SU1005791A1 - Method of including substances to liposoms - Google Patents

Abstract

METHOD FOR INCLUDING SUBSTANCES IN LIPOSOMS by adding an alcohol solution of phospholipids to an aqueous solution of the test substance, followed by separating liposomes from non-included material, characterized in that, in order to increase the internal volume of liposomes, the incorporation process of liposomes is carried out at a temperature of from -10 to -40 C for two or more days with posleuk psm heating to room temperature

Description

ate

with

The invention relates to biochemistry, in particular to methods for producing liposomes, and can be used in microbiology, pharmacology and medicine.

There is a known method for preparing liposomes by introducing an ethereal lipid solution into the aqueous phase containing the substance to be included, followed by evaporation of the ether by heating l 1.

However, this method is of limited use due to the high temperature of 55-60 ° C) of an aqueous solution, the substance to be included, and the impossibility of including thermolabile compounds in liposomes.

A known method for producing liposomes involves preparing an alcohol solution of phosphospides, introducing this solution into an aqueous solution of the substance to be switched on and separating the resulting liposomes from the non-included material by ultracentrifugation, dialysis or gel chromatography 23.

However, liposomes with a small internal volume (about 4 µl / µm mole of lipids) are obtained in a known manner - and, accordingly, with a low percentage of the test substance.

The aim of the invention is to increase the internal volume of liposomes.

The goal is achieved according to the method of introducing a substance into liposomes by adding an alcoholic solution of phospholipids to an aqueous solution of the test substance, followed by separating liposomes from non-included material and incorporating substances into liposomes at -10 to -40 ° C for two or more days. followed by warming to room temperature.

The method is carried out as follows.

PRI me R. In 1 ml of 96 ° ethanol

62 mg of dipalmitoylphosphagidylcholine, 23 mg of cholesterol and 1.6 mg are dissolved

diethyl phosphate, 560 μl of ethanol

a solution of lipids (80 mol) syringe

with a fine needle injected into 8 ml of glucose solution - C (50 µci / ml) in 0.01 M

phosphate buffer (pH 7.4). The formation of liposomes is judged by the sharp turbidity of the solution. The suspension is frozen at -40 ° C and stored in

 for two days. After this period, the preparation is thawed at room temperature and 2 ml applied onto a chromatographic column, mm), filled with Sephadex G-25 (coarse). The elution was carried out with 0, O1 M phosphate buffer pH 7.4, and collecting 5 ml fractions. The yield of liposomes is determined by turbidity of the solution visually and spectrophotometrically at 330 nm. Fractions containing; liposomes are pooled. The eluted free glucose-C 14 is detected by measuring radioactivity in fractions and is also pooled. The measurement of the radioactivity of glucose-C in preparations of liposomes and non-glucose glucose is carried out using a RZHS-2M liquid scintillation counter using a sc. The total internal volume of liposomes is determined by the efficiency of glucose-C incorporation into liposomes, expressed as a percentage. The calculation of the percentage of glucose-C incorporation into liposomes is based on the total radioactivity of glucose-C in both fractions. The percentage of glucose-C incorporation into liposomes was 8.06. The internal volume of liposomes was 8.06 uF

. 1 µm OLA lipids taken. For liposomes obtained by a known method, these values were 3.43% and 3.43 microns, respectively.

The method allows to obtain liposomes with a large internal volume; than in the well-known method. The table shows the data of comparative analysis to determine the internal volume of liposomes obtained by the proposed and in a known manner, i.e inclusion of glucose-C in liposomes before and after freezing and thawing.

Without freezing

(known method) 3016878О63,433,43

Freezing (1 day) 4537887254,864,86 Freezing 8О64 99556 (2 days) Freezing ia 8О19 82725 (3 days)

Two-day incubation at room temperature, without freezing, does not lead to an increase in the internal volume of liposomes.

Electron microscopic examination

Continuation table

, liposomes obtained by the proposed method, showed an increase in their size, compared with liposomes obtained by a known method, with the preservation of a monobiscular structure. 8, O6 8, O6 8.84 8.84

Claims (1)

METHOD FOR INCLUSION OF SUBSTANCES IN LIPOSOMES by adding an alcoholic solution of phospholipids to an aqueous solution of the test substance followed by separation of liposomes from unincorporated material, characterized in that, in order to increase the internal volume of liposomes, the process of incorporation of substances into liposomes is carried out at a temperature of from -10 to -40 ° C for two or more days, followed by warming to room temperature * 1 1005791