RU2356538C1 - Antigen-containing liposome preparation process - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine and biotechnology and concerns antigen-containing liposome preparation process. Substance of the invention includes mixing the dissolved H and D fractions of pseudocholera agent antigens, dissolving the neutral lipids in chloroform, blending the mixed dissolved antigen and lipids, preparing the liposomes by phase-inversion evaporation, removing unenclosed material vesicles by dialysis thereby using phosphate buffer 0.01 M, pH 7.2. The prepared liposomes contain 89-93% H and D fractions of pseudocholera agent antigens of size 600-800 nm.

EFFECT: advantage of the invention consists in liposome preparation of high load of pseudocholera antigen.

2 ex, 2 dwg

Description

The invention relates to medicine, and more specifically to the problem of creating a carrier containing antigenic material.

The problem of specific prophylaxis of melioidosis with the help of chemical vaccines that create intense immunity and are devoid of side effects is still far from a successful solution. In recent years it has been shown that certain fractions of surface melioid antigens have protective ability at low infectious doses of the causative agent of melioidosis (Piven N.N. Volgograd, 1997 .-- 296 p.); Viktorov D.V. Protein and antigenic composition of B. pseudomallei strains of different virulence: Diss ... cand. biol. sciences. - Volgograd, 1997 .-- 110 s). A number of authors have shown the possibility of increasing the efficiency of antigens of various pathogens immobilized in liposomes (Pseudomonas infection, plague, brucellosis) (Minukhin BV, Borodina NS, Tsyganenko A.Ya. et al. Immunogenic properties of the liposomal form of Pseudomonas aeruginosa // Vestnik of the USSR Academy of Medical Sciences. - 1990. - No. 8. - P.44-47; Byambaa A., Efremenko V.I., Taran T.V., Taran V.I., Overchenko V.V. Study of the protective properties of antigen-containing liposomes in case of plague // Diagnosis, treatment and prevention of dangerous infectious diseases. Biotechnology. Veterinary ia: Materials of the Anniversary Scientific and Practical Conference dedicated to the 70th anniversary of the Research Institute of Microbiology of the Ministry of Defense of the Russian Federation, November 30 - December 1, 1998. - Kirov, 1998. - P. 62-63; Kulikov V.N., Kashkin K.P. ., Dranovskaya E.A. Inclusion of the protective antigen of B. abortus in liposomes and immunogenic properties of antigen-containing liposomes // ZhMEI. - 1985. - No. 12 - P.69-72).

The prospects of liposomes are based on two of their main properties: similarity with natural membranes and universality. Universality lies in the fact that, due to the semi-synthetic nature of liposomes, they can be widely varied in size, surface characteristics, and composition. Liposomes are non-toxic and easily biodegradable. The possibility of using in lower doses, reducing the frequency of allergic and immunological reactions, the intracellular orientation of the vesicles and increasing the elimination time are the main advantages of using liposomes. However, the problem of emergency immunoprophylaxis of melioidosis is not sufficiently developed.

Preparation of monolamellar liposomes containing antigens by injecting an ethanol solution of phospholipids into the aqueous phase:

50 mg of a mixture of lipids (phosphatidylcholine, cholesterol, dicetylphosphate in a ratio of 7: 2: 1) is dissolved in 1 ml of 96 ° ethanol. An ethanolic lipid solution is drawn into a syringe with a needle and quickly injected into 15 ml of a solution of included 100 μg antigenic material in 0.01 M phosphate buffer pH 7.5. Unincorporated material is removed by centrifugation. The resulting liposomes comprise an average of 2.5% of the material in the internal volume. This method allows the inclusion of labile biological macromolecules without loss of specific activity. The disadvantage of this method is the limitation in the amount of lipids - 3.5 mg, which can be added to 1.0 ml of the aqueous phase, which in some cases requires subsequent concentration of the liposome preparation (Kremer J.M.N., Esker M.WJ, Pathmamanoharan C. , Wiersema, R.N., Vesicles of variable diameter prepared by a modified injection method. - Biochemistry, 1977, vol. 16, p. 3933-3935).

Preparation of liposomes by the method of "manual" shaking:

100 mg of a mixture of lipids (phosphatidylcholine, cholesterol, dicetylphosphate in a ratio of 7: 2: 1) was dissolved in 50 ml of chloroform in a 200 ml round bottom flask for a rotary evaporator, and the organic solvent was evaporated under reduced pressure. The end time of evaporation was determined by the disappearance of the smell of chloroform in the flask, while a thin lipid film was formed on its walls. The flask removed from the rotary evaporator was purged with nitrogen gas to prevent lipid oxidation and then 5 ml containing 100 μg of antigenic material in 0.01 M phosphate buffer pH 7.2 was added. After a 2-hour incubation at room temperature, several glass beads were placed in a flask to form a homogeneous suspension of liposomes and shaken vigorously for 5 minutes. Unincorporated antigenic material was removed by centrifugation. Mild cooking conditions and large liposome sizes allow this method to be used to incorporate large biological macromolecules without significant loss of activity. The disadvantage of this method is the low percentage inclusion of the material (1-3%) (Acuto O., Pugliese O., Muler M., Tosi R. Preparation of liposomes incorporating membrane components from human lymphoid cells // Tissue Antigenes. - 1979. - V. 14, No. 5. - P.385-397).

The closest analogue is the liposome preparation method proposed by Szoka F., Papahandjopoulos D. Procedure for preparation of liposomes with large internal space and high capture by reverse-phase evaporation // Proc. Nat. Sci. USA - 1978. - V.75, N 9. - P.4194-4198. According to the method, 30 mg of lipids (phosphatidylcholine, cholesterol, dicetylphosphate in a ratio of 7: 2: 1) are dissolved in 3 ml of a mixture of ether and chloroform 2: 1 and introduced into a round bottom flask of a rotary evaporator with a volume of 100 cm ³ 1 ml of a solution of the included material in 0.01 M phosphate buffer pH 7.5, add to the solution of lipids in the organic phase and sonication with 20 kHz, 200 W power for 5 minutes to achieve the formation of an emulsion of the type "water in oil". The emulsion flask is attached to a rotary evaporator and gradually lowering the pressure so that boiling does not occur and the organic solvent is completely removed. The end of evaporation is judged by the formation of gel in the flask and the disappearance of the smell of an organic solvent. During the evaporation process, the temperature of the mixture is maintained above the phase transition temperature of phospholipids (20-25 ° C). The flask was removed from the evaporator, 5 ml of 0.01 M phosphate buffer, pH 7.5, were added to the resulting gel and shaken until a homogeneous suspension of liposomes was formed. Liposomes obtained by this method are characterized by high inclusion efficiency - up to 50-60% and large sizes - up to 1.0-1.2 microns. Unincorporated material is removed by centrifugation. The disadvantages are the low stability of the emulsion and the gel type "water in oil", as a result of which, in each case, it is necessary to select the composition of the lipid mixture, the evaporation technique and skill in use, the use of a mixture of solvents with different boiling points, which leads to an inhomogeneous formation of phospholipid vesicles (temperature boiling of ether + 34.5 ° С, boiling temperature of chloroform + 61.2 ° С), as well as the toxic effect of titanium ions on antigenic material and phospholipids (G. Gregoriadis, A. Allison. Liposomes in biological systems topics. - M .: Medicine. - 1983. - S.28-29).

The aim of the invention is to develop a method for producing antigen-containing liposomes with a high amount of immobilization of fractions (H, D) of the antigen of the causative agent of melioidosis.

This goal is achieved by the fact that due to an increase in lipid load, the stability of the liposome membrane increases, their number and the percentage of encapsulation of antigenic melioid fractions (H, D) increases. The universality of the carrier is ensured by the neutral charge of the liposome membrane. Obtaining an emulsion on a rotary mixer eliminates the toxic effect of titanium ions (titanium probe of an ultrasonic disintegrator) on antigenic fractions. For this, 56.7 mg of a mixture of lipids (phosphatidylcholine, cholesterol in a ratio of 7: 3) was dissolved in 3.8 ml of chloroform. The included material, dissolved in 0.01 M phosphate buffer pH 7.2, in a volume of 1.5 ml was added to the lipid solution in the organic phase, and emulsion formation was achieved by treatment with a 500 W rotary mixer at 15,000 rpm for 1 min for 1 min . The organic solvent was completely removed under reduced pressure on a rotary evaporator. To the resulting gel was added 5 ml of 0.01 M phosphate buffer pH 7.2 and thoroughly mixed in a flask on a rotary evaporator. Removal of unincorporated antigenic material was carried out by dialysis. As a dialysis solution, a buffer taken for the preparation of liposomes (0.01 M phosphate buffer, pH 7.2) was used. Dialysis was carried out at + 4 ° C, constantly stirring the dialysate solution on a magnetic stirrer for 2 hours, after which the buffer was changed. For complete removal of antigenic material that is not included in the liposomes, 4 buffer changes are sufficient.

Due to the increase in lipid load, high stability of the emulsion and the water-in-oil gel has been achieved. The universality of the carrier is ensured by the non-neutral charge of the liposome membrane. The method used allows the use of one organic solvent with a specific boiling point. Obtaining an emulsion on a rotary mixer eliminates the toxic effect of titanium ions (titanium probe of an ultrasonic disintegrator) and reduces the processing time. This method allows you to encapsulate inside liposomes 89-93% of H and D fractions of the melioid antigen.

Examples of specific performance.

Example 1. Immobilization of liposomes of the H-fraction of the antigen of the causative agent of melioidosis.

For this, 56.7 mg of a mixture of lipids (phosphatidylcholine, cholesterol in a ratio of 7: 3) was dissolved in 3.8 ml of chloroform. 100 μg of the H-antigen fraction of the melioidosis pathogen, in 0.01 M phosphate buffer, pH 7.2, in a volume of 1.5 ml was added to the lipid solution in the organic phase and processed on a 500 W rotary mixer at 15,000 rpm for 1 min until the formation of the emulsion. The organic solvent was completely removed under reduced pressure on a rotary evaporator. To the resulting gel was added 5 ml of 0.01 M phosphate buffer pH 7.2 and thoroughly mixed in a flask on a rotary evaporator. Removing unincorporated material (fractions of H-antigen) was carried out by dialysis. As a dialysis solution, a buffer taken for the preparation of liposomes (0.01 M phosphate buffer, pH 7.2) was used. Dialysis was carried out at + 4 ° C, constantly stirring the dialysate solution on a magnetic stirrer for 2 hours, after which the buffer was changed. To completely remove antigenic material that was not included in the liposomes, 4 buffer changes were performed. The percentage of immobilization of the H-fraction was determined by chemiluminescence and the radioisotope method. He accounted for 89%. In this case, sorption of the H-fraction of antigen on the surface of the liposome membrane was not observed. The size of the liposomal preparation during electron microscopy was 600-800 nm (figure 1). The oxidation state of liposome membrane lipids was 0.8 ± 0.02 (Klein peroxide index) (Klein RA The detection of oxidation in liposome preparations // Biochim. Biophys. Acta. - 1970. - N 21. - P.486-489) .

Example 2. Obtaining the D-fraction encapsulated in liposomes of the antigen of the causative agent of melioidosis

Upon receipt of liposomes with a D-fraction of the antigen of the causative agent of melioidosis, the method of preparation of the drug is identical to that specified in example No. 1. The percent immobilization of the D fraction was 93%. In this case, sorption of the D fraction of antigen on the surface of the liposome membrane was not observed. The size of the liposomal preparation during electron microscopy was 600-700 nm (figure 2). The lipid oxidation state of the liposome membrane was 0.8 ± 0.02.

Thus, due to an increase in the load of lipids in the initial samples, high stability and ductility of the liposome membrane was achieved and their number increased. The preparation of an emulsion on a rotary mixer made it possible to eliminate the toxic effect of titanium ions on the antigenic material (titanium probe of an ultrasonic disintegrator) and to reduce the processing time. The universality of the carrier is ensured by the neutral charge of the liposome membrane. This method allows to obtain stable liposomes containing 89-93% of H- and D-fractions of antigens of the causative agents of melioidosis.

Claims (1)

A method for producing antigen-containing liposomes, including dissolving a portion of lipids in chloroform, preparing a solution of antigen fractions, creating an emulsion from a chloroform solution of lipids and antigenic material, preparing liposomes by evaporation with phase reversal, removing antigenic material that has not been incorporated into the vesicles by dialysis, transferring it to a solution for use and storage, characterized in that to obtain liposomes with a high content of native H and D antigens of the causative agent of melioidosis 56, 7 mg of a mixture of lipids (phosphatidylcholine, chol esterin in the ratio of 7: 3) is dissolved in 3.8 ml of chloroform, then 100 μg of the fraction of antigen dissolved in 0.01 M phosphate buffer pH 7.2 in a volume of 1.5 ml is added to the lipid solution in the organic phase and processed on 500 W rotary mixer at 15,000 rpm for 1 min until the formation of an emulsion is achieved, then the pressure in the rotary evaporator is reduced and the organic solvent is completely removed, up to 5 ml of 0.01 M phosphate buffer pH 7.2 is added to the resulting gel and mix thoroughly in a flask on a rotary evaporator, removing The cured material is carried out by dialysis, the buffer taken for liposome preparation (0.01 M phosphate buffer, pH 7.2) is used as the dialysis solution, dialysis is carried out at a temperature of + 4 ° C with constant stirring with a magnetic stirrer of the dialysis solution for 2 hours, after of which 4 buffer changes are made, the liposomes obtained in the chemiluminescent and radioisotope studies contained 89-93% of the H- and D-fractions of the melioid antigen, and in the electron microscopic examination, the sizes of antigen-containing liposomes were 600-80 0 nm.

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