RU2840703C1 - Method of producing selenium nanoparticles - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention can be used in production of biologically active additives with antihypoxic properties. Method of producing selenium nanoparticles involves mixing a solution containing: 0.001-1 wt.% of potassium or sodium selenite, 0.5-5 wt.% of stabilizer – chitosan, 0.0011-1.5 wt.% of reducing agent – ascorbic acid, 0.1-6 wt.% of solvent – solution of acetic acid, or lactic acid, or succinic acid, or their mixture, and water – balance up to 100%. Obtained solution is subjected to microwave radiation with apparatus power equal to 400-1000 W for 2-10 minutes.

EFFECT: invention simplifies process of synthesis of selenium nanoparticles and increases efficiency thereof, biocompatibility and bioavailability.

5 cl, 3 dwg, 1 tbl, 6 ex

Description

The proposed invention relates to biotechnology, medicine and the food industry, concerns a method for producing selenium nanoparticles, which can be used in the production of biologically active additives with antihypoxic properties for humans, as well as in the field of veterinary medicine and agriculture, in particular livestock and poultry farming, and can find application as a biologically active additive with high digestibility in feed rations.

Hypoxia is a condition in which tissues and organs do not receive enough oxygen to support normal functioning. If oxygen saturation is severely impaired, irreversible destructive processes begin in the tissues, and organs gradually lose their functions. The causes of hypoxia can be extremely varied: staying in a stuffy, unventilated room; staying at height without using oxygen equipment; suffocation, drowning; swelling of the bronchial mucosa; bronchospasm; pulmonary edema; destruction of red blood cells and anemia; myocardial infarction; heart defects; pneumonia (including complications of coronavirus infection); poisoning with carbon monoxide, toxins, heavy metal salts, etc.; intense muscle stress, when the need for oxygen exceeds its actual flow to the tissues; vasculitis; vascular pathologies in diabetes mellitus. Selenium is an essential microelement and a basic component of the body's antioxidant system - the selenium-containing enzyme glutathione peroxidase prevents the accumulation of peroxide metabolic products in cells. Selenium has anti-inflammatory, antimutagenic, anticancer, antiviral, antibacterial, antifungal, immunomodulatory effects, and stimulates metabolic processes. In addition, it is necessary for the synthesis and action of thyroid hormones, which are important regulators of the body's growth and development. This microelement is important for a non-specific immune response, and its low level is associated with weakened immunity. Introducing selenium into the diet reduces inflammatory activity in the body. Selenium deficiency is a risk factor for the development of various pathologies - thyroid disease, iodine deficiency, reproductive failure, increased susceptibility to inflammatory diseases, diseases of the skin, hair and nails, cardiopathy, growth retardation, atherosclerosis, liver disease, anemia.

Compared with organic and inorganic forms of selenium (oxyanions of selenate or selenite (selenomethionine, selenocysteine, methylselenocysteine and sodium selenite), selenium nanoparticles demonstrate lower toxicity. It is also extremely important to develop innovative systems for selenium transport that would increase the bioavailability of this element and facilitate its controlled release in the body, for example, using a biocompatible, bioavailable polysaccharide chitosan as a. The main advantage of nanoselenium, compared with other forms: much lower toxicity. Due to this complex of positive properties, selenium nanoparticles stabilized with chitosan are a promising biological antihypoxic supplement.

An antioxidant agent with a hepatoprotective effect based on nanostructured selenium and methods for its production and use are known (RU 2557992 C1), which is a water-soluble powder of red-orange color containing zero-valence selenium in an amount of 0.5 to 60% in the form of a nanocomposite with selenium particle sizes in the range of 1-100 nm. The nanocomposite is obtained from various selenium-containing raw materials directly in an aqueous solution of a natural galactose-containing hepatotropic polysaccharide (arabinogalactan, galactomannan, carrageenan, etc.) with reducing agents - sodium borohydride, hydrazine hydrate or hydrogen peroxide, then it is purified by precipitation in 96% ethyl alcohol or another alcohol, or acetone and dried. To obtain a ready-made antioxidant agent, dry selenium polysaccharide nanocomposite is dissolved in distilled water, in the following ratio of components, mass %:

selenium-containing nanocomposite 0.0001-1.0 distilled water the rest

The ratio of the nanocomposite and water is set in such a way that the concentration of selenium in the prepared antioxidant agent is close to the effective dose of selenium in sodium selenite used for the prevention and treatment of white muscle disease caused by selenium deficiency in the body.

The disadvantage of this invention is the use of sodium borohydride as one of the reducing agents, since boron compounds are toxic to the body and may not be purified during reprecipitation of the composite, since they will be adsorbed by the polysaccharide.

A method for obtaining a Pleurotus tuber-regium polysaccharide functionalized nanoselenium hydrosol with antitumor activity and a method for obtaining it are known (US 9072669 B2), where Pleurotus tuber-regium is a royal tuber mushroom. Obtaining said hydrosol includes the following steps: 1) adding a vitamin C solution to an aqueous solution of Pleurotus tuber-regium polysaccharides and uniformly mixing them at normal temperature and under normal pressure; 2) adding dropwise a selenium dioxide solution or a selenite solution to the solution described above (1) with uniform mixing; and 3) adding water to this solution to a given volume to obtain a nanoselenium hydrosol stabilized by polysaccharides until the deepening of the red color of the hydrosol ceases.

The disadvantage of the known method is the use of Pleurotus tuber-regium polysaccharides, since the source of polysaccharides are fungi growing in the tropics, which determines the high price and low prevalence of this reagent.

The method described in patent US 9624237 B2 is known, which we have chosen as a prototype. In this method, selenium nanoparticles are synthesized by following the following steps:

a) a solution of a selenium-containing compound (selenite, selenite salt, selenite acid, selenium thiosulfate, selenium dioxide or a combination thereof), a solution of oridonin, a solution of a reducing agent (ascorbic acid, gallic acid, asparagine, sodium sulfite or a combination thereof) and a solution of a stabilizer (chitosan, hyaluronic acid, sodium hyaluronate or a cellulose derivative or a combination thereof) are prepared. The aim of the invention is to use the resulting composition in anticancer and anti-inflammatory procedures. The oridonin-functionalized selenium nanoparticle according to the prototype is a two-layer structure formed by the oxidation-reduction reaction of selenium and oridonin in the presence of a stabilizer. Selenium forms spherical nanoparticles, and the stabilizer and oridonin are coordinated and adsorbed on the surface of the selenium nanoparticles. In one embodiment, chitosan is used as a stabilizer in this invention. Then, selenite is reduced in the presence of a reducing agent (e.g., ascorbic acid) to form selenium nanoparticles. The stabilizer is absorbed onto the surface of the selenium nanoparticle, resulting in the appearance of NH ₃ ⁺ groups on the surface of the nanoparticles. And at the same time, oridonin is incorporated onto the surface of the selenium nanoparticle according to the present invention to form an oridonin-functionalized selenium nanoparticle. Oridonin-functionalized selenium nanoparticles solve the problem of oridonin's insolubility in water.

The average size of selenium nanoparticles functionalized with oridonin in the prototype is 35-160 nm. However, the bulk of the particles, based on the attached Fig. 4 (size distribution of nanoparticles) and Fig. 6 (TEM of selenium nanoparticles functionalized with oridonin) are characterized by sizes from 50-60 nm. It is known that the larger the size of the nanoparticles, the lower their efficiency due to the decrease in their "size" effect. The preparation according to this invention is characterized by a narrowly targeted specific action.

From patent CN 114288412 A (class A61K33/04; A61K47/36; A61K9/08; A61P25/24; A61P3/02; A61P35/00; A61P37/04; A61P39/06; B82Y40/00; B82Y5/00, published 08.04.2022), a method for producing and using a hydrosol of chitosan-nanoselenium particles is known. The method includes mixing a food solution of chitosan with selenious acid H ₂ SeO ₃ and vitamin C. A method for obtaining a hydrosol of chitosan-nanoselenium particles is proposed, which includes the following stages: mixing a food solution of chitosan with H ₂ SeO ₃ , mixing the solution with the chitosan solution for 30 min, adding a solution of ascorbic acid as a stabilizer, stirring for 30 min at room temperature until an orange-red solution with a stable color is obtained, and, finally, filtering and removing impurities from the orange-red solution using a filter membrane of an aqueous system to obtain a hydrosol of chitosan-nanoselenium particles. A distinctive feature is the use of only acetic acid for dissolving chitosan, and in subparagraph 4 of the formula the acid content is indicated as only 1%. The application of the patent is narrowly targeted: reducing oxidative stress to counteract the neurotoxicity of fluorine. In the proposed invention, chitosan dissolution occurs in aqueous solutions of acetic, succinic and lactic acids in concentration ranges of 0.5-6 mass. % (if necessary, the ranges are variable from 1.2 mass. %). The method presented in this isouretion does not require removal of impurities from the solution using a filter membrane. In addition, the main amount of selenium nanoparticles in the method of the present invention (more than 50%) are less than 10 nm in size, therefore, they are more biologically active (based on the nature of the nanoparticles).

From the patent CN111434339A (class A23K20/163; A23K20/20; A23K50/80; A61K31/722; A61K33/04; A61K47/36; A61K9/06; A61P37/04, published on 21.07.2020) the “Nanoselenium chitosan hydrosol with the effect of enhancing immunity and its production, preservation and use” is known. The invention relates to the technical field of nanoselenium hydrosol, in particular to nanoselenium chitosan hydrosol with an immunity enhancing effect and its production, preservation and use, wherein the nanoselenium chitosan hydrosol with an immunity enhancing effect contains the following components: 0.5 mmol/l-5.0 mmol/l of nanoselenium and 100.0 mg/l-1000.0 mg/l of functionalized chitosan. Chitosan is functionalized with an organic acid. The amount of chitosan specified in the patent corresponds to 0.01 wt. % - 0.1 wt. % (a concentration of up to 0.12 wt. % appears in subclauses). The patent indicates that the organic acid solution can be any citric acid solution and acetic acid solution. In the proposed invention, the concentration of chitosan is within the range: 0.5-5 wt. %, which is higher than in the analogous patent, we use solutions of acetic acid or lactic acid or succinic acid or their mixtures as a solvent.

From the patent CZ 26981 U1 (class A01N25/00; A01N43/00; A01N43/72; A01N45/00; A01N59/02; A61K31/43; A61K31/65; A61K31/7048; A61K33/04; A61K9/70; A61L15/44; A61L2/16; B82B1/00; B82Y5/00; C08L5/08; C08L89/00, published on 26.05.2014) the “Complex of a biopolymer substance with selenium nanoparticles and antibiotic preparations exhibiting antibacterial activity” is known. The complex of selenium nanoparticles in the technical solution contains a biopolymer substance in an amount of 0.01% to 5%, selenium nanoparticles in a concentration of 10 to 1000 μM and at least one antibiotic drug in a concentration of 10 μM to 10 mM, or other substances. An amino acid, mercaptopropionic acid, is used as a selenium ion reducer to nanoparticles. Its cost is much higher than the cost of ascorbic acid, which is given in the present invention of the method for producing selenium nanoparticles stabilized with chitosan. The upper limit for selenium concentration is 1000 μM, in terms of mass fraction it is 0.00779 mass %. This is the upper limit specified in the prototype. In the present invention, the limits for selenium when recalculated from salt: 0.000385 mass % - 0.46 mass %.

Known is the patent KR 101120635 B1 (class A01G1/00; C05D11/00, published on 16.03.2012) "Method for growing high-quality and functional vegetable fruits". When growing fruit vegetables using the cultivation method according to the present invention, functional minerals (Ca, Fe, Se), which are in short supply in the human body, as well as such qualities as sugar content, hardness, color difference, texture. Production of selenium nanoparticles: (SeO ₂ ) (selenium oxide IV, 99.5%) was added and dissolved. Then 500 g of ascorbic acid were added to the flask at a rate of 20 g/min. The product in the round flask was centrifuged for 30 minutes using a high-speed centrifuge (5000 rpm), centrifuged and washed three times with 1000 g of distilled water to obtain a selenium oxide precipitate, and then dried in a vacuum at 25 ° C for 24 hours to obtain 15 g of selenium nanoparticles before obtaining. Selenium nanoparticles were analyzed for particle size using laser diffraction (Nano-ZS, MALVERN). As a result, it was confirmed that the average particle size of selenium nanoparticles w is 12 nm. According to this technique, selenium nanoparticles are not stabilized by any polymer, therefore, they will further aggregate to larger particles. In addition, chitosan is known to increase the bioavailability of the components associated with it, since it acts as a "transport" for them in the body. In the prototype, no polymers are used as a stabilizer and transport for nanoparticles.

Known patent CN 112010271 A (class B82Y30/00; B82Y40/00; C01B19/02; C05G3/50; C05G5/12, published on 01.12.2020). According to the invention, a low-heat solid-phase method is used, various Se sources and various reducing agents are used, Se nanoparticles are first rapidly synthesized, Se nanoparticles modified with various surface modifying agents are synthesized later under the action of ultrasound and surface modifying agents, and a centrifugal separation method is used to separate the products, so that spherical Se nanoparticles with different sizes are ultimately obtained. The ultrasonic wave is used to accelerate the nucleation rate of the experimental product, increase the nucleation number and reduce the particle size. The method described in the prototype uses energy-consuming, multi-stage industrially unprofitable processes for obtaining selenium nanoparticles - ultrasonic treatment, lyophilization and centrifugation. In addition, at the first stage, nanoparticles are obtained by mixing selenium salt and a reducing agent, which are then stabilized by adding a surface modifier. Such a process will not ensure uniform distribution of nanoparticles in the modifier volume. In addition, ultrasonic treatment of the mixture will lead to partial destruction of chitosan to low-molecular values, which can affect the properties of the product. It is not specified in what form chitosan is introduced, since if a high-molecular polymer is used, it can only be acid-soluble, but the examples do not use any acids in which chitosan would be soluble.

Known is patent CN 102283318 A (class A23K20/163, published on 21.12.2011) "Method for preparing and using a meat improver consisting of ultrafine chitosan particles containing selenium". The preparation method comprises the following stages: 1) adding chitosan to an acid solution to obtain a chitosan acid solution; 2) dripping an aqueous solution of sodium polyphosphate into a chitosan acid solution with magnetic stirring, stirring and obtaining a milky suspension; 3) adjusting the pH of the milky suspension from 3 to 5 to form an emulsion of particles; 4) centrifugal separation of the emulsion of particles to obtain particles, adding particles to a selenium salt solution, adjusting the pH of the solution in the range from 4.0 to 6.0, carrying out the reaction at room temperature for 2-10 hours and obtaining an emulsion; 5) adjusting the pH value of the emulsion in the range from 7.0 to 9.0, washing, filtering or centrifugal dehydration, collecting the solid product; and 6) drying and crushing the solid product and thereby obtaining a meat improver of ultrafine particles of chitosan bearing selenium. The meat improver with ultrafine particles of chitosan containing selenium is used as a feed additive for livestock and poultry or aquatic animals. This method is multi-stage, labor-intensive, and energy-inefficient. Nanoparticles obtained by this method are broadly dispersed and large (from 5 to 500 nm). The acid for dissolving chitosan is not specified.

The closest technical solution to the claimed invention is the synthesis of a biologically active chitosan-selenium nanoparticle composition (article "Synthesis of a biologically active chitosan-selenium nanoparticle composition" by K. V. Apryatina, E. I. Murach, S. V. Amarantov, E. I. Erlykina, V. S. Veselov, L. A. Smirnova // Applied Biochemistry and Microbiology, 2022, Vol. 58, No. 2, pp. 140-14), adopted as the closest analogue (prototype). Synthesis according to the prototype includes obtaining selenium nanoparticles in a chitosan solution with a molecular weight of 200 kDa, a deacetylation degree of 85% (produced by Bioprogress LLC, Moscow Region, Russia) without additional purification. 3 wt. % chitosan was dissolved in 2% succinic acid. Selenium nanoparticles were obtained from sodium selenite with a concentration of 2.1 wt. % with the addition of ascorbic acid in an equimolar concentration with the required additional exposure to UV irradiation under a UV lamp with a power of 1600 mW/ ^m2 at a fixed distance of the solution from the lamp of 15 cm at a temperature of 35°C. The bulk of the formed nanoparticles fell on size characteristics from 20 to 60 nm.

The advantages and common features with the developed method for obtaining selenium nanoparticles are:

- use of the same source of selenium, stabilizer, reducing agent, solvent;

- concentrations of components in similar ranges to the present invention.

However, the prototype is not without its drawbacks:

- firstly, the complexity of the technological process when using the stage with UV irradiation, for the implementation of which a special UV installation with wavelengths of 313, 330, and 366 nm is required;

- secondly, the average size of the obtained nanoparticles is 20 nm, which reduces their bioavailability and effectiveness;

- thirdly, the choice of chitosan with a certain molecular weight for stabilization in the process of synthesis of selenium nanoparticles excludes its interchangeability, which complicates the synthesis process.

The objective of the invention is to develop a new method for obtaining biocompatible selenium nanoparticles stabilized by chitosan, without the use of UV radiation, and additionally possessing an antihypoxic effect.

The technical result of using the invention consists in simplifying the synthesis process, increasing the efficiency, biocompatibility and bioavailability of the obtained selenium nanoparticles, the possibility of using them as a biocompatible bioactive additive with antihypoxic properties for humans, as well as animals and birds, both in the form of a solution immediately after synthesis, and in dried form - in the form of a powder, for example, in the composition of dietary supplements, or in compressed form in the composition of tablets, capsules.

This is achieved by the fact that in the method for producing selenium nanoparticles, which includes mixing a solution containing a selenium source, which is sodium selenite or potassium selenite in an amount of 0.001-1 wt. %, a stabilizer, which is chitosan with a molecular weight of 25 kDa to 400 kDa in an amount of 0.5-5 wt. %, a reducing agent, which is ascorbic acid in an amount of 0.0011-1.5 wt. %, and a solvent, which is a solution of acetic acid, or lactic acid, or succinic acid, or a mixture thereof in an amount of 0.1-6 wt. % and water in an amount of up to 100% when calculating the total mass of the solution, the resulting solution is subjected to microwave radiation at a device power of 400-1000 W for 2-10 min; ascorbic acid is introduced into a chitosan solution with continuous stirring and constant stirring is maintained for 1-2.5 hours; the synthesis time of selenium nanoparticles is monitored spectrophotometrically by the appearance and decrease to a constant value of the absorption band corresponding to ascorbic acid λ ~ 250-280 nm; nanoparticles are formed with sizes in the range of 1-22 nm, with an average nanoparticle size of 5 nm; the resulting selenium nanoparticles are used as a biologically active additive with antihypoxic properties.

Fig. 1 shows the absorption bands of ascorbic acid during the formation of selenium NPs in a chitosan solution: 1 - 0 min, 2 - 30 min, 3 - 60 min, 4 - 90 min.

Fig. 2 shows a typical size distribution of chitosan-stabilized selenium nanoparticles determined by small-angle X-ray scattering.

Fig. 3 shows a typical manifestation of biocompatible properties on the surface of the sample films after application to the surface of hTERT BJ-5ta cell line fibroblasts: λ is a chitosan-based composite containing ionic selenium, [Se ⁺⁴ ] = 0.012 mol/l; b is a chitosan-based composite containing selenium NPs, [Se ⁰ ] = 0.012 mol/l.

The proposed method for obtaining selenium nanoparticles is related to green chemistry methods, since it does not use toxic organic solvents, and ascorbic acid, taken in molar excess relative to selenite, is used as an ion reducing agent (Fig. 1).

The stabilized selenium nanoparticles obtained by the proposed method, in all examples, are formed with sizes in the range of 1-22 nm, have a narrow distribution with an average size of 5 nm, which indicates their higher digestibility compared to the prototype, high biocompatible properties (Fig. 3) and an effective antihypoxic effect (Table 1).

The biocompatible, non-toxic, hypoallergenic polysaccharide chitosan was selected as a stabilizer for selenium nanoparticles, ensuring the transportation of nanoparticles in the body. In addition, chitosan itself is capable of increasing the bioavailability of the drug. The use of chitosan with different molecular weights - from 25 kDa to 400 kDa is interchangeable according to the given formula.

Ascorbic acid, which acts as a reducing agent for selenium ions, participates in oxidation-reduction reactions in the body, has a non-specific general stimulating effect, adaptive abilities and resistance of the body to infections, and also promotes regeneration processes.

All substances are of chemically pure or highly pure grade.

The proposed method for obtaining selenium nanoparticles is carried out as follows.

An aqueous solution of sodium selenate (or potassium selenate) is added to the aqueous acid solution of chitosan, then, with continuous stirring, the required amount of ascorbic acid is added, exceeding the selenite content by 10-20 mole%. Then the solution is placed in a microwave oven, for example, in an LG MS2336GIN microwave oven, however, the brand of the oven does not affect the result. To obtain selenium nanoparticles, only the power of the unit is important - the required oven power is set in the range of 400-1000 W. The time of exposure of the solution to radiation depends on the power - from 2 to 10 minutes, respectively. Mixing of the components is carried out at room temperature in the stated ranges. The sizes of selenium nanoparticles are determined by the method of small-angle scattering of X-rays. The course of the reaction is monitored spectrophotometrically by the appearance and subsequent decrease to a constant value in time of the absorption band characteristic of ascorbic acid in the wavelength range of 250-280 nm. The constant value of the absorption band corresponding to ascorbic acid taken in molar excess relative to selenite indicates the complete reduction of selenium ions to nanoparticles. In this case, during the process of selenium ion reduction and nanoparticle formation, the color of the solution changed from pale yellow to red, characteristic of compositions containing selenium nanoparticles. The molecular weight of chitosan can vary from 25 kDa to 400 kDa.

The use of microwave radiation to initiate and accelerate the process of synthesis of selenium nanoparticles facilitates its simplification. The synthesis process is also simplified by the use of chitosan with different molecular weights, which ensures its interchangeability. The production of selenium nanoparticles with an average size of 5 nm, which have an antihypoxic effect, facilitates an increase in their efficiency, biocompatibility and bioavailability.

Below are examples of the implementation of the proposed group of inventions.

Example 1.

A solution of sodium selenite at a concentration of 0.001 wt % (as a precursor of selenium nanoparticles) was added dropwise to a solution of 2 wt % chitosan (molecular weight 400 kDa) in 2 wt % acetic acid, and the system was stirred for 5 minutes. Ascorbic acid (0.0012 wt %) was introduced into the chitosan solution with continuous stirring and constant stirring was maintained for 1 hour. The amount of water in the solution was 95.9978 wt %. The resulting solution was exposed to microwave radiation at a power of LG MS2336GIN - 1000 W for 2 min. The synthesis time of selenium nanoparticles was monitored spectrophotometrically by the appearance and decrease to a constant value of the absorption band corresponding to ascorbic acid λ ~ 250-280 nm. The constant value of the absorption band corresponding to ascorbic acid taken in molar excess relative to selenite indicates the complete reduction of selenium ions to nanoparticles. This is also indicated by the cessation of the change in the color of the solution.

Example 2.

A solution of sodium selenite at a concentration of 0.005 wt % (as a precursor of selenium nanoparticles) was added dropwise to a solution of 0.5 wt % chitosan (molecular weight 100 kDa) in 0.5 wt % succinic acid, and the system was stirred for 5 minutes. Ascorbic acid (0.006 wt %) was introduced into the chitosan solution with continuous stirring and constant stirring was maintained for 2.5 hours. The amount of water in the solution was 98.989 wt %. The resulting solution was exposed to microwave radiation at a power of 400 W for 5 min. The synthesis time of selenium nanoparticles was monitored spectrophotometrically by the appearance and decrease to a constant value of the absorption band corresponding to ascorbic acid λ ~ 250-280 nm. The constant value of the absorption band corresponding to ascorbic acid taken in molar excess relative to selenite indicates the complete reduction of selenium ions to nanoparticles. This is also indicated by the cessation of the change in the color of the solution.

Example 3.

A solution of potassium selenite at a concentration of 1 wt % (as a precursor of selenium nanoparticles) was added dropwise to a solution of 5 wt % chitosan (molecular weight 25 kDa) in 6 wt % lactic acid, and the system was stirred for 5 minutes. Ascorbic acid (1.5 wt %) was introduced into the chitosan solution with continuous stirring and constant stirring was maintained for 2.5 hours. The amount of water in the solution was 86.5 wt %. The resulting solution was subjected to microwave radiation at a power of 1000 W for 10 min. The synthesis time of selenium nanoparticles was monitored spectrophotometrically by the appearance and decrease to a constant value of the absorption band corresponding to ascorbic acid λ ~ 250-280 nm. The constant value of the absorption band corresponding to ascorbic acid taken in molar excess relative to selenite indicates the complete reduction of selenium ions to nanoparticles. This is also indicated by the cessation of the change in the color of the solution.

Example 4.

A solution of 0.2 wt % sodium selenite (as a precursor of selenium nanoparticles) was added dropwise to a solution of 3 wt % chitosan (molecular weight 100 kDa) in 1 wt % acetic acid and 0.5% lactic acid, and the system was stirred for 5 minutes. Ascorbic acid (0.26 wt %) was introduced into the chitosan solution with continuous stirring, and constant stirring was maintained for 1.5 hours. The amount of water in the solution was 95.04 wt %. The resulting solution was exposed to microwave radiation at a power of 700 W for 6 min. The synthesis time of selenium nanoparticles was monitored spectrophotometrically by the appearance and decrease to a constant value of the absorption band corresponding to ascorbic acid λ ~ 250-280 nm. The constant value of the absorption band corresponding to ascorbic acid taken in molar excess relative to selenite indicates the complete reduction of selenium ions to nanoparticles. This is also indicated by the cessation of the change in the color of the solution.

Example 5.

A solution of sodium selenite at a concentration of 0.01 wt % (as a precursor of selenium nanoparticles) was added dropwise to a solution of 4 wt % chitosan (molecular weight 230 kDa) in 1 wt % acetic acid and 1 % succinic acid, and the system was stirred for 5 minutes. Ascorbic acid (0.015 wt %) was introduced into the chitosan solution with continuous stirring and constant stirring was maintained for 1 hour. The amount of water in the solution was 93.975 wt %. The resulting solution was exposed to microwave radiation at a power of 1000 W for 5 min. The synthesis time of selenium nanoparticles was monitored spectrophotometrically by the appearance and decrease to a constant value of the absorption band corresponding to ascorbic acid λ ~ 250-280 nm. The constant value of the absorption band corresponding to ascorbic acid taken in molar excess relative to selenite indicates the complete reduction of selenium ions to nanoparticles. This is also indicated by the cessation of the change in the color of the solution.

Example 6.

A solution of sodium selenite at a concentration of 0.1 wt % (as a precursor of selenium nanoparticles) was added dropwise to a solution of 3 wt % chitosan (molecular weight 100 kDa) in 1.2 wt % acetic acid, and the system was stirred for 5 minutes. Ascorbic acid (0.12 wt %) was introduced into the chitosan solution with continuous stirring and constant stirring was maintained for 2 hours. The amount of water in the solution was 95.58 wt %. The resulting solution was subjected to microwave radiation at a power of 400 W for 10 min. The synthesis time of selenium nanoparticles was monitored spectrophotometrically by the appearance and decrease to a constant value of the absorption band corresponding to ascorbic acid λ ~ 250-280 nm. The constant value of the absorption band corresponding to ascorbic acid taken in molar excess relative to selenite indicates the complete reduction of selenium ions to nanoparticles. This is also indicated by the cessation of the change in the color of the solution.

Acetic, lactic and succinic acids, as well as sodium selenite and potassium selenite are interchangeable in the corresponding mass percentages.

The use of chitosan with different molecular weights - from 25 kDa to 400 kDa is interchangeable according to the given formula.

Shifting the concentration of components away from the specified limits complicates the process of obtaining the product, since the solubility of chitosan worsens and the yield of the target product decreases. Reducing the concentration of chitosan below the specified limits leads to a decrease in the stability of the dispersion of selenium nanoparticles in the polymer. The limits of acid concentration are optimally selected for better dissolution of the polysaccharide.

The microwave radiation power limits: 400-1000 W were chosen based on the fact that at a power of less than 400 W, the duration of nanoparticle synthesis increases, which is not highly profitable; at a power of more than 1000 W, the solution begins to boil away due to excessively high radiation.

Qualitative evidence of the formation of selenium nanoparticles with a yield of 100% and complete depletion of the selenium precursor is the spectrophotometrically recorded constant value of the absorption band corresponding to ascorbic acid taken in molar excess relative to selenite dissolved in an aqueous acid solution of the stabilizer (Fig. 1).

According to this method of the invention, selenium nanoparticles with an average size of 5 nm are obtained in all examples. The sizes of the remaining nanoparticles are within the range of 1-22 nm. This is confirmed by the data of small-angle scattering of X-rays (Fig. 2).

The obtained composition of selenium nanoparticles stabilized by chitosan is characterized by high biocompatibility, which is evidenced by the adhesion and proliferation of fibroblast cells as precursors of connective tissue after 24 hours of their incubation on the surface of the films of the composition. On the surface of the compositions of selenium nanoparticles stabilized by chitosan, washed from excess acid to a neutral pH of the medium, a uniform distribution of fibroblasts, their growth and division were observed, which indicated a high degree of biocompatibility of this sample. At the same time, on the composition containing ionic selenium in the same concentration, the cells died after their cultivation and are not visible. (Fig. 3)

According to this method of the invention, in all examples, compositions are obtained that have high antihypoxic activity. The antihypoxic properties of the chitosan-selenium nanoparticles composition were proven under hypobaric hypoxia modeling conditions, assessed by the activity of free-radical processes in blood plasma. An experiment to study the antihypoxic effect of the chitosan-selenium nanoparticles composition was conducted in vivo on white nonlinear rats. The animals were divided into 4 groups of 5 individuals each. The first group included intact animals, the second group was a hypoxia control, the animals of the third group were administered a chitosan succinate solution, and the fourth group was administered selenium nanoparticles stabilized with chitosan. The drugs were administered to the animals orally once a day for four days in a volume of 1 ml per animal using a metal probe. One day after the end of drug administration, all animals except the first group were placed in a pressure chamber and exposed to hypobaric hypoxia in the following mode: conditional altitude of 8000 m above sea level for 30 min. The pressure in the pressure chamber was changed gradually, the altitude was reduced to 3000 m at a speed of no more than 1 km/min. Immediately after the end of hypobaric hypoxia, the animals were sedated with chloroform and blood was taken by decapitation. Blood parameters were measured.

The results of blood parameters in the study of the antihypoxic properties of chitosan compositions and chitosan containing selenium nanoparticles in vivo under conditions of hypobaric hypoxia modeling, assessed by the activity of free radical processes in the blood plasma of experimental animals are presented in Table 1.

Table 1

Blood indices of experimental animals when introducing samples before exposure to hypobaric hypoxia

Indicators Intact Hypoxia Chitosan-
succinic acid Chitosan nanoparticles Se DK,
rel. units opt. pl. Fraction Isopropanol 0.452±
0.036 0.330±
0.046 0.309±
0.026 0.421±
0.038 TC,
rel. units opt. pl. 0.312±
0,017 0.190±
0.020 * 0.161±
0.032 * 0.282±
0.024 OSH,
rel. units opt. pl. 0.076±
0.008 0.028±
0.006 * 0.022±
0.008 * 0.069±
0.007 * ^#& DK,
rel. units opt. pl. Heptane 0.532±
0.084 0.255±
0.620 * 0.355±
0,017 0.406±
0.036 TC,
rel. units opt. pl. 0.421±
0.102 0.097±
0.029 * 0.080±
0.019 * 0.461±
0.023 * OSH,
rel. units opt. pl. 0.262±
0.083 0.073±
0.017 * 0.011±
0.002 * 0.333±
0.098 * Erythrocytes,
10 ¹² /l 10.57±
0.70 7.87±
0.22* 7.79±
0.10 * 9.62±
0.26 * HGB,
g/l 163.40±
7.65 129.00±
1.64* 127.00±
1.14 * 128.80±
3.29* Platelets,
10 ⁹ /l 291.40±
44.12 389.60±
25.32 546.00±
31.85* 491.60±
48.31* Leukocytes,
10 ⁹ /l 7.01±
0.28 6.11±
0.60 9.95±
0.74 * ^# 8.20±
0.73 ^& Lymphocytes,
10 ⁹ /l 3.63±
0.30 2.82±
0.51 6.04±
1.15 * ^# 4.08±
0.66 ^& Monocytes,
10 ⁹ /l 1.20±
0.16 0.85±
0.15 1.38±
0.29 1.47±
0.40 Neutrophils,
10 ⁹ /l 2.18±
0.31 2.44±
0.44 2.53±
0.63 2.79±
0.60

Statistically significant differences between groups:

* p < 0.05 - in relation to the "intact" group; # p < 0.05 - in relation to the "hypoxia" group; & p < 0.05 - in relation to the "chitosan-succinic acid" group

In animals, under hypoxia conditions, which enhances the formation of active forms of oxygen, the level of intracellular enzymes of the antioxidant system increases, which is associated with the mechanisms of maintaining the body's resistance to oxidative stress. Active forms of oxygen, in addition to promoting apoptotic cell death and inducing inflammatory processes in the body, also promote cell survival in hypoxic conditions and induce antioxidant protection. Thus, under hypoxia, the indicators of DC, TC and OS - diene conjugates, triene conjugates and Schiff bases, respectively - decrease.

However, the content of peroxidation products in animals fed with the composition "chitosan-selenium nanoparticles" after exposure to hypoxia increased compared to animals in hypoxic conditions, and was close to the indicators of intact animals. That is, the body of animals in hypoxic conditions when using the composition experienced much less stress and was practically no different from the state of animals in normal conditions.

The number of erythrocytes and hemoglobin content significantly decreased during hypoxia. No significant differences were found in the number of leukocytes and lymphocytes between the groups receiving selenium preparations compared with intact animals and the hypoxia group.

Thus, it can be concluded that the synthesized chitosan-selenium nanoparticle composition has high antihypoxic properties and leads to a decrease in oxidative degradation of lipids, which occurs mainly under the influence of free radicals.

Claims (5)

1. A method for producing selenium nanoparticles, comprising mixing a solution containing a selenium source, which is sodium selenite or potassium selenite in an amount of 0.001-1 wt. %, a stabilizer, which is chitosan with a molecular weight of 25 to 400 kDa in an amount of 0.5-5 wt. %, a reducing agent, which is ascorbic acid in an amount of 0.0011-1.5 wt. %, and a solvent, which is a solution of acetic acid, or lactic acid, or succinic acid, or a mixture thereof in an amount of 0.1-6 wt. %, and water in an amount of up to 100% when calculating the total weight of the solution, wherein the resulting solution is subjected to microwave radiation at a device power of 400-1000 W for 2-10 min. 2. The method according to item 1, characterized in that ascorbic acid is introduced into the chitosan solution with continuous stirring and constant stirring is maintained for 1-2.5 hours. 3. The method according to item 1, characterized in that the time of synthesis of selenium nanoparticles is monitored spectrophotometrically by the appearance and decrease to a constant value of the absorption band corresponding to ascorbic acid λ ~ 250-280 nm. 4. The method according to claim 1, characterized in that the nanoparticles are formed with sizes in the range of 1-22 nm with an average nanoparticle size of 5 nm. 5. The method according to paragraph 1, characterized in that the obtained selenium nanoparticles are used as a biologically active additive with antihypoxic properties.