JPS61274739A - Phospholipid vesicle and method for treating the same - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) This invention relates to liposomes. More specifically, the present invention involves encapsulating a quaternary ammonium hydroxide salt within its membrane, allowing the liposome to fuse with animal and plant cells, and transferring the encapsulation to animal and plant cells. This invention relates to liposomes that enable microinjection into the human body.

BACKGROUND OF THE INVENTION Liposomes, generally and as the term is used here, are vesicles made of phospholipid molecules (page 5). It has become generally understood that liposomes provide a means for introducing drugs or other modulating substances into certain animal or plant cells to modify cytoplasmic physiology. If the liposome is a drug or
Since it has been well understood that the introduction of substances that modify cytoplasmic physiological functions into cells can play an important role, it has become clear that drugs or drugs can be introduced into cells to modify cytoplasmic physiological functions. Natl, Acad. 8ci. ,
USA751978, pages 4194-4198, “
The procedure for preparation of liposomes with large internal aqueous space and high entrapment by reverse-phase evaporation is described in ``Procedure for preparation of liposomes with large internal aqueous space and high entrapment properties'', which allows the large internal aqueous space to confine the otherwise modified molecules that will be transferred and entrained. Various methods have been proposed for making or preparing liposomes, including liposomes with. Other methods are described in "Biotechnology" published in November 1984 (
Old o/+I'technology) No. 979 to 98
As described in "Dehydration-Rehydration Vesicles: A Simple Method for High-Yield Drug Incorporation into Liposomes" by KirbY and Gregoriadis (page 6), page 4. , is primarily directed to improving procedures for loading drugs or other substances into liposomes. U.S. Pat. No. 4,235,871 discloses a bilayer lipid obtained by providing a mixture of a number of synthetic biologically active substances and a lipid in an organic solvent, and encapsulating the mixture in a capsule. A method is disclosed for encapsulating an aqueous mixture of materials necessary to emulsify, remove the organic solvent, and suspend the synthesized gel in water. Furthermore, various applications of liposomes as a transfer vehicle have been demonstrated in a number of inventions. Thus, the aforementioned US Pat. No. 4,235,871 disclosed a number of compounds and formulations that can be encapsulated in liposomes for uptake into cells. U.S. Pat. No. 4,394,
No. 448 inserts deoxyribonucleic acid (DNA), or fragments thereof, into living cells, thereby
, or fragments are encapsulated in lipid vesicles that come into contact with the cell, thereby specifically targeting insertion. US Patent No. 411
No. 99.566, (Page 7) No. 4,201,767, No. 4,261,975, and No. 4,235,877 disclose positively charged materials, which may be quaternary ammonium halides. discloses the incorporation of viral or bacterial antigens into liposomes encapsulating surfactants with amine groups.

US Patent No. 4,483,929 discloses immunoreactive liposome reagents used to determine chemical compounds capable of entering into immunospecific reactions with known antibodies. Although in all applications known to the applicant phospholipid liposomes have been shown to be able to introduce their inclusions not only into cells of specific tissues of whole animals, but also into cultured cells;
These liposomes were found to be relatively poor carriers. These prior art liposomes are believed to introduce the carried substances, ie, drugs, into cells in a process such as endocytosis.

Therefore, only a small percentage of the molecules that are trapped in the liposomes reach the cytoplasm of the recipient cell. Furthermore, it has been demonstrated that when liposomes are injected into humans as well as animals, up to a certain concentration, phospholipid liposomes are inactive and not immune genes. Only at relatively high concentrations are liposomes immunogenic and toxic. Although these liposomes of the prior art are capable of agglutinating cells, they are not fusogenic and do not or cannot fuse with cell membranes or induce cell fusion from cells.

(Problem to be Solved by the Invention) The envelope of some animal viruses, such as those produced from Sendai virus, is fusogenic and [Biochemistry Biophysics Acta J 773 (1
984), pp. 181-188, by Vuinstein et al. It has been shown to be useful as a carrier. The fusogenic activity of ranoviruses is believed to be due to the presence of specific viral glycoproteins contained in the viral envelope. However, the presence of proteins containing H. enperozoni makes these vesicles immunogenic and therefore impractical for use in vivo. it is obvious. Animal meat injection of viral envelopes
It induces a composition of specific antiviral antibodies, a fact that limits its use as a biological carrier in vivo.

Thus, although the reported literature accepted phospholipid vesicles as a potentially important tool for the uptake of drugs and other cell modifiers into cells, the limitations of prior art liposomes Therefore, this technique has only had limited success.

Therefore, the main object of the present invention is to provide a liposome that is fusogenic, fuses with cell membranes, and induces cell fusion from cells.

Another main object of this invention is to provide a method for producing liposomes that fuse with cell membranes and induce cell fusion from cells.

(Means for Solving the Problem) (Page 1O) The object of the present invention is to provide a quaternary ammonium hydroxide salt,
This is achieved by incorporation into liposomes, either during liposome vI4Ii or after preparation. It has been found that the presence of quaternary ammonium hydroxide salts in the phospholipid vesicle membranes allows the vesicles to fuse with the cell membrane and induce cell-to-cell fusion. When a drug or other cell-modifying substance is encapsulated in a vesicle, the vesicle's encapsulated substance is microinjected into an animal or plant cell. The ability of quaternary ammonium hydroxide salts to convert phospholipid vesicles from non-fusodienic to fusodienic is sometimes surprising in that quaternary ammonium salts other than said ammonium hydroxide salts provide vesicles that are non-fusodienic. There is something. Thus, phospholipid vesicles encapsulating quaternary ammonium halide salts, quaternary ammonium acetate salts, and the like do not fuse with cells and therefore do not allow trace doses of the vesicles to enter cells. I found out that it doesn't inject.

These vesicles react similarly to known non-fusogenic vesicles and apparently take up the vesicle injectate into cells through processes such as (page 11) endocytosis. . In contrast, the fusogenic properties of the inventive noliposomes allow for the direct delivery of virtually any cell-modifying substance to plant or animal cells in virtually any amount that can be trapped within the phospholipid vesicles. This can be an appropriate means of incorporating it.

(Function) The quaternary ammonium hydroxide salt that is incorporated into the walls or membranes of phospholipid vesicles has the following chemical formula, where R1 is a large alkyl group or a salt that imparts surfactant properties to the salt. is a combination of alkyl and aryl groups, and R4 is a branched chain alkyl group of 1 to additional carbon atoms, or an aryl group, i.e., a uniform R
3 may both be 5jl or a 6-membered heterocyclic group, such as, for example, pyrrole or pyridine. especially,
Preferred compounds include the surfactants cetylbenzyldimethylammonium hydroxide, hexadecyltrimethylammonium hydroxide, cenaltrimethylammonium hydroxide, diisobutylcrenxyethoxyethyldimethylpenzylammonium hydroxide, and diimbutylphenoxyethoxyethyl Dimethylbenzyl ammonium hydroxide, methyldodecylbenzyltrimethylammonium hydroxide, methyldodecylxylene bis(trimethylammonium hydroxide), N alkyl (ela, C14, C1
6) Dimethylbenzyl ammonium hydroxide, and octerculenxyethoxyethyl dimethylpenlyl ammonium hydroxide. It is essential that the quaternary ammonium hydroxide salt has surfactant properties and encapsulates hydroxy groups so as to impart fusodigenic properties to the phospholipid vesicles. The amount of quaternary ammonium hydroxide salt encapsulated in the vesicles typically ranges from about 5 to 75 mm
0 μV, and preferably about 1 to 250 μf of quaternary ammonium hydroxide salt per inch of phospholipid.

(Page 13) Liposomes useful according to this invention can be any of the prior art liposomes. Illustrative liposomes include natural and synthetic phosphocholines, including monolipidic acid chains of 12 to additional carbon atoms, cholesterol and its congeners, as well as simyristoylphosphatylylcholine, It encapsulates a lipid having dioleoylphosphatidylcholine, dinoqlumitoylphosphatidylcholine, distearoylphosphatidylcholine, and one fatty acid chain of at least 8 carbon atoms embodied in phosphatidylcholine and sphingomyelin. Liposomes can be prepared by any method known in the art such as sonication of a phospholipid suspension, back evaporation, or
It can also be prepared by dehydration of a dry double coat of phospholipid molecules. Appropriate techniques include the above-mentioned ``Procedure for preparation of liposomes with large internal aqueous spaces and high entrapment properties'' by Ntska and others and the above-mentioned ``Procedure for preparation of liposomes with large internal aqueous spaces and high entrapment properties'' by Ntsuka and others, and ``Procedure for the preparation of liposomes with large internal aqueous spaces and high entrapment properties by reversed-phase evaporation''.
Dehydration-rehydration vesicles: A simple method for high-yield drug incorporation into liposomes" as well as other known techniques such as the use of detergents (Sock and pQ) j Annu, Rev, Biophy
s+Bioerig,.

9 (1980) J, pp. 467-480]. Moreover, it can be entrapped in liposomes and
Cell-modifying substances that can be microinjected into animal or plant cells through fusion with the liposome of this invention may be any of the substances proposed above. Substances that can be encapsulated in liposomes and microinjected into cells according to this invention include DNA-?, as well as other substances that can influence the physiology of animal and plant cells.
These include DNA fragments, naturally occurring and synthetically occurring pharmaceutically active compounds such as carbohydrates, nucleotides and polynucleotides, and their combinations, as well as influenza vaccines and antigens.

Having described the invention in general terms, the following two examples demonstrate the now preferred method of preparing the fusodiene liposomes of this invention.

Examples Example 1 (Page 15) Incorporation of quaternary ammonium hydroxide salt into liposome membrane during liposome preparation Uncharged phospholipid, phosphotezylcholine (PC) from 2 to 2
and 1 m9 of cholesterol were produced by drying these chloroform baths. The dry film made of PC and cholesterol was made soluble in ly+J riether ether, and the resulting aqueous solution was encapsulated with 2 octercresoxyethoxyenaldimethylbenzyl ammonium hydroxide in an acetate buffer. B-H7 in suspension
．． Added at 0. The resulting suspension is then vigorously stirred and briefly sonicated in a bath sonicator. Back-evaporated, large, unilamellar liposomes can be produced using the method described in Sotsu and others, supra, ``Procedure for re-arranging ribbons with large internal aqueous spaces and still trapping properties by reverse-phase evaporation.'' Adjust according to the method you are using.

Excess unincorporated Q-salts were removed using the technique defined by Vuinstein and others supra [``A New Method for the Reconstitution of Advanced Viral Envelopes''].
It is removed by adding 8M-2 biobeads. Thus, about 80 to 80 8M-2 biobeads were gently incubated with the phospholipid and Q-salt at room temperature for about 40 to (a) minutes in a final volume of 1 tri acetate buffer. It is kept warm by vibrating it. Quantitative estimates indicate that approximately 40% to 40% of the added Q-salt has a phospholipid double coating (pc
It has been shown that 300 to 400 μV of Q-salt (Q-salt) can be taken up per 1 μV of Q-salt. Preparation of liposomes encapsulating any component of interest, such as a drug, enzyme, RNA or DNA, can be carried out using Liposomes with Internal Aquatic Spaces and High Capacity - Preparation Procedures'' are added to a buffer in which the phospholipids are suspended.

As described in Example 1, the method of preparation of fusogenic ribbon molecules is not recommended when the charged molecules are incorporated into liposomes. Under certain conditions and at certain BHs, any actively charged components will be synthesized into quaternary ammonium hydroxide salts and precipitated with it. When the quaternary ammonium hydroxide salt is positively charged, it is a neutral peech and does not react with any passive molecules, such as nucleic acids, exemplified by DNA or RNA, which are strongly passively charged. It also interacts electrostatically with charged molecules. The composition of such complexes, between actively charged quaternary ammonium hydroxide salts and passively charged molecules, determines the entrapment efficiency (number of nine molecules entrapped within each liposome) and the liposome decreases the fusodienic activity of. Therefore, in Example 2, a second technique for the preparation of fusodienic liposomes is described, in which direct contact between the quaternary ammonium hydroxide salt and the entrapped molecules is avoided.

Example 2 Q-Salt Insertion into Prepared Encapsulated Liposomes Encapsulated liposomes are prepared either as described in Example 1 or by the method previously published above.

(Page 18) However, contrary to the method described in Example 1, Q~salt is
It is not added during preparation of the liposomes, but after preparation of the encapsulated pomone, it is added to the suspension that encapsulates the re-encapsulated liposomes. When Q-salt is incubated with a suspension of resealed liposomes, Q-salt is added to the liposomal phospholipid bilayer.
-Results in the incorporation of the hydrophobic portion of the salt.

The steps are as follows: 2) n? of PC and phospholipid vesicles (liposomes) containing 11 gv of cholesterol.
The suspension, suspended in 400 μj of complex salt buffer, was added to a tube containing 31 v of Q-salt and added μj
of complex salt buffer is added. The f mixture is
Stir vigorously and then keep warm at 37 degrees Celsius for 10-15 minutes with gentle shaking. Excess free and unincorporated Q-salt is removed by adsorption onto 8M-2/l Iobads as described above. According to Ashiori estimation,
It was again shown that the addition of approximately nod/(-cent Q-salt was incorporated into the phospholipid bilayer).

This method of Example 2 includes encapsulation within liposomes (19th
For some preparations, the molecules are trapped inside the liposome prior to the addition of the Q-salt.
This may be advantageous compared to Example 1, and as a result no interaction between the encapsulating substance and the added surfactant can occur. In addition, the 7 zodiac properties of the Q-salt carrying liposomes prepared by the method of Example 2 are superior to any of the liposomes prepared by the technique of Example 1.

As described in Example 2, the ability of liposomes consisting of phosphatidylcholine, cholesterol (pc/cholesterol liposomes) to induce cell fusion from cells was demonstrated by incubation with human red blood cells and liver cancer tissue culture cells (
HTC) was studied in comparison to restriction. The Q-salt, which is octylcresoxyethoxyethoxyethyldimethylbenzyl ammonium hydroxide, the C-salt, which is octylcrenoxyethoxyethoxyethyldimethylbenzyl ammonium chloride, and the B-salt, which is penlyldimethylhexadecyl ammonium bromide, were As mentioned in Example 2, it was already incorporated into liposomes, which were provided with biological characteristics. In such experiments, the free C-salt (5~IOμ
? ), free C-salt (5-101tt), free JvIB
- salt (5~lOμf) or these ammonium quaternary molecules (20μt PC) at 15°C at 37°C.
The cell suspension (106 cells) was incubated for 1 minute. Cell agglutination and cell-to-cell fusion were observed under a phase microscope. The results are tabulated in Table 1.

Table 1 PCI cholesterol liposome...
--Free mq-salt (OH-salt) 10
Juyu dC-salt (cl-salt)...
10 -Free B-Salt...
10 - (Page 2)) PCI cholesterol liposome... -
-Free Q-salt (OH2 salt piece... 10
+Free C-salt (C6-salt)...
10 -B-Salt...
- The results summarized in Table 1 confirm that: Cell adhesion or cell-to-cell fusion was not affected.

(2) Whether in free form or incorporated into liposomes,
All of the ammonium quaternary (page 22) molecules used (Q-salt, C-salt and B-salt) were capable of inducing cell agglutination from cells. This is believed to be due to the attachment of positively charged quaternary ammonium salts to passively charged cell membranes.

(3) Is the Q-salt, i.e., the hydroxide salt, the only free form?
Even when incorporated into liposomes, cell fusion could be induced from cells. The induction of cell fusion from cells by liposomes carrying Q-salt was much higher than by free Q-salt. Fourth, ammonium chloride and ammonium bromide salts have the ability to bind to cell membranes, as can be inferred from their ability to induce cell agglutination. I didn't give any guidance.

Fusion of liposomes carrying quaternary ammonium hydroxide in cells has also been shown to have the ability to microinject the encapsulated material into the cytoplasm of animal and plant cells. Fusion of liposomes and their inclusions that carry Q-salt in living cells.
Three schemes were used to confirm (page 23) microinjection into living cells: (1) Toxin lysine chains were encapsulated within liposomes, and These encapsulated liposomes contain HTO
Incubated with cells in culture such as.

Ricin A suppresses cellular protein synthesis, resulting in
It kills cells only when they appear intracellularly. The lysine chain cannot enter the cell on its own because it is opposed to the entire molecule of ricin (which encapsulates both the mu and B subunits). Therefore, when it appears outside the cell, it does not have any fatal effects.

f2) eV, o-DNA, i.e. virus eV
, o was encapsulated in liposomes. S.V.

- Liposomes encapsulating DNA were incubated in culture HTO. S.V. - SV, which is a specific protein by which DNA is microinjected into HTO cells. - It was found by the appearance of Tm antigen. Synthesis of 5v4o-T-antigen is sv. -DN
A is induced only when introduced into the cytoplasm of the cell,
It is then transcribed into the cell nucleus.

The following should also be specified: Suhiko Wachi, (a)
874 (The appearance of 1-T-antigen occurs only in living cells because it requires active protein synthesis; (1)) Free F3V, o-DNA molecules, when incubated in living cells. , it is not possible to enter the cell and, as a result, these free molecules do not induce the synthesis of the T-antigen.

(3) A fluorochrome-labeled protein molecule, ie, fluorochrome-labeled bovine serum albumin (BAA), was encapsulated within liposomes. The encapsulated liposomes are kept warm in a suspension of plant protoplasts, water, or plant cells. Fusion-mediated microinjection was demonstrated by the expression of intracellular fluorescence described below.

(a) Fusion-mediated microinjection of lysine chains by liposomes The results summarized in Table 2 show that only liposomes encapsulating lysine chains and carrying a quaternary ammonium hydroxide salt (Q, -salt) , which caused a strong inhibition of protein synthesis and a high degree of killing with HTO. As seen (page 25), liposomes carrying quaternary ammonium chloride salts (〇-salts) have no or virtually no liposome cell fusion, with very little overkill. This was caused by what was shown.

Table 2 PO/cholesterol liposomes... 00 free ricin A chain... 54 ricin A encapsulated PO/cholesterol), 6 full liposome lysine chain IJ with aqueous spaces and high entrapment properties are first entrapped within PO/cholesterol liposomes using the technique described in ``Procedure for Somesome Preparation'' (page 26), followed by quaternary ammonium salts (page 26). Either OH'' salts or Cl- salts) were incorporated into the liposomal double coat as described above. In experiments, 10-μm liposomes were incubated at 37 degrees Celsius for minutes in 106 cells. during which time the liposomes interacted with the cells. At the end of the incubation period, the cells were washed and incubated for an additional 12 hours in growth medium to allow protein synthesis (
(IHO icine uptake button) and cell viability (as shown by trivan blue staining) were evaluated.

(b) Melt-mediated microinjection of l1lIv4o-T)NA The results in Table 3 show that the fusodiene liposomes
1) Cells in culture, such as TO, are injected with an active gene (Dl
This shows that it can be used for microinjection of iA molecules). All experimental conditions and incubation with H'l'O and ricin A are as described in Table 2. S
V. - The DNA is entrapped within the liposomes as described in Sokka et al., supra, ``Procedure for preparing liposomes with large internal aqueous spaces and high entrapment properties'' by reverse-phase evaporation. eV,. -τ (page 27) The appearance of one antigen was assessed using specific fluorochrome-labeled anti-sv, o-T-antigen antibodies as described above.

Table 3 PCl cholesterol liposome...
0-specific T-antigen encapsulates 5y4o-DNA and Q
- Appeared in the nuclei of 10 to 154 cells insulated with liposomes carrying (OH-salt). T-antigen is SV,
.

It did not appear in cells insulated with po/cholesterol carrying C-salt (OJ-salt) containing -DN mu.

(o) Fusion-mediated injection of fluorescent B8A into Vetunia hybrida plant protoplasts and plant cell suspensions. Adjusted by conventional methods. Fluorescent BsA was encapsulated within liposomes as described for the ricin A chain, where Q-salt was incorporated into the membrane of the encapsulated liposomes as described above.

In experiments, encapsulated liposomes of 10 to 50 μm were
10' protoplasts or incubated with plant cells. After incubation for 1 to 0 minutes at temperature Celsius, intracellular fluorescence expression was detected by fluorescence microscopy.

Table 4 POl cholesterol　　　　　・4... river.

Q-salt (aH-dan) transportation pa/cholesterol...
8゜POl cholesterol...
・A... OQ-salt (oH-salt) transportation p
a/Cholesterol... 6゜Looking at the results in Table 4, it is clear that liposomes carrying Q-salts (OH-salts) (page 29) can also inject small amounts of their inclusions into plant protoplasts. is clear. Expression of fluorescence can only occur through fusion of the encapsulated liposomes with plant protoplasts. In other processes.

Another form of fluorescence is observed. The results in Table 4 show that in addition to fusion into plant protoplasts (microinjection), the encapsulated liposomes were able to fuse and microinject their inclusions into plant cells surrounded by cell walls. I know. It is clear that due to the presence of the highly charged Q-salt, liposomes are able to cross the cell wall and fuse with the cell membrane.

Application of Liposomes Carrying Quaternary Ammonium Hydroxide Salts The illustrations illustrate the preparation of liposomes that encapsulate quaternary ammonium hydroxide salts, and also vesicles of liposomes that encapsulate quaternary ammonium hydroxide salts in the vesicle membrane. The ability to fuse with cells and the ability to induce cell fusion from cells while microinjecting inclusions into cells has been explained by example. Explain the application of

(1) Use of liposomes carrying free Q-salt (OH-) or Q-salt (OH-) as reagents for inducing cell fusion from cells. , cell-to-cell fusion is of paramount importance in gene-protein synthesis research. Furthermore, plant protoplast fusion has numerous commercial applications and can lead to the development of new oak species with improved properties.

(2) Use of Fusodienic liposomes to inject microscopic amounts of various components into animal and plant cells grown in culture Encapsulated Fusodiene liposomes, DNA and RNA
It can be used to microinject proteins (enzymes and hormones) and drugs into cells in culture. Additionally, they can be used for microinjection of any organelles or other components or molecules that can be encapsulated within liposomes. Microinjection of DNA or (310) RNA or organelles into cells, particularly plant protoplasts or plant cells, has substantial commercial potential, as does the entire field of genetic engineering of animal and plant cells. The above has application. Furthermore, based on experiments in animal and plant cells, IJ and trsomes carrying quaternary ammonium hydroxide salts can fuse and microinject their inclusions into cells, which can help keep them warm. It is possible. Therefore, these liposomes are capable of fusion with bacteria, yeast, and algae protoplasts7. .

(3) The use of fusolionic liposomes to transport various components into cells in vivo, that is, the tissues or cells of whole animals. Afterwards, it can be used as a vehicle for the delivery of drugs, hormones, enzymes, and proteins such as DNA. This method can be used for drugs, enzymes, and even gene therapy.

Non-fusogenic, encapsulated liposomes are currently used as carriers for therapeutic drugs. However, fusolienic liposomes can be much more effective than non-fusolienic liposomes as biocarriers because they inject their inclusions directly into the cytoplasm of cells.

(4) Use of fusodientric bombomes for the development of a highly sensitive analytical method for diagnostic isotopes Liposomes carrying quaternary ammonium hydroxide salts can be used for
? Fuse with Soum. Fusion #j between liposomes leads to leakage of ζ straw inclusions. This leakage is essentially due to interactions and fusion between liposomes. Due to the presence of actively charged quaternary ammonium hydroxide salt molecules, liposomes carrying these ammonium quaternary molecules
They act on Ai W and reject each other in Hiko. As mentioned above, they interact and fuse only with liposomes that leak quaternary ammonium hydroxide salts.

However, due to the high affinity, both of the two molecules that interact with each other can overcome the rejection force of the active charge and transfer the quaternary ammonium hydroxide salt to the liposome. Force them to merge with each other. Therefore, when a %-specific antigen is incorporated into a liposomal salt carrying a quaternary hydroxide, a carrier tailored for such anti-contaminants forces the liposome to interact with each other. Let it work. Pairing between liposomes carrying quaternary ammonium hydroxide salts leads to fusion and release of liposomal encapsulates. Any observation of the release process (release of the fluorochrome or any other reagent) is a measure of antigen/antibody interaction. Thus, fusion between liposomes carrying quaternary ammonium hydroxide salts can be used to predict the interaction between an antibody and its appropriate hormone and its appropriate safeter. Therefore, this method is often used to estimate any small amount of antibodies or hormones present in any biological fluid.

Claims (11)

[Claims] (1) The phospholipid vesicles incorporate a quaternary ammonium hydroxide salt into the vesicle membrane, and the salt is present in an amount sufficient to render the vesicle fusodiemic. consisting of phospholipid vesicles. (2) The quaternary ammonium hydroxide salt includes cetylbenzyldimethylammonium hydroxide, hexadecyltrimethylammonium hydroxide, cetyltrimethylammonium hydroxide, diisobutylcresoxyethoxyethyldimethylbenzylammonium hydroxide, and diisobutylphenol It is one element of the group consisting of ethoxyethyldimethylbenzyl ammonium hydroxide, methyldodecylbenzyltrimethylammonium hydroxide, methyldodecylxylenebis(trimethylammonium hydroxide), and octylcresoxyethoxyethyldimethylbenzylammonium hydroxide. A phospholipid vesicle according to claim 1, characterized in that: (3) the phospholipid vesicles incorporate a quaternary ammonium hydroxide salt into the vesicle membrane, and the salt is present in an amount sufficient to render the vesicle fusocienic; A phospholipid vesicle, wherein the quaternary ammonium hydroxide salt is octylcresoxyethoxyethyldimethylbenzyl ammonium hydroxide. (4) The phospholipids include phosphatidylcholine, dimyristoylphosphatidylcholine, dioleoylphosphatidylcholine, dipalmitoylphosphatidylcholine, listearoylphosphatidylcholine, and sphingomyelin. , cholesterol, and a mixture thereof.
Phospholipid vesicles as described in Section. (5) the vesicle has DNA or one DNA in it;
Claim 1 consisting of a sealed fragment of A
Phospholipid vesicles as described in Section. (6) The phospholipid vesicle of claim 1, wherein the vesicle comprises a pharmaceutically active compound or composition entrapped therein. (7) A method of making phospholipid vesicles fusodigenic, which comprises incorporating a quaternary ammonium hydroxide salt into the phospholipid vesicle membrane. (8) The quaternary ammonium hydroxide salts include cetylbenzyldimethylammonium hydroxide, hexadecyltrimethylammonium hydroxide, cetyltrimethylammonium hydroxide, diisobutylcresoxyethoxyethyldimethylbenzylammonium hydroxide, and diisobutylphenoxylated ammonium hydroxide. One element of the group consisting of ethoxyethyldimelbenzyl ammonium hydroxide, methyldodecylbenzyltrimethylammonium hydroxide, methyldodecylxylenebis(trimethylammonium hydroxide), and octylcresoxyethoxyethyldimethylbenzylammonium hydroxide The method according to claim 7, characterized in that: (9) The phospholipid vesicles are made into fusodiens by incorporating a quaternary ammonium hydroxide salt into the phospholipid vesicle membrane, and the quaternary ammonium hydroxide salt is octylcresoxyethoxyethyldimethylbenzyl ammonium hydroxide. A method consisting of. (10) The method of claim 7, wherein the quaternary ammonium hydroxide salt is incorporated during the production of the phospholipid. (11) The method according to claim 7, wherein the quaternary ammonium hydroxide salt is incorporated into the vesicle membrane after phospholipid formation.