RU2789327C1 - Method for production of hemostatic porous composite material - Google Patents

Abstract

FIELD: pharmaceutical industry.

SUBSTANCE: invention relates to the pharmaceutical industry, namely to a method for the production of hemocoagulating aerogel of bacterial cellulose and chitosan. The method for the production of hemocoagulating aerogel of bacterial cellulose and chitosan includes obtainment of bacterial cellulose with Medusomyces gisevii culture, purification with 1% sodium hydroxide solution at 80°C for 2 hours, followed by multiple flushing from a reagent with five volumes of distilled water until pH value of 5.5, homogenization on ER-10 homogenizer for 5 minutes at 6000 rpm until a gel-like structure and dynamic viscosity of 10-15 Pa*s, mixing until a homogenous consistency in a ratio of 1:0.5 with gel obtained by dissolution of chitosan with a deacetylation degree of 80% in 0.1 M acetic acid until a concentration of 1 mol/l for 12 hours, freezing in aluminum trays, followed by lyophilic drying under certain conditions.

EFFECT: above-described method allows for expansion of the range of hemostatic materials based on a complex of bacterial cellulose and chitosan having an expressed hemostatic effect.

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Description

The invention relates to the pharmaceutical industry and medicine and is aimed at creating an effective hemostatic material that can be used to stop external bleeding of varying intensity arising from open injuries and wounds and is a hemostatic airgel of bacterial cellulose and chitosan with a deacetylation degree of at least 80%.

Materials designed to stop bleeding can be made from natural biodegradable polymers and synthetic materials. These materials, depending on their composition and form of release, cause an acceleration of blood clot formation through the activation of various blood coagulation mechanisms. The nature of the components of hemostatic materials will determine both the effectiveness of hemocoagulation, which will determine the possibility of using these materials to stop not only capillary bleeding or venous bleeding, but also, possibly, arterial bleeding, and the appearance of possible side effects. Currently, hemostatic materials based on natural polymers such as cellulose and chitosan are widely used.

Known hemostatic sponge obtained by freeze-drying a mixture of a solution of chitosan in acetic acid and fibrinogen solution (Patent RU 2618896, A61K 9/18, A61L 15/28, A61L 15/32, A61L 15/20, A61L 15/44, A61P 7/04 , published 05/11/17). A hemostatic sponge was obtained by dissolving 1.0 g of chitosan, 1.0 g of acetic acid in 500 ml of distilled water with stirring on a shaker for 60 minutes, followed by the addition of 500 ml of distilled water and 0.1 g of fibrin monomer. The resulting mixture was stirred until a homogeneous mass was obtained, then poured into trays with a layer from 2 to 10 mm thick, which were placed in a freeze-drying chamber at t to -50°C for 48 hours until a sponge was formed.

The disadvantage of the known method is the difficulty of obtaining fibrin monomer, as well as the need to use a large amount of blood to isolate fibrin monomer.

A known method for producing a hemostatic mixture based on long and short cellulose fibers (RU 2731371, A61L 24/06, A61L 24/04, A61L 24/00, publ. 02.09.20). The hemostatic composition was obtained by grinding the material based on cellulose with the formation of long fibers and small fibers and mixing long and small fibers in a ratio in the range of 5-25% of the mass. and 95-75% of the mass. respectively, thus obtaining a hemostatic fiber composition. To give the material hemostatic properties, the composition included divalent cations, a positively charged peptide and/or polysaccharide, omega-aminocarboxylic acid, individually or in combination with each other.

The disadvantage of this method is the difficulty in selecting the conditions for obtaining fibers with the required range of linear dimensions.

A known method for producing a hemostatic agent based on oxidized cellulose using microwave exposure (RU 2563279, C08B 15/04, C08B 15/02, A61L 15/42, A61P 7/04, publ. 20.09.2015). Medical gauze or powdered cellulose was oxidized with a solution of nitrogen dioxide in an inert solvent under the influence of microwave radiation with a power of 60-100 W for 1-5 hours at a temperature of 40-50 ° C, while using constant external cooling of the vessel with air current, then the solution is removed in vacuum water jet pump, the oxidized fabric or powdered cellulose is washed and dried.

The disadvantage of this method is the complexity of the procedure for the oxidation of cellulose during its oxidation, as well as one-component hemostatic agent.

A known method for producing hemostatic powder from oxidized regenerated cellulose (RU 2014152706, A61L, publ. 20.07.2016). The hemostatic material is an oxidized regenerated cellulose (ORC) powder that contains particles ranging in size from 1.75 µm to 116 µm with a median size of 36 µm, having an average aspect ratio of from about 1 to about 18, a bulk density after compaction of at least 0.67 g/cm ³ and a fluidity of at least 7.5 cm/s. The hemostatic material can be used alone or contain additives, which are carboxymethyl cellulose (CMC) or other polysaccharides, a calcium salt, an anti-infective agent, a hemostasis activator, gelatin, collagen, or combinations thereof.

The disadvantages of the known method are the steps of mechanical processing of oxidized regenerated cellulose by pressing it to obtain a powder until the proportionality factor of the specified powder reaches a value from about 1 to about 18.

The closest in technical essence to the claimed invention is a method for producing a hemostatic agent based on chitosan airgel (RU 2743425, A61K 9/70, A61K 31/722, A61L 15/22, publ. 18.02.2021). The hemostatic material was a single or multilayer nonwoven material, with a surface density of 70 g/m ² , obtained from cellulose fiber (69%) and staple fiber (31%), impregnated with a chitosan solution, with a molecular weight of 125 and 343-420 kDa and the degree of deacetylation of 75% and 90%, with a concentration of 1 to 4 mol/l to form a chitosan gel for at least 6 hours, followed by supercritical drying.

The disadvantage of the prototype is that only chitosan hemostatic material is present in the form of an airgel. In addition, the hemostatic material has low efficiency, and there is also a possibility of wound infection due to shedding of chitosan particles.

The technical result of the claimed hemostatic material and the method of its production is to expand the range of hemostatic materials based on chitosan and bacterial cellulose with a pronounced hemostatic effect aimed at stopping bleeding that occurs during injuries and wounds.

The technical result is achieved by creating a hemostatic airgel with a pronounced hemostatic effect, consisting of bacterial cellulose isolated from a culture of Medusomyces gisevii and chitosan with a deacetylation degree of 80%, obtained from chitin of shrimp shells.

The essence of the invention is that a hemostatic sponge in the form of an airgel is obtained from bacterial cellulose isolated from a culture of Medusomyces gisevii by chemical treatment with 1% sodium hydroxide solution at a temperature of 80°C for 2 hours, followed by repeated washing of the polymer from the reagent with five volumes of distilled water to a pH value of 5.5 and subsequent homogenization of the bacterial cellulose film gel on an ER-10 homogenizer for 5 minutes at 6000 rpm, followed by mixing with chitosan gel with a concentration of 1 mol/l, obtained by dissolving chitosan with a deacetylation degree of 80%, isolated from chitin of shrimp shells during a sequential process of demineralization, deporteinization and deacetylation in the presence of 7% HCl, 7% NaOH and 50% NaOH for 1.5 hours at a temperature of 90-145°C, in 0.1 M acetic acid, freezing the mixture obtained and freeze-dried to a moisture content of 0.1%. The hemostatic airgel obtained in this way has a developed porosity, which reaches 99%, which allows this material to absorb the liquid part of the blood, increasing the rate of formation of a blood clot that fills the wound and stops bleeding.

Bacterial cellulose to obtain a hemostatic sponge was obtained using a symbiotic cultureMedusomyces giseviiaccording to the previously described method (Gladysheva E.K., Skiba E.A. Biosynthesis of bacterial cellulose by cultureMedusomyces gisevii. Bulletin of the Voronezh State University of Engineering Technologies. 2015. No. 3, P 149-156). Purification of bacterial cellulose from bacterial cells was carried out by treating it with 1% sodium hydroxide solution at 80°C for 2 hours, followed by repeated washing of the polymer from the reagent with five volumes of distilled water until a pH value of 5.5 was reached. Bacterial cellulose was subjected to homogenization on an ER-10 homogenizer for 5 minutes at 6000 rpm until a gel-like consistency was reached, having a dynamic viscosity in the range from 10 Pa*s to 15 Pa*s. chitosan obtained by dissolving chitosan with a degree of deacetylation of 80% in 0.1 M acetic acid to obtain a concentration of 1 mol/l for 12 hours. The resulting composite hydrogel, consisting of bacterial cellulose and chitosan, was transferred into aluminum trays of the required shape until a hydrogel layer of 2 cm was obtained and frozen at -60°C for 24 hours, followed by freeze drying until a moisture content of 0.1% was reached at a pressure of 0.035 mbar within 48 hours. The resulting material was a hemostatic airgel, which had a high porosity, reaching 99%, which allowed this material to absorb the liquid part of the blood, increasing the rate of formation of a blood clot, which fills the wound and stops bleeding.

The advantages of the proposed invention lie in the fact that the hemostatic airgel consists of nanosized bacterial cellulose fibers that closely interact with chitosan, thereby providing a strong hemostatic effect. Features of the technological process for obtaining a hemostatic airgel significantly increase the hemostatic activity of the biopolymer material formed by the claimed method. The resulting hemostatic airgel has a high hemostatic activity due to the ability to absorb the liquid part of the blood with a developed porous structure, as well as due to the interaction of blood with active hemocoagulant components of the material.

Examples of the implementation of the claimed method.

Example 1

A hemocoagulant airgel with a porosity of 99% was obtained from bacterial cellulose synthesized by a symbiotic culture of Medusomyces gisevii . Bacterial cellulose was obtained by purifying the gel film from the symbiotic culture by treating it with 1% NaOH solution at 80°C for 2 hours, followed by repeated washing of the polymer from the reagent with five volumes of distilled water until a pH of 5.5 was reached. Bacterial cellulose was subjected to homogenization on an ER-10 homogenizer for 5 minutes at 6000 rpm until a gel-like consistency was formed, having a dynamic viscosity in the range from 10 Pa * s to 15 Pa * s. The resulting hydrogel, consisting of bacterial cellulose, was transferred to aluminum trays of the required shape until a hydrogel layer of 2 cm was obtained and frozen at -60°C for 24 hours, followed by freeze drying until a moisture content of 0.1% was reached at a pressure of 0.035 mbar for 48 hours. The resulting material was a hemostatic airgel that absorbs the liquid part of the blood well, increasing the rate of formation of a blood clot, which fills the wound and stops bleeding.

Example 2

A hemocoagulating airgel with a porosity of 99% was obtained from chitosan with a degree of deacetylation of 80% isolated from the chitin of shrimp shells. Chitosan with a degree of deacetylation was obtained by sequential treatment of shrimp shell chitin during the processes of demineralization, deproteinization and deacetylation in the presence of 7% HCl, 7% NaOH and 50% NaOH for 1.5 hours at a temperature of 90-145°C, respectively. To obtain a gel, chitosan with a degree of deacetylation of 80% was dissolved for 12 hours in 0.1 M acetic acid to obtain a concentration of 1 mol/l. The resulting chitosan hydrogel was transferred into aluminum trays of the required shape until a hydrogel layer of 2 cm was obtained and frozen at -60°C for 24 hours, followed by freeze drying until a moisture content of 0.1% was reached at a pressure of 0.035 mbar for 48 hours. The resulting material was a hemostatic airgel that effectively absorbs the liquid part of the blood, increasing the rate of formation of a blood clot that fills the wound and stops bleeding.

Example 3

A hemocoagulating airgel with a porosity of 99% was obtained from bacterial cellulose synthesized by the symbiotic culture of Medusomyces gisevii and chitosan with a degree of deacetylation of 80% isolated from the chitin of shrimp shells. Bacterial cellulose was obtained by purifying the gel film from the symbiotic culture by treatment with 1% NaOH solution at 80°C for 2 hours, followed by repeated washing of the polymer from the reagent with five volumes of distilled water until a pH of 5.5 was reached. Chitosan with a degree of deacetylation was obtained by sequential treatment of shrimp shell chitin during the processes of demineralization, deproteinization and deacetylation in the presence of 7% HCl, 7% NaOH and 50% NaOH for 1.5 hours at a temperature of 90-145°C, respectively. Bacterial cellulose was subjected to homogenization on an ER-10 homogenizer for 5 minutes at 6000 rpm until a gel-like consistency was formed, having a dynamic viscosity in the range from 10 Pa*s to 15 Pa*s. chitosan in a ratio of 1:0.5, obtained by dissolving chitosan with a degree of deacetylation of 80% in 0.1 M acetic acid to obtain a concentration of 1 mol/l for 12 hours. The obtained composite hydrogel, consisting of bacterial cellulose and chitosan, was transferred into aluminum trays of the required shape until a hydrogel layer of 2 cm was obtained and frozen at -60°C for 24 hours, followed by freeze drying until a moisture content of 0.1% was reached at a pressure of 0.035 mbar within 48 hours. The resulting material was a hemostatic airgel that effectively absorbs the liquid part of the blood, increasing the rate of formation of a blood clot that fills the wound and stops bleeding.

Example 4

A hemocoagulating airgel with a porosity of 99% was obtained from bacterial cellulose synthesized by the symbiotic culture of Medusomyces gisevii and chitosan with a degree of deacetylation of 80% isolated from the chitin of shrimp shells. Bacterial cellulose was obtained by purifying the gel film from the symbiotic culture by treatment with 1% NaOH solution at 80°C for 2 hours, followed by repeated washing of the polymer from the reagent with five volumes of distilled water until a pH of 5.5 was reached. Chitosan with a degree of deacetylation was obtained by sequential treatment of shrimp shell chitin during the processes of demineralization, deproteinization and deacetylation in the presence of 7% HCl, 7% NaOH and 50% NaOH for 1.5 hours at a temperature of 90-145°C, respectively. Bacterial cellulose was subjected to homogenization on an ER-10 homogenizer for 5 minutes at 6000 rpm until a gel-like consistency was formed, having a dynamic viscosity in the range from 10 Pa*s to 15 Pa*s. chitosan in a ratio of 1:1, obtained by dissolving chitosan with a degree of deacetylation of 80% in 0.1 M acetic acid to obtain a concentration of 1 mol/l for 12 hours. The obtained composite hydrogel, consisting of bacterial cellulose and chitosan, was transferred into aluminum trays of the required shape until a hydrogel layer of 2 cm was obtained and frozen at -60°C for 24 hours, followed by freeze drying until a moisture content of 0.1% was reached at a pressure of 0.035 mbar within 48 hours. The resulting material was a hemostatic airgel that effectively absorbs the liquid part of the blood, increasing the rate of formation of a blood clot that fills the wound and stops bleeding.

Claims (1)

A method for producing a hemocoagulative airgel from bacterial cellulose and chitosan, including obtaining bacterial cellulose by a Medusomyces gisevii culture, purifying it by treating it with a 1% sodium hydroxide solution at 80°C for 2 hours, followed by repeated washing of the reagent with five volumes of distilled water until a pH value of 5 is reached ,5, homogenization on an ER-10 homogenizer for 5 minutes at 6000 rpm until a gel-like structure is formed and a dynamic viscosity of 10-15 Pa * s is reached, mixing until a homogeneous consistency is obtained in a ratio of 1: 0.5 with a gel obtained by dissolving chitosan with a degree of deacetylation of 80% in 0.1 M acetic acid to obtain a concentration of 1 mol/l within 12 hours, freezing in aluminum trays of the required form of a hydrogel layer equal to 2 cm at -60°C for 24 hours, followed by freeze drying at a pressure of 0.035 mbar for 48 hours to a moisture content of 0.1%.