JPH02273539A - Preserving method for liposome - Google Patents

Abstract

PURPOSE:To strengthen the film structure, control agglomeration to a large extent and preserve liposome to be utilized for a a carrier for carrying an enzyme and an anticancer drug, immunological field, reciprocal action with cells stably for a long period of time by adding amount acid into liposome- preserved liquid. CONSTITUTION:Amino acid is added into liposome-preserved liquid to make agglomeration little after preserving, strengthen the film structure and preserve liposome stably. This method is effective on the preservation of liposome com posed of lipid dispersed into water. As for acid to be added, neutral amino acid of low molecule basic amino acid, acid amino acid salt or the like is suit able. Further, a film structure strengthening agent such as sterol, cholesterol or the like can be added to a constituting component of liposome film, if desired. Also, an electron donor (for example, stearic acid, olein acid or the like) can be added therein. The liposome can be utilized as a cell separating agent, a carrier for chemicals or the like.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to a method for preserving liposomes, and more specifically,
This article relates to a preservation method by adding abanoic acid to a preservation solution.

(Prior Art) A liposome is a closed endoplasmic reticulum consisting of a lipid bilayer membrane. Natural biological membranes are said to have a co-molecular structure of lipids, and liposomes are widely used as model biological membranes to study their physicochemical properties. In addition, liposomes can trap various substances in their internal aqueous layer or membrane, and are used as carriers to transport substances into living organisms because they fuse with cells or are taken up by cells. .

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. etc. are 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.

RlI 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, and the leakage of inclusions, association of ribbon atoms, and aggregation occur, leading to the formation of precipitates. It is.

Therefore, as methods for strengthening liposome membranes, there have been conventional methods for producing liposomes coated with multi-layered shields (%Kaisho t/-72ROI issue) and phospholipids whose structure is strengthened by hydrogen bonding (Journal of the Chemical Society of Japan, p. 142, 2017). , and ascorbyl in lipids/
Method of mixing M ester (% Kaisho 4O-JJ1601
No.), method of increasing external osmotic pressure of preservation solution (#Kaishobuλ
- Kota l! 6r) 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 storage solution, which is most important for practical use. It wasn't.

Recently, a method for inhibiting aggregation using glycerol has been developed (JP-A-Sho,
4 (7-7FJ, 2) was disclosed, but? Method 1 1j2
, which is complex and removes lipids, cholesterol, etc. at high temperatures (
Dispersible in glycerol at 60° to 0c) and can be used with inclusions ii: It also has the problems of being severely limited.

(Objectives of the Invention) Therefore, one object of the present invention is to provide a method for storing stable liposomes with a strengthened membrane structure and less aggregation during storage.

(Structure of the Invention) The object of -OE was achieved by a method for preserving microcapsules, which is characterized by adding amino acids to a liposome preservation solution.

The present inventors, from the viewpoint of aggregation during liposome storage,
After extensive research, we discovered that aggregation can be greatly suppressed by adding all amino acids to the liposome storage solution.

The amino acid used in the non-exploitable amino acid is preferably a low-molecular-weight neutral amino acid or a salt of a basic amino acid or an acidic amino acid.

The amino acid S' appropriate addition lid used in the present invention is preferably (Ds4/Q/θ) (preferably 3Q/-7%) with respect to the storage solution lid.

All specific examples of suitable amino acids are listed below, but the invention is not limited to these.

A.-7 The liposome membrane-forming lipid in the present invention is not limited in terms of its properties, and as long as it forms liposomes, it can be natural,
In addition, synthetic lipids (1, lipid and glycolipid) can be used.

Examples include lecitate/% phosphatidylethanolamine, phosphatidic acid, phosphatidyl seri/, phosphatidylinositol, phosphatidylglycerol, sphingomyelin, cardiolipi/, and those obtained by hydrogenating these in accordance with a conventional method. You can also use 1.

Furthermore, it is preferable to use a membrane structure-strengthening agent such as sterol or cholesterol as a component of the liposome membrane, if desired. Charge-imparting substances (eg stearic acid, oleic acid, linoleic acid, lyric/acid) can also be added to the solution.

The liposome of the present invention can be used in a wide variety of fields, either by itself or by incorporating various substances.

For example, it can be used as a cell separation agent by utilizing its affinity for biological membranes.

They can also be used as carriers for drugs that are unstable in vitro or in viv, are released gradually in the body, or are desired to be quickly distributed to specific organs. Examples of hydrophilic drugs include anti-cancer drugs such as adriamycin, adriamycin, mitomycin, /-β-arabino-7-lacyl cytosi/, pleomycin, and cisprasin, antiviral drugs such as interferon, aminoglycosides (e.g., gentamicin), β-lactams (e.g. sulpenicillin).

Antibiotics such as cefotiam, cefmetuxime), TRH
, peptide hormones such as lyubrolide and insulin, enzymes such as lysozyme, asnoq-laginase, and glycosidase 7, and muramyl dipeptide.

Examples include immunostimulants such as muramyl dipeptide, immunoglobulins, and proteins such as various toxins.

Other materials other than drugs are not particularly limited as long as they are effective when administered in vivo, such as markers, plasmids, DNA, and RNA. The liposomes of the present invention are produced by methods known per se.

That is, the portic swing method (A, D, Bangham
J, Mo1. Biol,,ti,,23r(/ritritrit.

Sonication@(C, Huang, Biochem,
.

1,! ≠≠ (lri4ri)], prevesicle method [HoTr
auble, Neurosci, Res, Prog.

13u11. , F, J7J (/ri71)], Ethanol injection method C8, Batzri, Biochem, Biop
hys.

Acta,,-ta1-,10/J(/ri73)],7L
/ inch press extrusion method (Y, Barenhollz, F
E.B.S.

Lett, F, 210 (/79)], Chonic acid removal method CY, Kagawa, J, Biol, Chem.

24At, Jlfi77 (/ta7l)],
Torito yX-100 batch method (W, J, Ger
ritsen, Eur.

J,Biochem,,rJ,λj! (/ri7t):
) rCa” + fusion method CD, PaPahadjopoul
os.

Biochem, Biophys, Acta, sup
, 4'iJ (/ri77)], ether injection method CD, De
amer.

Biochem, Biophys, human cta,, 4'4
'J, tkota (lli74)), annealing method (
R.

Lawaczeck, Biochem, Biophys
, Acta.

4AIIJ, J/J (/ri7t)], freeze-thaw fusion method (
M., Kasahara, J., Biol, Chem.

2! , 2,73Ilfi (/ri77)), W10/W
- Marujo/Method (S, Matsumoto, J,
Co11oidInterface Sci,,A
2, / 4'ri (lri77)'J.

Reverse phase evaporation method [F, 5zoka, Proc, Na1l.

Many methods are known, such as Acad, Sci, USA, 7j, Hirouri 4A (/ri 7r)], but
In the present invention, any of the above-mentioned preparation methods may be used, and the present invention is not limited thereto.

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

Example 16 Liposomes having the composition shown in Table 1 were prepared using the reverse phase method (Proc.

Natl, Acad, Sci, USA, 7j (ri)≠li≠ (/ri77) JP-A No. 11-1114111).

A solution of a predetermined amount of phos-7-atedylcholine and cholesterol dissolved in chloropormuco0ILt! Place in a Qd eggplant-shaped flask. The solvent is distilled off using a rotary evaporator to form a thin film on the inner wall of the flask.

Then, diethyl ether solution was added to dissolve the lipids, and a further amount of HEPES buffer (pH=7.t) was added.

Then, after replacing the gaseous phase in the eggplant flask with t-N2 gas, while keeping the eggplant flask at 100C, keep stirring until a homogeneous suspension is formed. Ultrasonic irradiation was performed using a probe for a minute.

Next, use the rotary evaporator! Most of the diethyl ether was distilled off under reduced pressure at 0C.

After the gel-like lipid thus prepared was mixed with a portex for 20 seconds, the solvent was completely removed under reduced pressure of 0-21''C.

HEPE8 buffer ljd was added to this (pH near, and ultrasonic irradiation was performed for 20 minutes at Riko O0C. 5ephar
After passing Gel F in ose Hiro B, the average particle diameter and phospholipid concentration (manufactured by Wako; using a phospholipid measurement kit) for each 7 lactation (manufactured by Nikkaki Co., Ltd.; using Coulter Ng-submicron particle analyzer) The division was determined. Then, the phosphorus concentration was adjusted to 3.0X/θ MK using Fujiction with an average particle size of O8/-0.2μ. There are various kinds of preparations! Amino acids were added once to bring the total amount to /Q.

Example-9 The aggregation test was carried out by storing samples with compositions 1 to 7 in a constant temperature room at 100C for a long period of time, and measuring the particle size and visually observing the samples. (The particle size is recorded after applying a latex for λ minutes before measuring the particle size.

) From the results in Table 1, it can be seen that by using the method of the present invention, there is less aggregation of liposomes than when no additive is used, and the particle size hardly changes.

Similar trends were observed even when the amount of abanoic acid was varied between 7% and 10%.

(Effects of the Invention) The method of the present invention suppresses aggregation of liposomes formed when lipids are dispersed in water during storage.

It is effective for

The inventors added various additives to the liposome storage solution and after long-term test results, they discovered that amino acids have a greater-than-expected aggregation inhibiting effect.

Patent applicant: Fuji Photo Film Co., Ltd.

Claims (1)

[Claims] A method for preserving liposomes, which comprises adding amino acids to a liposome preservation solution.