salt In The Membrane And A Procedure For Its Preparation - Google Patents

Description

464 448 Furthermore, a variety of uses of liposomes as transfer agents have been proposed in numerous patents. Thus, the aforementioned patent discloses 4,235,871 numerous active compounds and preparations encapsulated in the liposome for incorporation into cells. U.S. U.S. Patent 4,394,448 is particularly directed to the introduction of deoxyribonucleic acid (DNA) or fragments thereof into a living cell, wherein the DNA or fragment thereof is encapsulated in a lipid vesicle and contacted with a cell so that the introduction occurs . U.S. Patents 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 may be a quaternary ammonium halide salt. U.S. U.S. Patent 4,483,929 discloses an immunoreactant liposome reagent capable of involved in an immunospecific reaction with a known antibody. Although it has been shown that phospholipid liposomes are capable of introducing their contents into cultured cells as well as into cells of specific tissues in whole animals, these liposomes have been known to be only relatively poor carriers in all known uses. It is believed that these known liposomes introduce the substances carried by them, ie the drugs, into the cells by endocytosis-like processes. Consequently, only a small proportion of the molecules trapped in the liposomes will reach the cytoplasm of the recipient cells. Furthermore, it has been found that when liposomes are injected into animals, including humans, the phospholipid liposomes are inert and non-immunogenic up to a certain concentration. Only at relatively high concentrations do the liposomes become immunogenic and toxic. Although these prior art liposomes are capable of agglutinating cells, they are not fusogenic and are unable to fuse with a cell membrane or induce fusion of cells with each other. 464 448 It has also been found that the envelopes of certain animal viruses, such as those derived from Sendai viruses, are fusogenic, and have been shown to serve as an effective carrier for the introduction of molecules into cultured cells, as set forth in "A New Method for Reconstitution of Highly Fusogenic Sendai Virus Envelopes ”, by Vainstein et al., Biochimica et Biophysica Acta, 773 (1984), pp. 181-188. The fusogenic activity of these viruses is likely to be due to the presence of specific viral glycoproteins in the virus envelopes However, the presence of a protein in the fusogenic viral envelopes appears to render these vesicles immunogenic and therefore impractical for use in vivo. Injection of the viral envelopes into animals induces the formation of specific antiviral antibodies, which limits their usefulness. as a biological carrier in vivo.

The reported literature therefore shows that atrophospholipid vesicles have been recognized as a potentially important tool for incorporating drugs and other cell modifying materials into cells, this technique due to the limitations of the known liposomes has had only limited success.

Accordingly, it is the principal object of the present invention to provide liposomes which are fusogenic and which fuse with a cell membrane and induce fusion of cells with each other.

It is another object to provide a process for producing liposomes which fuse with a cell membrane and induce fusion of cells.

The objects of the present invention are achieved by incorporating a quaternary ammonium hydroxide salt into a membrane of a liposome either during the preparation of the liposome or after the preparation. It has been found that the presence of the quaternary ammonium hydroxide salt in the membrane of the phospholipid vesicle allows the vesicle to fuse with a cell membrane and induce fusion of cells. If the vesicle is loaded with a drug or other cell-modifying substance, the contents 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 given that quaternary ammonium salts other than the hydroxide salts yield non-fusogenic vesicles. Thus, it has been found that phospholipid vesicles containing quaternary ammonium halides, quaternary ammonium acetate and the like, do not fuse with a cell and thus do not microinject the contents of the vesicles into a single cell. These vesicles react similarly to the known non-fusogenic and viscous contents. in the cells through an endocytosis-like process. The fusogenic properties of the liposomes of the present invention, on the other hand, offer a convenient means of incorporating any cell-modifying substance that can be entrapped in 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 R 1 - N - R 3 OH wherein R 1 is a large alkyl group or a combination of. an alkyl and aryl radical which imparts surfactant properties to the salt, and R 2, R 3 and R 2 are branched chain alkyl radicals having 1 to 20 carbon atoms or an aryl radical or R 2 and R 3 together may be a 5-membered or 6-membered heterocyclic radical , such as pyrrole or pyridine. And in particular preferred compounds are the surfactants cement tylbensyldimetylammoniumhydroxid, hexadecyltrimetylam- hydroxide, cetyltrimetylammoniumhydroxid, diisobutyl tylkresoxietoxietyldimetylbensylammoniumhydroxid, diisopropyl butylfenoxietoxietyldimetylbensylammoniumhydroxid, methylene tyldodecylbensyltrimetylammoniumhydroxid, metyldodecyl- xylenbis (hydroxide), N-alkyl (C12, C1u, - C16) -dimethylbenzylammonium hydroxide and octylcresoxy-oxyethyldimethylbenzylammonium hydroxide. It is essential that the quaternary ammonium hydroxide salt has surfactant properties and contains the hydroxy radical so that it imparts fusogenic properties to the phospholipid vesicle. The amount of quaternary ammonium hydroxide salt in the vesicles normally ranges from about 5 to 750 ug per 1 mg of phospholipid and preferably from about 50 to 250 pg of quaternary ammonium hydroxide salt per 1 mg of phospholipid.

The liposomes useful in the present invention may be any of the known liposomes. Exemplary liposomes include the natural and synthetic phosphocholine-containing lipids having a fatty acid chain of 12 to 20 carbon atoms and a fatty acid chain having at least 8 carbon atoms, exemplified by dimyristoylphosphatidylcholine, dioleoylphosphatidylcholine, dipalcataphylphylylphosphonylpholinylphosphonylpholinyl as well as cholesterol and the like. The liposomes can be prepared by any of the well-known published methods, such as sonication of phospholipids, reversed evaporation or removal of dried layers of phospholipid molecules. Suitable methods are described in "Procedure for Preparation of Liposomes with Large Internal Aqueous Space and High Capture by Reverse-Phase Evaporation", by Szoka et al., Cited above; "Dehydration-Rehydration Vesicles: A Simple Method for High Yield Drug Entrapment in Liposomes", by Kirby and Gregoriadis, cited above, as well as other known 464,448 methods, such as the use of surfactants (see Szoka and Papahadjopoulos, Annu. Rev. Biophys. Bioeng., 2 (1980), pp. 467-480). In addition, the cell-modifying substance that can be entrapped in the liposome and microinjected into animal or plant cells by fusion with the liposomes of the present invention may be any of the previously proposed substances. It has been found that substances which can be encapsulated in the liposome and micro-injected into a cell of the invention include DNA and DNA fragments, pharmaceutically active compounds and preparations thereof such as carbohydrates, nucleotides, polynucleotides, both natural and synthetic, influenza. vaccines and antigens as well as other substances which may affect the physiology of animal and plant cells.

The following examples illustrate preferred methods of preparing the fusogenic liposomes of the invention.

Example 1 Incorporation of quaternary ammonium hydroxide salt into liposome membranes during liposome preparation 2 mg of uncharged phospholipid, phosphatidylcholine (PC), and 1 mg of cholesterol are dried from chloroform solutions. The dry layer obtained by PC and cholesterol is solubilized with 1 ml of diethyl ether, and the resulting solution is added to a suspension containing 2 mg of octylcreoxyethoxyethyldimethylbenzylammonium hydroxide (Q-salt) in acetate buffer at pH 7.0. The resulting suspension is then stirred vigorously and sonicated briefly in a bath sonicator. Reverse-evaporated large single lamellar liposomes are then prepared by 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 unincorporated Q salt is removed by the addition of SM-2 Bio-beads according to the method described in "A New Method for Reconstitution of Highly Fusogenic Sendai Virus Envelopes" by Vainstein et al, 464 448 above. Thus, about 50-80 mg of SM-2 Biobeads are incubated with the above-described 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 estimate shows that about 30-40% of the added Q-salt is incorporated into the phospholipid bilayer (300-400 pg of Q-salt per mg of PC). To prepare liposomes loaded with a 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 procedure set forth in Example 1 for the preparation of fusogenic liposomes is not recommended when charged molecules are trapped in the liposomes. Under certain conditions and at a certain pH, each negatively charged component becomes complexed to the quaternary ammonium hydroxide and precipitates thereby. Because the quaternary ammonium hydroxide salt is positively charged, it interacts electrostatically 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 reduces the capture efficiency (the number of molecules captured within each liposome) and the fusogenic activity of the liposomes. Accordingly, Example 2 discloses a second method for preparing fusogenic liposomes in which direct contact between the quaternary ammonium hydroxide salt and the entrapped molecules is avoided. 464 449 Example 2 Insertion of Q-salt into already prepared charged liposomes Charged liposomes are prepared as described in Example 1 or according to the previously published methods described above. Unlike the method described in Example 1, the Q salt is not added during the preparation of the liposomes, but after the addition of the charged liposomes to a suspension containing charged resealed liposomes. Incubation of the Q salt with a suspension of resealed liposomes results in the incorporation of the hydrophobic portion of the Q salt molecule into the phospholipid bilayer of the liposome.

The procedure is performed as follows: A suspension containing phospholipid vesicles (liposomes) prepared from 2 mg PC and 1 mg cholesterol, suspended in 400 μl acetate buffer, is added to a tube containing 3 mg Q salt in 20 μl acetate buffer. The resulting mixture is stirred vigorously, after which it is incubated for 10 - 15 minutes at 37 ° C with slow shaking. excess free unincorporated Q-salt is disposed of by adsorption on SM-2 Bio-beads, as described above. A quantitative estimate again showed that about 30% of the added Q salt was incorporated into the phospholipid bilayer. The process of Example 2 may be advantageous for certain preparations over the process of Example 1, in that molecules entrapped in the liposome are trapped within the liposome prior to the addition of the Q salt; therefore, no interaction can occur between the entrapped material and the added surfactant. In addition, the fusogenic properties of the Q salt-bearing liposomes prepared by the method of Example 2 are superior to those of the liposomes prepared by the method of Example 1.

The ability of the liposomes (PC / Cholesterol Liposomes) of phosphatidylcholine and cholesterol described in Example 2 to study cell fusion was studied by incubation with human erythrocytes and with hepatoma tissue culture cells (HTC) in comparison with controls. Q-salt, which is octylcresoxyethoxyethyl-dimethyl-benzylammonium hydroxide, C-salt, which is octylcreoxyethoxyethyldimethyl-benzylammonium chloride, and B-salt, which is benzyl-dimethyl-hexadecylammonium bromide, were incorporated into the already formed liposomes 2, as described in I. experiments, either free Q-salt (5 - 10 μg) or free C-salt (5 - 10 ng) or free B-salt (5 - 10 pg) or liposomes bearing these quaternary ammonium molecules (20 pg PC) were incubated with a cell suspension ( 106 cells) for 15 minutes at 3700. Agglutination of cells and cell fusion were monitored 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 Human erythrocytes incubated Agglutination Cellfusion with: PC / cholesterol liposomes .... - - Free Q-salt (OH_-salt) .... + + PC / cholesterol liposomes bä - edge Q-salt (OH_-salt) .... + +++ Free c-salt (c1 "-sa1t) .... + - PC / cholesterol-C-salt (Cl-- salt)... ........... + + Free B-salt ..... =.;..... .. + - PC / cholesterol liposomes bearing B-salt ............... + - 464 448 Table I (continued) HTC-incubated with: Agglutination Cellfusion PC / cholesterol liposomes .... - - Free Q-salt (oH'-salt) .... + + PC / cholesterol liposomes bearing Q-salt (OH-salt) .... + +++ Free C-salt (Cl - salt) .... + - PC / cholesterol-C-salt (Cl_-salt).................. .. + - B-salt ......... ......... + - PC / cholesterol liposomes bearing B-salt.......... ... + - The results summarized in Table I show the following: : (1) Liposomes composed of PC / cholesterol but without quaternary ammonium molecules had no effect on agglutination or cell fusion of human erythrocytes or HTC cells. (2) All the quaternary ammonium molecules used (Q-salt, C-salt and B-salt), either in free form or incorporated into liposomes, were capable of inducing cell agglutination. This is likely due to the binding 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, could induce cell fusion. The induction of cell fusion with liposomer-free Q-salt was much higher than with free Q-salt. The quaternary ammonium chloride and bromide salts did not induce cell fusion although they can bind to cell membranes, as can be deduced from their ability to induce cell agglutination.

Fusion of liposomes bearing a quaternary ammonium hydroxide salt with cells is also shown by their ability to microinject their contents into the cytoplasm of animal and plant cells. Three systems have been used to detect fusion of Q-salt-bearing liposomes with living cells and microinjection of their contents, as follows: (1) The toxin ricin-A chain was entrapped in the liposomes, and these charged liposomes were incubated with cells in culture, such as HTC. Ricin A inhibits cell protein synthesis and therefore kills cells only when present inside the cells. Unlike the entire ricin molecule (ricin containing both the A and B units), the ricin A chain cannot penetrate the cells on its own. When it occurs outside the cell, it thus has no lethal effect. (2) SVQÛ DNA, ie DNA extracted from the SVQO virus, was entrapped in the liposomes. Liposomes loaded with SVQO DNA were incubated with cultured HTC. Microinjection of Svro DNA into the HTC cells observed through the appearance of a specific protein, SVQO-T antigen. Synthesis of SV40-T antigen is induced only when SV40 DNA is introduced into the cytoplasm of the cell from where it is transferred to the cell nucleus.

It is also to be noted that (a) appearance of SVQO-T antigen occurred only in living cells, since it requires active protein synthesis; and (b) free SV40 DNA molecules are unable to enter the cells upon incubation with living cells, and thus these free molecules do not induce synthesis of the T antigen. (3) Fluorescently labeled protein molecules, namely fluorescently labeled bovine serum albumin, BSA, were included in the liposomes. Charged liposomes were then incubated with either plant protoplasts or a suspension of plant cells. Fusion-mediated microinjection was observed by monitoring the occurrence of intracellular fluorescence. 464 448 12 (a) Fusion-mediated microinjection of ricin-A chain with fusogenic liposomes The results summarized in Table II show that only liposomes charged with ricin-A chain and bearing the quaternary ammonium hydroxide salt (Q salt) developed a strong inhibition of protein synthesis and a high-level killing of HTC. No killing was observed either with uncharged liposomes or with free ricin A chain. Apparently, very little killing was induced beyond the controls of liposomes bearing the quaternary ammonium chloride salt (C-salt), indicating no or substantially no fusion of liposomes and cells.

Table II Ability of fusogenic liposomes loaded with ricin A to inhibit protein synthesis and kill HTC HTC incubated with Inhibition of HTC killed protein synthesis (%) (%) PC / cholesterol liposomes._ .. 0 Free ricin A chain. . . . . . . . .. 5 PC / cholesterol liposomes loaded with ricin A. . . . . . . . . .. 6 7 PC / cholesterol liposomes carrying Q-salt (OH - salt) .... 5 8 PC / cholesterol liposomes loaded with ricin A chain and carrying Q-salt zon'-sawfy .. a 90 95 PC / cholesterol liposomes loaded with ricin-A chain and bearing C-salt (Cl_-salt) .. 17 14 Ricin-A chain was first captured in PC / cholesterol liposomes by the method described in "Procedure for the Preparation of Liposomes with Large Internal Aqueous Space and High Capture by Reverse-Phase Evaporation", UV) 464 448 13 by Szoka et al., supra, after which quaternary ammonium salt (either the OH_ or Cl - salt) was incorporated into In the experiments, - 20 pg of liposomes were incubated with 106 cells for 30 minutes at 37 ° C, during which time the liposomes reacted with the cells. At the end of the incubation period, the cells were washed and incubated for another 12 h in a culture medium, after which protein synthesis ( by incorporation of 3 H-leucine) and cell viability (by staining with trypan blue) were assessed. mediated microinjection of SV1,0 DNA The results in Table III show that fusogenic liposomes can be used for microinjection of active genes (DNA molecules) into cells under culture, such as HTC.

All experimental conditions and incubation with HTC were as described in Table II for ricin A. SVQO 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 The occurrence of Svro-T antigen was estimated as described above using specific fluorescently labeled anti-SV ~ O-T antigen antibody.

Table III Ability of charged fusogenic liposomes (Q-salt-bearing liposomes) to microinject Svro DNA HTC-incubated with liposomes SVQO-T antigen-positive charged with Svgo DNA cells (t of all; PC / cholesterol liposomes). .... 0 PC / cholesterol-bearing Q-salt (oH'-salt)............. ._ PC / cholesterol-bearing C-salt (c1'-salt). The specific T antigen was synthesized in the nucleus of - 15% of cells incubated with liposomes loaded with SV40 DNA and bearing Q-salt. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. ... (OH - salt) No T-antigen was found in cells incubated with PC / cholesterol loaded with SVQU DNA, but bearing C-salt (Cl - salt). (C) Fusion-mediated injection of fluorescent BSA in protoplasts and plant cell suspension of Petunia hybrida Protoplasts of cells of Petunia hybrida were prepared by conventional methods as described in the literature, fluorescent BSA was entrapped in liposomes as described for ricin A chain, and then Q-salt was incorporated into the charged li the membranes of the posomes, as described above. In the experiment, 10 - 20 pg of charged liposomes were incubated with 5 x 105 protoplasts or plant cells. After -60 minutes of incubation at 28 ° C, the occurrence of intracellular fluorescence was monitored using fluorescence microscopy.

Table IV Fusion-mediated injection of fluorescent bovine serum albumin into plant protoplasts of plant cell suspension of Petunia hybrida Petunia protoplasts incubated with liposomes loaded with fluorescent BSA PC / cholesterol ......... . . . . . . . . _.

PC / cholesterol bearing Q-salt (OH_-salt). . . . . . . . . . . . . . . . . . . . ..

Petunia cell suspension incubated with liposomes loaded with fluorescent BSA PC / cholesterol. . . . . . . . . . . . . . . . . ..

PC / cholesterol-bearing Q-salt (o fl f-salt). . . . . . . . . . . . . . . . . . . . ..

Presence of intracellular fluorescence (% of all cells) 80 60 (å 464 448 The results in Table IV show that liposomes bearing Q-salt (OH_-salt) can also microinject their content in plant protoplasts. of fluorescence can result only from fusion of the charged liposomes with the plant protoplasts.Every other process would lead to a different kind of behavior of fluorescence.The results in Table IV show that in addition to fusion (or microinjection) with plant protoplasts charged liposomes could fuse with and microinjecting their contents into plant cells surrounded by a cell wall.

Due to the presence of the highly charged Q salt, the liposomes appear to be able to pass through the cell wall and fuse with the cell membranes.

Uses of the quaternary ammonium hydroxide salt bearing liposomes Since the preparation of liposomes containing quaternary ammonium hydroxide salts has been illustrated and the quaternary ammonium hydroxide salt in the vesicle membranes containing the ability of the liposomes to fuse with cells and induce microcellular fusion, various uses of the liposomes of the invention are indicated. (1) The use of free Q-salt (OH-) or liposomes bearing Q-salt (OH-) as reagents to induce cell fusion Animal cell fusion, as has been demonstrated for the present liposomes, is of paramount importance for gene expression studies. Furthermore, plant protoplast fusion has many commercial applications, which can lead to the development of new plant species with improved properties. (2) Use of fusogenic liposomes for microinjection of various components in cultured animal and plant cells Charged fusogenic liposomes can be used for microinjection of DNA, RNA, proteins (enzymes, hormones) * 464 44s 16 and drugs in cultured cells. In addition, they can be used for microinjection of organelles or other components or molecules that can be entrapped in the liposomes.

Microinjection of DNA, RNA or organelles into cells, especially in plant protoplasts or plant cells, has a significant commercial significance as does the whole field of genetic engineering with plant and animal cells. Furthermore, on the basis of experiments with plant and animal cells, it can be said that liposomes bearing a quaternary ammonium hydroxide salt can fuse with and microinject their contents into any cells with which they can be incubated. Thus, these liposomes can also fuse with bacterial, yeast and algal protoplasts. (3) Use of fusogenic liposomes for the delivery of various components in cells in vivo, namely tissue or cells of whole animals Fusogenic liposomes can be used as carriers for the delivery of drugs, hormones or proteins after injection into laboratory animals or humans. , such as enzymes, and DNA. This method can be used for drug, enzyme and gene therapy. Non-fusogenically charged liposomes are currently used as carriers for drugs for therapy. However, fusogenic liposomes can be much more effective than non-fusogenic liposomes as biological carriers, as they inject their contents directly into cell cytoplasm. (4) Use of fusogenic liposomes for the development of a new non-isotopically sensitive assay system for diagnostic purposes Liposomes bearing quaternary ammonium hydroxide salts fuse with other liposomes. Fusion between the liposomes leads to leakage of their contents. This leakage is specifically due to the interaction and fusion between the liposomes. Due to the presence of the positively charged molecules of quaternary ammonium hydroxide salt, liposomes bearing these quaternary ammonium molecules do not interact with each other, but repel each other.

As mentioned above, they can only interact and fuse together with liposomes lacking the quaternary ammonium hydroxide salt. However, two molecules capable of interacting with each other with high affinity force liposomes bearing the quaternary ammonium hydroxide salt to fuse with each other by overcoming the repulsive forces of the positive charge. Thus, if a specific antigen is incorporated into the quaternary ammonium hydroxide salt-bearing liposome membranes, an antibody produced against that antigen will drive the liposomes to interact.

Interaction between liposomes bearing quaternary ammonium hydroxide salt leads to fusion and release of the liposome content. A quantitative estimate of the release process (release of fluorescent dye or other reagent) becomes a measure of the interaction between antigen and antibody. Thus, fusion between liposomes bearing a quaternary ammonium hydroxide salt can be used to quantitatively assess the interaction between antibody and its antigen or hormone and its appropriate receptor.

This system can therefore be used to determine small amounts of antigen or hormone in a biological fluid.

Claims (9)

464 448 18 PATENT REQUIREMENTS Phospholipid vesicle, characterized in that it contains a quaternary ammonium hydroxide salt in the membrane of the vesicle, which salt is present in sufficient quantity to make the vesicle Fusogen. 2. A phospholipid vesicle according to claim 1, characterized - in that the quaternary ammonium hydroxide salt is a member of the group consisting of ammonium hydroxide cetylbensyldimetyl-, hexadecyltrimetylammoniumhydroxid, cetyl trimethyl ammonium hydroxide, diisobutylkresoxietoxietyldimetyl- bensylammoniumhydroxid, diisobutylFenoxietoxietyldimetylben- sylammoniumhydroxid, metyldodecylbensyltrimetylammoniumhydroxid, metyldodecylxylenbis (trimethylammonium) and octylcresoxyethoxyethyl dimethylbenzylammonium hydroxide. Phospholipid vesicle according to claim 1, characterized in that the quaternary ammonium hydroxide salt is octylcresoxyethoxyethyl dimethylbenzylammonium hydroxide. Phospholipid vesicle according to claim 1, characterized in that the phospholipid is a member of the group consisting of Phosphatidylcholine, dimyristoylphosphatidylcholine, dioleoylphosphatidylcholine, dipalmitoylphosphatidylcholine, disteapholinylcholine, disteapholinyl. A phospholipid vesicle according to claim 1, characterized in that DNA or DNA fragments are encapsulated therein. Phospholipid vesicle according to claim 1, characterized in that a Pharmaceutically active compound or preparation is encapsulated therein. 7. A process for making a phospholipid vesicle fusogenic, characterized in that a quaternary ammonium hydroxide salt is incorporated into the membrane of the vesicle during the formation or after the formation of the phospholipid vesicle in an amount sufficient to make the phospholipid vesicle fusogenic. 8. A method according to claim 7, c h k nnetec carbonate of the quaternary ammonium hydroxide salt is a member of the group consisting of cetylbensyldimetylammonium- hydroxide, hexadecyltrimetylammoniumhydroxid, cetyltrimetyl- ammonium hydroxide, diisobutylkresoxietoxietyldimetylbensyl- WW 464 443 19 ammonium hydroxide, diisobutylfenoxietoxietyldimetylbensylammo- hydroxide, metyldodecylbensyltrimetylammoniumhydroxid , methyldodecylxylenebis (trimethylammonium hydroxide) and octylcresoxyethoxyethyl dimethylbenzylammonium hydroxide. 9. A process according to claim 7, characterized in that the quaternary ammonium hydroxide salt is octylcresoxyethoxyethyldimethylbenzylammonium hydroxide.