JPH0374323A - Preservation of liposome - Google Patents

Abstract

PURPOSE:To reinforce the membrane structure and remarkably inhibit aggregation during storage by adding an aminoalkanesulfonic acid to a liposome dispersion. CONSTITUTION:A liposome containing encapsulated substances such as an anti- cancer agent, an anti-viral agent, an antibiotics, a peptide hormone and a phospholipid is prepared and the resultant liposome dispersion is preserved by adding an aminoalkanesulfonic acid (e.g. compound such as one represented by formula I) thereto. The above-mentioned method is utilized especially in case of a liposome composed of a phospholipid and/or a glycolipid and cholesterol. The amount of the aminoalkanesulfonic acid added is suitably 0.2-15 pts.wt. based on 100 pts.wt. liposome dispersion. This preservation method is effective to inhibit aggregation in storage of a liposome produced by dispersing a lipid into water in a liquid state.

Description

[Detailed Description of the Invention] (Industrial Application Field) The present invention relates to a method for preserving liposomes, and more specifically, c.
This invention relates to a preservation method in which aminoalkanesulfonic acid is added to a liposome dispersion.

BACKGROUND OF THE INVENTION Liposomes (Liposon-+e) are closed endoplasmic reticulum consisting of a lipid bilayer membrane. Natural biological membranes are said to have a bilayer structure of lipids, and liposomes are widely used as model membranes for biological membranes in studies of their physicochemical properties. In addition, liposomes can confine various substances within their internal water layer and membrane, allowing them to fuse with or be taken into cells! It is used as a carrier to transport monoga into the living body.

Research using liposomes spans a wide range of fields, including biology, medicine, and pharmacy, including use as carriers for enzymes and anticancer drugs, use in the field of immunology, interaction with cells, and use as drug delivery systems. end·
is being studied.

As mentioned above, liposomes have an extremely wide range of applications, but the fragility of their membrane structure has been pointed out as a problem.

That is, this is a phenomenon in which the orientation of the membrane is disturbed due to chemical or physical changes in lipids, which are membrane-forming substances, resulting in leakage of inclusions, association and aggregation of liposomes, and eventually formation of a precipitate.

Therefore, as a method for strengthening liposome membranes, conventional methods for producing liposomes coated with polysaccharides (JP-A-61-61-6) and phospholipids whose structure is strengthened by hydrogen bonding (Journal of the Chemical Society of Japan! p. 62, 1983) have been proposed. Also, a method of mixing ascorbic acid ester with lipids (JP-A No. 60-23/608')
How to increase the external osmotic pressure of the storage solution (% opening/closing x, z-,
zoirir) have been proposed, but all of them are mainly aimed at suppressing the leakage of contained substances, and no technological progress has been made in suppressing aggregation in the preservation solution, which is important in practical use. There wasn't.

Recently, a method for producing liposomes using propylene glycol, glycerin, etc. (Japanese Unexamined Patent Publication No. 6O-7932) has been disclosed, but this method is complicated and involves the use of lipids, cholesterol, etc. at high temperatures (40-/J O'C). These polyhydric alcohols are dispersed in the polyhydric alcohol at the bottom, and there have been problems in that what can be used as inclusions is extremely limited.

Furthermore, in the case of preparation using this method, dialysis, gel filtration, centrifugation, etc. are generally performed to remove unencapsulated substances, so that most of the polyhydric alcohol initially used is removed. Therefore, it cannot be expected that the final liposome dispersion will be stabilized in terms of dispersibility by the residual polyhydric alcohol.

As mentioned above, a method for stably preserving liposomes having an encapsulating member in liquid form has currently been discovered.

(Problems to be Solved by the Invention) Therefore, an object of the present invention is to provide a complete method for preserving liposomes with a strengthened membrane structure and less aggregation during storage.

(Means for Solving the Problems) In order to achieve all of the above objects, the present invention consists of the configurations shown in the following items.

(1) A method for preserving liposomes, which comprises adding aminoalkanesulfonic acid to a liposome dispersion having an encapsulating member.

(2) The method for preserving liposomes according to (1) above, wherein the liposomes are liposomes made of phospholipids and/or glycolipids and cholesterol.

(3) The method for preserving liposomes according to (1) above, wherein the amount of aminoalkanesulfonic acid added to the liposome dispersion is from 7.2 to 15 parts by weight per 100 parts by volume of the liposome dispersion.

The present inventors have conducted intensive research on methods for strengthening the membrane structure of liposomes from the viewpoint of leakage of inclusions and aggregation of liposome particles. As a result, the inventors have found that by adding aminoalkanesulfonic acid to the liposome dispersion, aggregation can be prevented. We found that it can be greatly suppressed.

What is particularly surprising is that the present invention has been developed because it has been found that aggregation is significantly suppressed compared to when polyhydric alcohols such as propylene glycol and glycerin used in the above-mentioned Japanese Patent Application Laid-Open No. 60-7932 are added. completed.

The aminoalkanesulfonic acid that can be used in the present invention is an alkanesulfonic acid having an unsubstituted amino group, has a solubility in water of 3% or more at 20°C, and does not form cells. It is something. The number of carbon atoms in the alkane moiety is preferably r or less, particularly t or less.

Next, specific examples of compounds will be given, but the invention is not limited thereto.

Compound example (1) Nl2-CH2-CH2-8O3H (2)
Nl2 CH2CH2・CH3-8O3H (3)
CH3-CH-CH2-803HH2 (4) Nl2 CH2-CH2-CH2-CH2
-803H(5) (CHa) 2-C-C)12
-8O3HH2 There is no particular restriction on the form in which these aminoalkanesulfonic acids are added, and they may be added in the form of solid 13-1 or an aqueous solution.

There are no particular limitations on the liposome membrane-forming lipid in the present invention, and any natural or synthetic lipid can be used as long as it forms liposome gold.

Examples thereof include phosphatidylcholine, phosphatidylethanolamine phosphatidic acid, phosphatidylserine, phosphatidylinositol, phosphatidylglycerolsphingomyelin, calciolipin, and hydrogenated products of these according to conventional methods, and these can also be used in appropriate combinations.

Furthermore, a membrane structure reinforcing agent such as cholesterol or a negative charge imparting agent (for example, stearic acid, oleic acid, linoleic acid, lylunic acid, dicetyl phosphate, stearylamine) can be added to the constituent acids of the liposome membrane, if desired. .

As the encapsulating member used in the present invention, either a hydrophilic drug or a lipophilic drug, or both can be used simultaneously. Examples of such hydrophilic drugs include adriamycin, adriamycin, mitomycin, /-β-
anticancer drugs such as acibinof-2 ciluntocin, pleomycin, and cisplatin, antiviral drugs such as interferon, antibiotics such as aminoglycosides (e.g., gentamicin), and β-lactam compounds (e.g., sulpenicillin, cefotiam, and cefmenoxime); TRH, ryuglolide, peptide hormones such as insulin, lysozyme,
Enzymes such as asparaginase and glycosidase, immunostimulants such as muramyl dipeptide and muramyl tripeptide,
Examples include proteins such as immunoglobulin and various toxins.

Examples of lipophilic drugs include anticancer drugs such as ansamitocin and T Ml) -1,A (Gann 741
(,2)/F2-tyr<tyg3)), MTP-PE
Examples include immunostimulants such as (JP-A SHOTA P-/1331) and phospholipid derivatives (JP-A SHOTA-P-/633).

Other materials other than drugs are not particularly limited as long as they are useful when administered in vivo, such as markers, plasmids, DNA, and RNA.

Next, the filling liquid uses water as a medium, and an aqueous solution in which an appropriate water-soluble substance is dissolved is used. In some cases, the drug may simply be dissolved in water. Water-soluble substances include various buffers (e.g., phosphate buffer, citrate buffer), various salts (e.g., sodium chloride, sodium phosphate, disodium phosphate), sugars (e.g., glucose),
Amino acids (eg, l-arginine) and the like can be used alone or in combination.

A preservative (eg, parabens, etc.) may be added to this encapsulating liquid, if necessary.

The ribosome containing the encapsulating member used in the present invention is prepared by known methods.

That is, the portic swing method (A, D.

Bangham J, Mo1. Biol, / 3,
．． 231 (7Y/, j), 7=K'/Yo 7 method (C, H
uang.

Biochem,,I,3111/L(/P1.de)
], prevesicle method (H, Trouble, Neuro
sci, Res.

Prog, Bull,,? , 2 7
3 (/5'7/') Ethanol injection method [5-Batzri, Biochem.

Biophys, Acta,,2! P! ,/o/
z (/Y73)], French press extrusion method [Y,
Barenhollz,.

-ter FEB8. Lett,,yy,2to(t!Pyy)
).

Cholic acid removal method CY, Kagawa, J, BioI.

Chem,, 24A A, Evening! 77 (/ri7/)
],]Triton X-700 patch method W, J, Ge
rritsen.

Eur, J-Biochem,,I! , 2 Tata (
/971)].

Ca2+ fusion method [D, PaPahadjopoul
os.

Biochem, Biophys, Acta, 3y
1Lt, xi v 3 (177ta)], Ether Injection Method CD, Deamer.

Biochem, Biophys, Acta, 414
t3. 627 (17t)), annealing method [Rolawaczeck, Biochern, Bio
phys-Acta.

lψ3,3/3C/ri76)], freeze-thaw fusion method [M,
Kasahara, J, Biol, Chem,...
Lujon method [8, Matsumoto, J, Co11o
idInterface Sci,,62,/I
Iri (ly77)).

Reverse phase evaporation method (F, 5zoka, Proc, Natl, A
cad.

Scr -USA + 7ta, 4t/ri! (/j7
7, S')).

High pressure emulsification method (E-Mayhew, Biochem, Bi
ophys.

Acta, 77, t, / liri (/yia
)), -/ θ JP-A No. 4 O-7F3, No. 60-7 33, No. 1 O-7
Although many methods are known, such as those described in 3ψ, 60-/2/27, and 62-1213/, any of the above preparation methods may be used in the present invention. It is not limited to.

The liposome solution prepared by the above method may be stored with aminoalkanesulfonic acid added thereto while still containing unencapsulated components.

On the other hand, after removing the components not encapsulated inside the liposome, aminoalkanesulfonic acid may be added and stored. Furthermore, after adding aminoalkanesulfonic acid, the members not encapsulated inside the liposome may be removed, and after removal, further aminoalkanesulfonic acid may be added for storage. Examples of methods for removing unencapsulated members include dialysis, filtration (eg, gel filtration), and centrifugation.

The amount of aminoalkanesulfonic acid added is 0.2~/!/10θ volume parts of the liposome dispersion. Parts by weight. More preferably, it is 7 parts by weight of O, S.

It is added in at least an equimolar amount to the amount of lipid in the liposome dispersion. If the amount is less than these amounts, the agglomeration prevention effect will be low, and if it is more than these amounts, various problems will occur in practice (eg, precipitation).

Next, the effects of the present invention will be explained in more detail with reference to Examples.

Example 1 Liposomes having the following composition were subjected to reverse phase evaporation method (Proc
, Natl, Acad, 8ci, USA, 7j (Y
)tt/91t(/97g'), JP-A-11-//Il
l/! No.). Carboxyfluorescein was used as the encapsulating material. Each ψ μml of egg yolk fosone atidylcholine, cholesterol total chloroform. 20
The solution dissolved in CC was placed in an eggplant-shaped flask for OQC. The solvent was distilled off using a rotary evaporator to form a thin film on the inner wall of the flask.

Then, 20 CCf of diethyl ether was added to dissolve the lipids, and then HEPES buffer (p)]==7,1)3
After adding cc'l and replacing with gas phase iN2 gas in the eggplant flask, a homogeneous gas solution can be made while keeping the eggplant flask f20'c. Probe-type ultrasonic irradiation was performed for minutes.

Next, use a rotary evaporator for 200~! Most of the diethyl ether was distilled off under reduced pressure at 0C. After mixing the gel-like lipid thus prepared by vortexing for 20 seconds, the gel-like lipid f was 20-2! The solvent was completely removed under reduced pressure of "C". To this was added HEPES buffer t, zcc. 2
08epha subjected to ultrasonic irradiation for 20 minutes at 0 C.
After gel filtration with
using a submicron particle analyzer) and phospholipid concentration (using a submicron particle analyzer)
All measurements were carried out (using Wako Niphospholipid measurement kit). Then, the phospholipid concentration was adjusted to jXIO'μ using 7-lactone with an average particle size of 0.2μ.

As shown in Table 1, 0.2 g of each powder of Compound Example 1.3 of the present invention was added and dissolved in the adjusted solution, tCC. After dissolution, the particle size was measured and the appearance was inspected.

Same sample solution again! After refrigerated storage at 0C for one month, the particle size and appearance were measured. For comparison, the same tests were conducted on a sample without additives, a sample containing glycerin and propylene chloride, and a sample containing t) and zcc, respectively.

The results are shown in Table 1.

-/4I- From the results in Table/, it can be seen that by using the method of the present invention, there is almost no aggregation of liposomes and there is almost no change in the average particle size, compared to when no additive is added or when polyhydric alcohol is added. I understand.

A similar effect was seen with liposomes consisting of phosphatidylcholine/cholesterol/cisetyl phosphate.

(Effects of the Invention) The method of the present invention is effective in suppressing aggregation when storing liposomes in liquid form formed when lipids are dispersed in an aqueous liquid.

Claims (3)

[Claims] (1) A method for preserving liposomes, which comprises preparing liposomes having an encapsulating member and then adding aminoalkanesulfonic acid to the liposome dispersion. (2) The method for preserving liposomes according to claim 1, wherein the liposomes are liposomes made of phospholipids and/or glycolipids and cholesterol. (3) The method for preserving liposomes according to claim 1, wherein the amount of aminoalkanesulfonic acid added to the liposome dispersion is 0.2 to 15 parts by weight per 100 parts by volume of the liposome dispersion.