RU2754849C1 - Method for fractionation of niosomes - Google Patents

Abstract

FIELD: biotechnology.SUBSTANCE: invention relates to the field of biotechnology, in particular, to a method for obtaining fractions of niosomes. A method for fractionation of niosomes, including dilution of a suspension of niosomes with a phosphate-buffered saline, centrifugation at 2,000 rpm, followed by transferring the supernatant to a clean test tube and centrifuging at 12,000 rpm, the resulting niosome residue is resuspended with a phosphate-buffered saline, obtaining niosomes with a size of 200±50 nm, under certain conditions.EFFECT: above method is simple in execution and provides a possibility to obtain niosomes of a considerably uniform size in large amounts.1 cl, 1 dwg, 3 ex

Description

The technical field to which the invention relates

The invention relates to the field of biotechnology, in particular to a method for producing fractions of niosomes, and can be used for the study of vesicles and for therapeutic purposes.

State of the art

A known method of obtaining nanoparticles of low molecular weight chitosan, including the preparation of a solution, pre-purified low molecular weight chitosan in filtered 1-2 wt. % aqueous acetic acid, adding solutions of alkali metal or ammonium hydroxides for 2 hours, dispersing the system using a mechanical stirrer at a speed of 200-300 rpm at a temperature of 20 ° C to pH 6.9-7.0, at the end of the process, dispersion centrifuged at a speed of 10000 rpm and the resulting solid residue is redispersed in a bidistillate with mechanical stirring at a speed of 200-300 rpm at a temperature of 20 ° C (see patent RU 2428432).

The considered method is characterized by the following disadvantages: the long duration of the dispersion process, the complexity of the hardware design and the multistage process of the formation of chitosan nanoparticles.

There is a known method of obtaining exosomes from blood, including the separation of blood into acellular and cellular fractions by centrifugation, followed by obtaining exosomes by ultracentrifugation, while the blood is separated into plasma and cellular fraction, then blood cells are sequentially, in two stages, treated first with a buffer solution of PBS, containing 5 mM EDTA, then an equal volume of 0.15-0.35% trypsin solution in PBS, then the plasma and the resulting supernatants are combined, cell debris and impurities of non-exosomal origin are removed by centrifugation at 15000-20000 g for 10-20 minutes and filtration through filters with a pore diameter of 0.1 μm, and the total pool of exosomes is precipitated by ultracentrifugation at 100,000-160000 g for 60-120 minutes. In the method, treatment with a PBS buffer solution containing 5 mM EDTA is carried out for 5 minutes, followed by centrifugation for 20 minutes at 300 g and collecting the supernatant. In the method, the treatment with 0.15-0.35% trypsin solution in PBS is carried out for 5 minutes, followed by the addition of an enzyme inhibitor, stirring, precipitation of blood cells by centrifugation for 20 minutes at 300 g and collecting the supernatant (see US Pat. No. 2556825).

The disadvantage of this method is the length and complexity of obtaining exosomes from blood in a wide laboratory and diagnostic practice.

A known method of isolation of highly dispersed fragments of purple membranes containing bacteriorhodopsin from halophilic bacteria by the method of disintegration, by repeated procedures of centrifugation, washing the precipitate and resuspension in double distillate or saline solutions, while in order to reduce the cost and accelerate the process, as well as to obtain homogeneous and small fragments of purple from 50 to 400 nm, the disintegration of an aqueous suspension of biomass of microorganisms is carried out by the method of cyclic extrusion under a pressure of 10 to 150 MPa from 1 to 5 cycles, and the subsequent procedure of centrifugation and washing in bidistilled water is carried out at 25000 g, 100000 g for 30 minutes each until until the ratio of the optical densities of the suspension of fragments of purple membranes at wavelengths of 280 and 560 nm becomes equal to from 1.9 to 2.1. In the method, the procedure of centrifugation and washing in bidistilled water is carried out in two stages, first at 25,000 g for 30 minutes, and then at 100,000 g for 30 minutes alternately (see US Pat. RU No. 94033539).

The disadvantages of this method is the long duration of the ultrasonic dispersion process; low productivity of the process; the inability to regulate the average size of fragments from the magnitude of the ultrasonic effect.

The closest invention to the described method in technical essence is a method for separating liposomes by density by gradient centrifugation of glycerol. After centrifugation, liposomes selected from several positions in such a gradient migrate as narrow bands at positions close to their original positions, indicating that the distribution of liposomes in the first gradient is the result of density-based fractionation. Molecular sieve chromatography, turbidity and trapped volume measurements show that the density of liposomes is qualitatively related to their size, with larger liposomes being denser than smaller ones. Sizes obtained by electron microscopy indicate that fractionation is effective for liposomes ranging from 200 to 600 A with maximum efficiency in the 200-300 A range, where most liposomes are found (Goormaghtigh,. Density-Based separation of liposomes by glycerol gradient centrifugation / E. Goormaghtigh, GA Carborough // Analytical biochemistry. - 1986. - No. 159. - P. 122-131).

The considered method has a number of disadvantages, the main of which are: labor intensity, duration of the process, low productivity of the process.

Disclosure of invention

The objective of the invention was the development of such a method for fractionation of niosomes, which is simple in execution, makes it possible to obtain niosomes of a sufficiently uniform size in large quantities and in high concentrations.

The technical result, which can be obtained with the help of the present invention, is reduced to a reduction in the duration of the process, an increase in its productivity, and a decrease in the labor intensity of obtaining fractions of nios.

The technical result is achieved using a method of fractionation of niosomes, in which centrifugation is performed, while 10 ml of a suspension of niosomes is diluted with 35 ml of chilled 0.01 Μ phosphate-buffered saline (pH 7.2) in a Falcon-type test tube, centrifuged at 2000 rpm in for 3 minutes at 4 ° C, then the supernatant is transferred into a clean Falcon tube, centrifuged at 12000 rpm for 1 hour at 4 ° C, the sediment of niosomes is resuspended in 0.01 Μ phosphate-buffered saline (pH 7.2) to a volume of 10 ml, store at a temperature of (2-8) ° C.

The essence of the method of fractionation of niosomes, including centrifugation, while 10 ml of a suspension of niosomes is diluted with 35 ml of chilled 0.01 Μ phosphate-buffered saline (pH 7.2) in a Falcon tube, centrifuged at 2000 rpm for 3 minutes at 4 ° C, then the supernatant is transferred into a clean Falcon tube, centrifuged at 12000 rpm for 1 hour at 4 ° C, the niosome sediment is resuspended in 0.01 Μ phosphate-buffered saline (pH 7.2) to a volume of 10 ml, stored at a temperature of (2-8) ° C.

Brief description of drawings and their materials

FIG. 1, a method for fractionation of niosomes is given, a micrograph of particles of the finished niosomal dispersion, obtained on an EVO LS 10 Carl Zeiss device.

Implementation of the invention

Examples of specific implementation of the method for fractionation of nios.

Example # 1.

The niosomal dispersion is purified from large particles (> 1 μm) and residues of niosome components as follows: 6 ml of the niosome suspension are diluted with 30 ml of chilled 0.01 Μ phosphate-buffered saline (pH 7.2) in a Falcon tube, centrifuged at 1000 rpm / min for 1 minute at 2 ° C, then the supernatant is transferred into a clean Falcon tube, centrifuged at 8000 rpm for 1/4 hour at 2 ° C, the niosome sediment is resuspended in 0.01 Μ phosphate-buffered saline ( pH 7.2) to a volume of 6 ml, store at a temperature of (2-8) ° C.

The particle size is 500 ± 130 nm. The presence of particles larger than 500 nm can probably be explained by the formation of fragile associates of niosomes in solution. The formation of fragile associates in solutions containing niosomes was confirmed during the study of samples after preliminary filtration of dispersions through a filter with a pore size of 220 nm by probe and electron microscopy.

Example # 2.

It is carried out analogously to example 1, but 8 ml of a suspension of niosomes is diluted with 33 ml of chilled 0.01 Μ phosphate-buffered saline (pH 7.2) in a Falcon tube, centrifuged at 1500 rpm for 2 minutes at 3 ° C, then supernatant transferred into a clean Falcon tube, centrifuged at 10,000 rpm for 0.5 hour at 3 ° C, the Niosome sediment is resuspended in 0.01 Μ phosphate-buffered saline (pH 7.2) to a volume of 8 ml, stored at temperature (2-8) ° С.

The particle size is 350 ± 95 nm. Also, as in example 1, the formation of fragile associates was observed in solutions containing niosomes.

Example No. 3.

It is carried out analogously to example 1, but 10 ml of a suspension of niosomes is diluted with 35 ml of chilled 0.01 Μ phosphate-buffered saline (pH 7.2) in a Falcon tube, centrifuged at 2000 rpm for 3 minutes at 4 ° C, then supernatant transferred to a clean Falcon tube, centrifuged at 12000 rpm for 1 hour at 4 ° C, the Niosome sediment is resuspended in 0.01 Μ phosphate-buffered saline (pH 7.2) to a volume of 10 ml, stored at a temperature ( 2-8) ° C.

The particle size is 200 ± 50 nm (Fig. 1).

Thus, example 3 is optimal. The considered method makes it possible to obtain niosomes of a sufficiently uniform size in large quantities and in high concentrations, and is also simple to execute.

The proposed invention in comparison with the prototype and other known technical solutions has the following advantages: reducing the duration of the process, increasing its productivity, reducing the labor intensity of obtaining fractions of nios.

Claims (1)

A method of fractionation of niosomes, including centrifugation, characterized in that 10 ml of a suspension of niosomes is diluted with 35 ml of chilled 0.01 Μ phosphate-buffered saline pH 7.2 in a Falcon tube, centrifuged at 2000 rpm for 3 minutes at 4 ° C , then the supernatant is transferred into a clean Falcon tube, centrifuged at 12000 rpm for 1 hour at 4 ° C, the resulting niosome sediment is resuspended in 0.01 Μ phosphate-buffered saline pH 7.2 to a volume of 10 ml to obtain niosomes of size 200 ± 50 nm.

Images