RU2602534C2 - Method of producing colloidal silver nanoparticles - Google Patents

Abstract

FIELD: nanotechnology.

SUBSTANCE: invention relates to methods of producing highly dispersed colloidal particles or nanoparticles of metallic silver, which can be used in biotechnology, medicine and veterinary science as part of preparations with antimicrobial action. Described is a method of producing colloidal silver nanoparticles, involving dissolution in water of a medical polymer and a silver salt, reduction of silver ions, wherein for reduction of silver ions obtained solution is subjected to electron-beam treatment by passing through it a beam of accelerated electrons at absorbed dose of 5-30 kGy.

EFFECT: technical result is high antimicrobial activity, low toxicity and high stability of aqueous solution of silver nanoparticles.

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Description

The invention relates to methods for producing highly dispersed colloidal particles or metallic silver nanoparticles, which can be used in biotechnology, medicine and veterinary medicine as part of preparations with antimicrobial activity.

A known method of producing silver nanoparticles in reverse micelles (patent RU No. 2147487, IPC B22F 9/24, publ. 04/20/2000). The method consists in forming a reverse-micellar solution (dispersion) and in a controlled reduction of silver ions in micelles. Water-soluble silver salts (nitrate, acetate) are used as ionic silver, various reducing agents are used as reducing agents - formaldehyde, hydrazine, borohydride, hydrogen, hypophosphite, hydroquinone, tartrate, citrate and others.

The disadvantages of this method are the low processability, the use of organic solvents, reducing agents and surfactants, which are subsequently difficult to get rid of, and whose impurities increase the toxicity of the resulting silver nanoparticles.

A known method of producing silver nanoparticles by reduction from a solution of its salts in a gel cation exchange matrix (patent RU No. 2385293, IPC C01G 5/00, B82B 3/00, publ. 03/27/2010). Obtained by this method, silver nanoparticles are immobilized in the matrix.

The disadvantage of this method is the low bioavailability of silver nanoparticles.

A known method of producing nanosized particles of silver from an array of metallic silver (RU No. 2403317, IPC C23C 16/44, B82B 3/00, publ. 10.11.2010).

In the vacuum chamber of the reactor for evaporation and pyrolysis containing one or more polymers, a reactor for controlled evaporation of a metal containing one or more metals and a substrate are placed. Carry out simultaneous heating of the reactor for evaporation and pyrolysis for the decomposition of the polymer to a precursor gas and the reactor for controlled evaporation of the metal. A supersonic or subsonic jet is formed as the precursor gas passes through the nozzle of the reactor for evaporation and pyrolysis. Then, the resulting stream flows onto a heated wire mesh mounted on the nozzle of the reactor for evaporation and pyrolysis, and the resulting stream is fed onto the substrate. The substrate is polymerized to form a polymer material. At the same time, a carrier gas is fed into the heated reactor for the controlled evaporation of the metal. Then, a jet of carrier gas, together with vapors of one or more metals and / or their oxides, is supplied to the substrate and the vapors of one or more metals and / or their oxides are mixed with the resulting polymer on a rotating substrate to form a metal-polymer material.

The disadvantages of this method are the high energy intensity, the use of sophisticated equipment and special conditions (vacuum, inert gas). Silver nanoparticles obtained by this method are mainly used for technical purposes. The low stability of aqueous solutions of silver nanoparticles obtained by this method and their unsatisfactory pharmacological parameters (in terms of toxicology, specific antibacterial activity) make it difficult to use such nanoparticles in biotechnology, medicine, and veterinary medicine.

A known method of producing silver nanoparticles in an aqueous medium by electrochemical anodic dissolution of metallic silver (patent RU No. 2390344, IPC A61K 33/38, C01G 5/00, B82B 3/00, publ. 05.27.2010).

The method includes dissolving stabilizers in distilled water, placing an anode made in the form of a silver plate and a cathode made in the form of a stainless steel plate in the resulting solution, electrochemical dissolution of the anode when a constant electric current is passed through the solution.

The disadvantage of this method is that a significant part of silver is presented in oxidized form, which can adversely affect the pharmacological parameters of the resulting nanosilver. Note that the toxicological and bactericidal characteristics of aqueous solutions of silver nanoparticles obtained by this method are not given.

Known methods for producing silver nanoparticles in which silver ions are reduced to metal using solvated electrons generated in solution by ionizing radiation. In patent US 7348365, publ. 2008, a method for producing a colloidal solution consists in reducing silver from its salts, including silver nitrate, in aqueous solutions in the presence of the following polymers as a stabilizer - polyvinylpyrrolidone, polyacrylonitrile, polymethylmethacrylate, polymethylacrylate, followed by bubbling through a solution of nitrogen or argon. To restore silver ions into metal nanoparticles, gamma radiation was used.

There is another method for producing a colloidal solution of silver nanoparticles (patent RU No. 2474471, IPC B01J 13/00, C09D 1/00, B82B 3/00, publ. 02/10/2013), comprising dissolving silver nitrate AgNO ₃ and a stabilizing polymer in water, bubbling an inert gas through the resulting solution and subsequent gamma irradiation of the solution with the restoration of silver ions in silver nanoparticles. Carboxymethylchitin is used as a stabilizing polymer at a concentration of 0.1-3 wt.% In an aqueous solution. %, the dissolution of AgNO _{3 is} carried out to a concentration of 3.5-10.1 mm in a solution of carboxymethylchitin, and the specified gamma irradiation with a dose of 2-12 kGy (kilogray). Carboxymethylchitin with a weight average molecular weight of 50,000-450000 daltons is used. To suppress radical side reactions, alcohols are added to the solution: isopropyl alcohol, or ethanol, or ethylene glycol.

The disadvantages of these methods are the difficulty of scaling, low manufacturability, the presence of adverse radical reactions, the products of which can increase the toxicity of the drug. Note that the toxicological characteristics of aqueous solutions of silver nanoparticles obtained by this method are not given.

Known commercial bactericidal water-soluble drug collargolum (Mashkovsky M.D. - Medicines / Moscow, New Wave, 2008). Collargol is a colloidal composition containing highly dispersed metallic silver stabilized by protective high molecular weight polymers of protein nature - casein and / or albumin hydrolysates.

The method of producing collargol is the closest technical solution to the claimed one and includes the following basic operations (Blagitko E.M., Burmistrov V.A., Kolesnikov A.P., Mikhailov Yu.I., Rodionov P.P. - Silver in medicine / Novosibirsk, Science Center, 2004).

1. Preparation of a reducing mixture of protein hydrolysates by alkaline hydrolysis of casein and albumin (treatment with sodium hydroxide solution when heated).

2. Preparation of silver oxide by its quantitative precipitation from an aqueous solution of silver nitrate with a solution of sodium hydroxide.

3. Reduction of silver oxide by treatment with a reducing mixture in an aqueous solution by heating and stirring.

4. Reprecipitation of the obtained colloidal silver product with sulfuric or acetic acid with washing the precipitate with distilled water in order to remove foreign substances. Then the precipitate is transferred into the solution by adding sodium hydroxide solution, the solution is concentrated by evaporation, the precipitate is again precipitated and the precipitate is separated, which is dried.

In appearance, the pharmacopeia collargol is a greenish or bluish-black plate with a metallic sheen. Contains 70% silver. It dissolves in water with the formation of a colloidal solution.

The disadvantages of the known method for producing colloidal metallic silver are low manufacturability and the difficulty of obtaining the target product with reproducible properties due to the inconsistency of the composition and heterogeneity of the original protein raw materials and products of its hydrolysis. In addition, aqueous solutions of collargol are unstable, which does not allow the release of collargol in the form of ready-to-use dosage forms with long shelf life. Collargol is available in the form of a substance (powder), from which extemporaneous forms are prepared in the formulations of pharmacies or clinics in the form of a 1-2% aqueous solution, the shelf life of which is no more than 30 days. This complicates the widespread use of collargol in medical and veterinary practice. In general, we note that collargol no longer fully meets the increased requirements for modern medical and veterinary drugs both in terms of release and pharmacological parameters (specific activity, toxicity).

The objective of the invention is to develop a method for producing a water-soluble composition of colloidal particles (nanoparticles) of silver with improved indicators of antimicrobial activity, toxicology and stability of an aqueous solution.

The problem is solved using the method of producing silver nanoparticles, including dissolving in water a polymer for medical purposes and silver salts, the restoration of silver ions.

To restore silver ions, the resulting solution is subjected to electron beam processing by passing through it a beam of accelerated electrons in an absorbed dose of 5-30 kilograms.

Preferably, polymers with the detoxifying effect of polyvinylpyrrolidone or dextran are used as a medical polymer.

Preferably, silver nitrate, silver acetate, silver carbonate, silver sulfate are used as the water-soluble silver salt.

Preferably, a polymer solution with a concentration of 5-40 wt. %

Preferably, a silver salt solution is introduced into the polymer solution based on a final silver concentration of not more than 3% and with a silver / polymer ratio of 1 / 10-1 / 20.

Preferably, the resulting polymer solution and silver salts are mixed and poured into a flat container until a layer thickness of not more than 5 cm is formed.

In the inventive method, nanosilver is obtained by electron beam treatment of an aqueous solution containing a water-soluble stabilizing polymer and a water-soluble silver salt. Electron beam processing consists in passing through a solution a beam of accelerated electrons obtained in a facility (linear accelerator) of the ILU-10 type. To ensure the recovery of silver ions, a dose of 5-30 kGy (kilogray) or 0.5-3 mRad (megarad) is used. At the time of processing by accelerated electrons, the process of formation and formation of stable nanosized particles of silver is in progress. In the proposed solution, the authors saw a difference in the properties of nanosilver particles obtained by the restoration of silver ions using gamma radiation and accelerated electron flux. The advantage of the stream of accelerated electrons is that the stream of accelerated electrons has a softer biogenic effect compared with hard gamma radiation, does not cause side processes. The flow of accelerated electrons can be more accurately metered depending on the concentration of silver and polymer salts. Currently, linear accelerators of the ILU type are approved for use in the medical industry, they are used for sterilization of medical preparations and products, for electron beam synthesis of pharmaceuticals.

For the preparation of aqueous solutions of silver salts, reagents of the brand OSCH, ChDA, deionized or distilled water are used.

In distilled water, a solution of a polymer stabilizer with a concentration of 5-40 wt. % As a stabilizing polymer, medical polymers with a detoxifying effect are used: medical low molecular weight polyvinylpyrrolidone (FSP 42-0345-4368-03) or medical dextran.

In addition to these polymers, other polymers can be used with certificates for their use in the manufacture of medical, pharmaceutical and cosmetic products.

In a separate container, a solution of silver nitrate with a concentration of 0.1-15 wt. % The silver salt solution is introduced into the polymer solution based on the final silver concentration of not more than 2.5-3 wt. % and the ratio of silver / polymer 1 / 10-1 / 20 (per 1 weight part of silver 10-20 parts of polymer). The resulting solution is mixed and poured into a flat container made of glass or a polymeric material so that the thickness of the layer of the solution subjected to electron beam processing does not exceed 5 cm. This thickness allows high-quality processing of the entire volume of the solution.

Next, the solution is exposed to a beam of accelerated electrons in an installation of the ILU-10 type so that the absorbed dose is 5-30 kGy (kilograms). The dose value correlates with the silver concentration used. For low silver concentrations (up to 0.3%), 5 kGy is sufficient; for high concentrations (3%), 30 kGy. During processing, the solution becomes dark brown due to the formation of colloidal silver nanoparticles.

The completeness of the transition of ionic silver to colloidal metallic silver is checked by a quality drop sample with a saturated NaCl solution (USSR State Pharmacopoeia, X edition, p. 746). When chloride ions are added, ionic silver forms a characteristic white precipitate of silver chloride, insoluble in nitric acid, but soluble in an excess of ammonia solution. After processing, the solution is poured into a measuring container, adjusted with distilled water or a polymer solution to the desired silver concentration. The finished solution is poured into a container of opaque material. Store the solution in a hermetically sealed form in a dark place.

The technical result of the claimed invention is to increase specific antimicrobial activity, reduce toxicity and increase the stability of an aqueous solution of silver nanoparticles.

The invention is further illustrated by examples.

Example 1. Description of the method for producing colloidal silver nanoparticles.

In a vessel equipped with a stirrer, a solution of polyvinylpyrrolidone with a concentration of 20 wt. %, vigorously stirred until complete dissolution.

In another vessel, a silver nitrate concentrate with a concentration of 8 wt. %, stirred at room temperature until completely dissolved. The resulting silver salt solution is introduced into a vessel with a polymer solution per 15 parts by weight of silver; 15 parts of the polymer are intensively mixed and poured into a flat glass container with a solution layer thickness of 5 cm. The solution in the vessel is exposed to an accelerated electron beam with an absorbed dose of 15 kGy .

After processing, the solution is poured into a new container and diluted with distilled water to a silver concentration of 2.0 wt%.

Example 2. The production method according to claim 1, only medical dextran is used instead of polyvinylpyrrolidone.

Example 3. The production method according to p. 1, only silver acetate is used instead of silver nitrate.

Example 4. The production method according to claim 1, only instead of 15 kGy, the solution is exposed to a beam of accelerated electrons in an absorbed dose of 5 kGy. Doses of less than 5 kGy may not ensure complete recovery and conversion of ionic silver to colloidal silver.

Example 5. The production method according to claim 1, only instead of 15 kGy, the solution is exposed to a beam of accelerated electrons in an absorbed dose of 30 kGy. Doses greater than 30 kGy can lead to radiation degradation of the stabilizer polymer.

Example 6. The production method according to p. 1, only prepare a solution of polyvinylpyrrolidone at a concentration of 40 wt. %, and add a solution of silver salt based on the calculation of 1 weight part of silver 20 weight parts of a polymer (silver / polymer ratio 1/20). Higher polymer concentrations are difficult to dissolve and can lead to the formation of large colloidal particles and aggregates.

Example 7. The production method according to p. 1, only prepare a solution of polyvinylpyrrolidone at a concentration of 5 wt. %, and add a solution of silver salt based on the calculation of 1 weight part of silver 10 weight parts of a polymer (silver / polymer ratio 1/10). The use of lower concentrations is unproductive for economic reasons.

Example 8. Comparison of electron microscopic images of drugs.

In fig. 1 presents typical electron microscopic images of collargol and a drug obtained by the present method. It can be seen that, compared with collargol (prototype), the drug obtained by the present method is finer and more uniform.

The smaller particle size positively affects the sedimentation and aggregative stability and stability of the aqueous solution of the drug and its antimicrobial activity.

Example 9. Comparison of antimicrobial activity of drugs.

A comparative study was conducted of the antibacterial (bactericidal and bacteriostatic) activity of collargol (prototype) and the drug obtained by the present method. Aqueous solutions of the preparations before the study were standardized by the silver content, which was 1 wt. % (10 mg / ml). The following bacterial cultures were used as test strains:

- Escherichia coli - gram-negative non-spore-forming bacterium;

- Staphylococcus aureus - gram-positive non-spore-bearing bacterium;

- Candida albicans - yeast, eukaryotic type of cell structure. Research Methodology

Materials and reagents:

- Saline solution of 0.9%;

- L-broth - standardized nutrient medium (medium composition: tryptone - 10 g / l, yeast extract - 5 g / l, sodium chloride - 10 g / l, 0.6% glucose, pH 7.2);

- Fish nutrient agar, RPA.

The antimicrobial activity of the preparations was determined by counting the number of viable bacteria in colony forming units (CFU / ml) during cultivation of the test strain in a liquid nutrient medium in the presence of dilutions of the studied drugs. As a control, there was a nutrient medium with the addition of a test strain, but without the addition of the drug.

To determine the antimicrobial activity of the preparations, suspensions of bacterial test cultures with a concentration of 10 ² -10 ³ CFU / ml were used, which were obtained by diluting a freshly prepared daily culture in L-broth. In tubes with 10 ml of a suspension of bacteria, the calculated amount of dilution of the preparation was added, and incubated in a thermostat on a shaker (180 rpm) at a temperature of 37 ° C. After 24 hours, suspension samples were taken which were triturated to determine viable bacteria. To calculate the concentration of viable bacteria (colony forming units, CFU), ten-fold dilutions of the samples were made in saline and plated on RPA plates, which were incubated in an incubator at 37 ° C, and after 20 hours the results were taken into account - the number of grown colonies was calculated and the concentration of viable bacteria was calculated in CFU / ml suspension.

The results are presented in table 1. Samples of preparations at a dilution of 10 ^-2 had a bactericidal effect (0, lack of bacterial growth of the test strain), therefore, are not shown in the table.

As can be seen from the data presented in the table, the preparation according to the claimed method has a higher antimicrobial activity.

Example 10. Comparative assessment of acute toxicity with intraperitoneal administration of drugs.

A comparative assessment of acute toxicity was carried out with intraperitoneal administration of drugs. The study was conducted on adult white mice weighing 20-22 g. Aqueous 25% solutions of collargol and the drug according to the present method in increasing doses were administered intraperitoneally to mice until death. The calculation of lethal doses was done in terms of the dry matter of the preparations in mg per kg of animal weight. LD ₅₀ caused the death of 50% of the mice, LD ₁₀₀ caused 100% death of the mice. The results are shown in table 2.

As can be seen from the data presented in the table, the preparation according to the claimed method is less toxic.

Example 11. A comparative assessment of the stability during storage of aqueous solutions of drugs.

A comparative assessment of the stability during storage of aqueous solutions of the preparations was carried out. Storage stability was assessed visually by the presence of sediment and the color change of the solution, as well as by the preservation of antibacterial activity. The method for determining antibacterial activity is given in paragraph 3. Solutions of collargol were prepared from a substance (dry powder) at a concentration of 1% (0.7% in silver), an aqueous solution of the drug according to the present method in the same concentration was prepared from a concentrated solution stored in a refrigerator , by dilution with water. The prepared solutions were stored at room temperature in a closed form, in a dark place for 3 months. A visual assessment showed that after storage in the collargol solution a loose precipitate formed, the solution became more cloudy, changed color from dark brown to dark gray-brown. In the solution of the drug prepared by the present method, the precipitate was not observed, the color of the solution remained practically unchanged. For microbiological testing, freshly prepared solutions and solutions after storage were used.

As can be seen from the data presented in the table, the antimicrobial activity in an aqueous solution of collargol significantly decreased after storage, the drug obtained by the present method, has not changed.

The advantage of the proposed method for producing a water-soluble composition of colloidal silver (nanosilver) is to increase antimicrobial activity, reduce toxicity and increase the stability of an aqueous solution.

Claims (6)

1. A method of producing colloidal silver nanoparticles, comprising dissolving a medical polymer and a silver salt in water, reducing silver ions, characterized in that the resulting solution is subjected to electron beam processing to recover silver ions by passing through it a beam of accelerated electrons in an absorbed dose of 5- 30 kilogrey. 2. The method according to p. 1, characterized in that the polymers with the detoxifying effect of polyvinylpyrrolidone or dextran are used as a medical polymer. 3. The method according to p. 1, characterized in that as a water-soluble silver salt using silver nitrate, silver acetate, silver carbonate, silver sulfate. 4. The method according to p. 1, characterized in that they prepare a polymer solution with a concentration of 5-40 wt. % 5. The method according to p. 4, characterized in that a solution of silver salt is introduced into the polymer solution based on the final silver concentration of not more than 3 wt. % and with a weight ratio of silver / polymer of 1 / 10-1 / 20. 6. The method according to any one of paragraphs. 1-5, characterized in that the resulting polymer solution and silver salts are mixed and poured into a flat container until the thickness of the layer subjected to irradiation, not more than 5 cm

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