RU2310663C1 - Method for preparing liposome composition with gel synthesized from cysteine and silver nitrate - Google Patents

Abstract

FIELD: biotechnology, chemical technology.

SUBSTANCE: invention relates to a method for preparing liposome-containing compiositions. Method involves placing phosphatidylcholine in an organic solvent into rotor evaporator and solvent is distilled off to obtain phospholipid film on rotor evaporator flask walls. Then film is transferred to aqueous dispersion and liposomes containing incorporated additive are prepared. Then aqueous dispersion of liposomes is subjected for dialysis and an aqueous solution containing gel-forming components is added. Ethyl alcohol is used as an organic solvent. Solvent is distilled off at 40^oC and phospholipid film is transferred to dispersion in a shaking device. Cysteine and silver nitrate are used as gel-forming components. Invention provides excluding using toxic organic solvent, elimination of sterilization of liposome dispersion and addition of special antibacterial additives to the ready preparation. Invention can be used in medicinal and cosmetic practice.

EFFECT: improved preparing method.

2 dwg, 1 ex

Description

The invention relates to the field of production of liposome compositions that are carriers of biologically active substances and are immobilized on a gel matrix, can be used in medical and cosmetic practice.

A known method of producing protein-containing liposomes (German patent No. 4402867 class. C07K 17/04, prototype), including

- preparation of a solution of lipid in an organic solvent;

- obtaining a lipid film on the walls of a rotary evaporator by distillation of the solvent;

- preparation of an aqueous dispersion of liposomes;

- mixing the dispersion with an aqueous solution of protein;

- sterilization of the finished composition by filtration through a polymer membrane;

- immobilization of the obtained liposomes on a gel cellulose matrix: sodium carboxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose.

The disadvantages of this method:

- a toxic organic solvent is used - chloroform, as a result of which an additional operation becomes inevitable - drying the lipid film in vacuum (residual pressure - 0.7 kPa);

- the need to sterilize the finished composition: the dispersion of protein-containing liposomes is filtered through a polymer membrane - polycarbonate with a pore width of 200 nm - when using a microextruder;

- the need to introduce biocidal additives in the finished composition to prevent microbiological contamination during storage.

The technical result of the present invention lies in the fact that the method of immobilizing liposomes on a gel matrix eliminates the use of a toxic organic solvent and allows us to consider the production process of the drug as environmentally friendly, and also that sterilization of the liposome dispersion and the introduction of special antibacterial additives in the finished drug are not required .

The technical result is achieved by the fact that the claimed method uses an organic solvent with low toxicity, ethyl alcohol, and the fact that liposomes that are carriers of biologically active substances are immobilized on a gel matrix based on cysteine and silver nitrate.

A study of the prior art found that no methods for immobilizing liposomes on a gel matrix based on cysteine and silver nitrate were found.

The known method (German patent No. 4402867, class C07K 17/04). However, a comparison of the properties of the totality of the features of the known method and the claimed shows that

- in the known method uses a toxic organic solvent - chloroform, and in the inventive - with low toxicity ethyl alcohol;

- in the known method, the dispersion of liposomes is obtained at room temperature, and in the claimed - in the temperature range from room temperature to 40 ° C;

- in contrast to the known in the claimed method, the preparation of the composition (see Disclosure of the invention) is carried out on the basis of a gel formed by the interaction of cysteine and silver nitrate in an aqueous solution, the frame structure of which is loose, rarefied, which significantly reduces the amount of substance for gelation;

- in contrast to the known inventive method correspond to compositions having antibacterial properties.

Therefore, the claimed method of immobilization of liposomes meets the criterion of "Significant differences".

The method is illustrated in graphic materials (Fig.1-2).

Figure 1 shows the structure of a liposome - a carrier of a biologically active substance (BAS) immobilized on a gel matrix.

A is a liposome; B - gel matrix.

In aqueous solutions, phospholipid molecules self-assemble into a bilayer membrane, and then the membranes are closed with the formation of lipid vesicles - liposomes.

Figure 2 presents an electron microscopic photograph of liposomes (electron microscope brand REM "DSM-962" Zeiss, resolution - 1: 12800, contrasting with uranyl acetate) embedded in a matrix of a gel obtained on the basis of cysteine and silver nitrate.

Disclosure of the invention

A method of producing a gel-liposome composition relates to the field of nanotechnology, supramolecular and colloid chemistry, namely: to the preparation of compositions of supramolecular objects, one of which is liposomes, and the other is gels based on cysteine and silver nitrate.

Liposomes are formations of phospholipids that are able to be structured in solutions in such a way that they form membrane capsules, within which a specific substance is embedded. Thus, liposomes can serve as "microcontainers" for the inclusion of substances of a certain chemical nature. Liposomes are studied in colloidal and supramolecular chemistry as a model of artificial cells. Currently used in the production of biomedical preparations and in cosmetology.

The matrix for liposomes, in particular, are gels; natural and synthetic polymers of various nature are used as gelling agents. Gels as three-dimensional disordered structures are the object of study of colloid chemistry, chemistry of high molecular weight compounds and are often used in the practice of thickening and structuring compositions of complex composition.

In world practice, there is a tendency to use liposomes filled with biologically active substances in the development of pharmacological and cosmetic preparations (Liposomes in biological systems. M: Medicine, 1983).

Since the aqueous dispersion of liposomes is not aggregatively stable, they are fixed in the gel matrix.

The disadvantage of compositions based on polymer gels is the fact that gelation is observed at high polymer concentrations in solution, from a few percent or more. The polymer in the composition is a ballast substance, and the technology for producing liposome compositions based on polymer gels is a complex technological process that requires expensive equipment.

In the inventive method, the matrix for liposomes is a new type of gel: a gel synthesized based on cysteine and silver nitrate. This kind of gel in its structure is a combination of fractal clusters with a low fractal dimension, as a result of which the gelation point is reached at low concentrations of cysteine and silver nitrate.

The bactericidal properties of silver cations, which are fixed in the gel structure, reliably protect the composition from microbiological contamination. The composition does not change for two years when stored at room temperature.

The method is as follows.

A solution of fofatidylcholine in ethanol is placed in a flask of a rotary evaporator, a vacuum is created at a residual pressure of not more than 2 kPa, and when the solution is heated to a temperature of 40 ° C, the solvent is distilled off. Get a film of phospholipid on the walls of the flask of the evaporator. Then a small amount of distilled water is poured into the flask and placed in a shaker. The result is an aqueous dispersion of liposomes. A biologically active substance is introduced into the dispersion and dialyzed. Liposomes become carriers of a biologically active substance. In a gelation vessel, aqueous solutions of cysteine and silver nitrate are mixed, the mixture is kept at room temperature for 15-24 hours, after which a liposome dispersion is introduced into it.

An example of the method.

1.3 ml of a 10% ethanol solution of phosphatidylcholine is placed in a round-bottom flask, the flask is fixed in a rotary evaporator, the evaporator is turned on and alcohol is distilled off when the solution is heated to 40 ° С. Then, 15 ml of an aqueous prednisone solution is poured into the flask and placed in a shaker for 20 minutes. An aqueous dispersion of liposomes is obtained which is dialyzed to free from prednisone not included in the liposomes.

To 3.2 ml of a 1% aqueous dispersion of liposomes - carriers of prednisone - add 0.6 ml of an aqueous 0.03 molar solution of cysteine, and then, after stirring, 0.9 ml of an aqueous 0.03 molar solution of silver nitrate. The composition is mixed and left to stand in a dark place for 15 hours.

In the composition thus obtained, in comparison with the prototype, the content of gelling components is significantly reduced and, thereby, the relative content of liposomes is increased.

In the inventive method used reagents and equipment of domestic production. The technology of immobilization of liposomes containing biologically active substances on a cysteine - silver nitrate gel matrix is not complicated and can be implemented at any enterprise specializing in the production of pharmacological and pharmaceutical preparations.

Therefore, the claimed method meets the criterion of "Industrial applicability".

Claims (1)

The method of immobilizing liposomes on a gel matrix containing biologically active substances, namely, that a solution of phosphatidylcholine in an organic solvent is placed in a rotary evaporator, the solvent is distilled off to obtain a phospholipid film on the walls of the flask of a rotary evaporator, the film is transferred to an aqueous dispersion, obtaining liposomes with the added biode after which the aqueous liposome dispersion is dialyzed and introduced into an aqueous solution containing gelling components, characterized in that as an organic Ethyl alcohol is used in the solvent, the solvent is distilled off at 40 ° С, the phospholipid film is dispersed in a shaker, and cysteine and silver nitrate are used as gelling components.

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