RU2481848C1 - Carbon sorbent with antibacterial properties and method for preparing it - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions refers to a method for preparing a carbon sorbent with the antibacterial properties, and to the carbon sorbent with the antibacterial properties prepared by this method. The declared method involves impregnation of the carbon hemosorbent granules in an initiator solution in N-vinylpyrrolidone at pH 7.0-7.5 and a residual pressure of 15-20 mm Hg. The hemosorbent : initiator solution in N-vinylpyrrolidone ratio is 1:1.4-2.0. Then the temperature is raised to 65-75°C, kept at that temperature for 0.5-8 hours in an inert medium and washed in water from the residual monomer at room temperature.

EFFECT: carbon sorbent with the antibacterial properties prepared by the specified method represents the round granules, contains polyvinylpyrrolidone in an amount of 4,5-5,5% and is characterised by a specific surface adsorption of less than 50 m²/g and total pore volume of less than 0,30 cm³/g.

2 cl, 2 tbl, 1 dwg, 7 ex

Description

The invention relates to a technology for producing carbon sorbents with antibacterial properties based on porous carbon adsorbents and is intended for use in medicine and veterinary medicine.

The development of effective materials with antibacterial properties is still an urgent task. One of the most effective methods in efferent therapy for the treatment of infectious diseases is application sorption (vulnerosorption) - a method of removing toxic components through the wound surface or focus of inflammation. When a sorbent is applied, the wound contents or the purulent cavity are cleaned and the transport of certain substances from the blood is accelerated with their subsequent sorption.

It is known that carbon materials exhibit high sorption ability against bacteria and bacterial toxins.

A manifestation of the sorption ability of activated carbon with respect to staphylococcal toxin was established in the works of NN Kaplin, V.F.Serkov, V.N. Alekseev and others (Kaplina NN [et al.] Method for increasing the sorption capacity of activated carbon coal in relation to staphylococcal toxin // Laboratory work. 1979. No. 9. P.546). It was also found that the nature and magnitude of adsorption of bacterial cells depends on the pore volume of the carbon sorbent (G. Kovalenko [et al.] // Carbon materials as adsorbents for biologically active substances and bacterial cells. 1999. V. 61. No. 6. S.787-795).

The carbon materials SUMS-1 (based on the mineral carrier Al ₂ O ₃ ) and SKN-1K, SKN-4M (based on synthetic polymers) capable of sorbing Staphylococcus aureus cells (Wood-46 strain), pneumococcus, streptococcus from the lymph are known. (Samsonov K.V. Comparative efficiency of sorption of bacteria and bacterial toxins by carbon and carbon-mineral sorbents // Bulletin of physiology and pathology of respiration. 2008. Issue 29. P.48-50). According to the results of studies, the carbon-mineral sorbent SUMS-1 deposited on its surface the largest number of studied pneumotropic microorganisms, which is explained by the presence on its surface of various chemical nature centers: polar and non-polar. Staphylococcus precipitated to a greater extent with this sorbent, pneumococcus and streptococcus somewhat less.

To increase the antibacterial properties of the carbon hemosorbent SUMS-1, the antiprotozoal and antimicrobial preparation metronidazole was adsorbed on its surface (active ingredient: 1- (b-hydroxyethyl) -2-methyl-5-nitroimidazole) (RF patents Nos. 2121842, 2127595, 2144797). This drug is active against Trichomonas vaginalis, Gardnerella vaginalis, Giardia intestinalis, Entamoeba histolytica, obligate anaerobic bacteria: Bacteroides spp. (including Bacteroides fragilis, Bacteroides distasonis, Bacteroides ovatus, Bacteroides thetaiotaomicron, Bacteroides vulgatus), Fusobacterium spp., Veillonela spp .; some gram-positive bacteria: Eubacterium spp., Clostridium spp., Peptococcus spp., Peptostreptococcus spp., as well as Helicobacter pylori.

The effect of this drug has not been investigated in relation to pathogenic bacteria - pathogens of sepsis. In addition, there are contraindications for the use of metronidazole: hypersensitivity, impaired hematopoiesis, active diseases of the central nervous system, as well as a significant number of side effects.

Thus, to increase the effectiveness of the antibacterial properties of carbon sorbents, it is necessary to use a modifier - a drug that is active against broad-spectrum pathogenic microflora, but also does not have a negative effect on the body.

It is known that polymers based on N-vinylpyrrolidone (VP) are widely used as hydrophilic non-toxic materials in medicine and biology. They are approved for use in contact with biological media of a living organism and can perform a variety of functions in medical supplies (Chernikova E.V., Terpugova P.S., Filippov A.N. Controlled radical polymerization of N-vinylpyrrolidone and N-vinylsuccinimide in conditions of reversible transmission of the chain according to the attachment-fragmentation mechanism. // Housing and communal services. 2009. T.82. No. 10. S.1730-1737). VP-based polymers are used to modify anionic surfactants with high bactericidal activity (Afinogenov G.E., Panarin E.F. Antimicrobial polymers. St. Petersburg: Hippocrates, 1993, 263 pp.). A number of VP copolymers with ionogenic comonomers have their own antimicrobial activity, immunostimulating and immunomodulating effects. To remove toxic substances from the body, poly-N-vinylpyrrolidone (PVP) with a molecular weight of 10000-15000 is usually used.

Low molecular weight PVP is widely used as an auxiliary substance in the creation of various dosage forms with antibacterial effects (RF patents No. 2281773, 2279278); when creating eye films in ophthalmology (patent No. 2286170); as a solubilizer in the creation of a disinfectant (patent No. 2308292); as a biologically active substance and builder in wound dressings (patent No. 2331444); as a conjugate in creating an antibacterial drug (patent No. 2371447); as a stabilizer in an antiviral drug (patent No. 2438697); as an immunomodulator in preparations for the prevention and treatment of respiratory and gastrointestinal infectious diseases of the bacterial and viral etiology of farm animals (patent No. 2286171), etc.

It is known the use of PVP in the composition of ointments for the treatment of infected wounds with a content of up to 5.5% (patents No. 2146127, 2233652). According to the research of V.N. Maltseva and others (V.N. Maltseva, V.A. Strelnikov, Ya. Ya. Fedorovsky. The effect of polyvinylpyrrolidone on microbial cells // Journal of Microbiology. 1987. N 4. P.9-11) PVP begins to exhibit antibacterial properties at a concentration in the solution of the order of 1.0%.

The application of PVP to the surface of the carbon sorbent makes it possible to increase its detoxification properties due to the migration of PVP macromolecules into the biological fluid. In this case, as an additional positive effect, an increase in the wettability of the carbon material is noted due to lyophilization of its surface and a decrease in surface energy.

Closest to the proposed sorbent is the carbon mesoporous hemosorbent VNIITU-1, which consists of granules with a size of 0.5-1.0 mm, is characterized by high chemical purity (carbon content of at least 99.5%) and a specific adsorption surface of 300-400 m ² / g (Technical specifications TU 9398-002-71069834-2004 Carbon hemosorbent in physiological saline, VNIITU-1; advertising brochure VNIITU-1 of the Institute for Hydrocarbon Processing SB RAS, 2011).

Closest to the proposed method for producing a carbon sorbent is a method of modifying a carbon sorbent by impregnating granules of carbon hemosorbent with an aqueous solution of aminocaproic acid with a concentration of 5-20% at a ratio of hemosorbent: solution of modifying acid 1: 10-1: 20 for 2-4 hours at a temperature 25-90 ° С, separation of the hemosorbent from the impregnating solution, drying of the impregnated hemosorbent at 105 ° С to constant weight, polycondensation of the aminoc applied to the surface of the hemosorbent Ronova acid in an inert environment at a temperature of 120-350 ° C for 0.25-6 hours with agitation, boiling in distilled water for 1-3 hours and drying the resulting product in air at room temperature (RF Patent №2440844).

The aim of the invention is the development of a carbon sorbent with pronounced antibacterial properties.

The proposed method for producing a carbon sorbent with antibacterial properties includes impregnating granules of carbon hemosorbent with a solution of a modifier and drying the product, and characterized in that the granules are impregnated with a 0.2-1.0% initiator solution in N-vinylpyrrolidone at pH 7.0-7.5, residual pressure of 15-20 mm Hg and the ratio of hemosorbent: initiator solution in N-vinylpyrrolidone 1: 1.4-2.0 for 15-30 minutes, followed by a rise in temperature to 65-75 ° C, holding at this temperature for 0.5-8 hours in an inert atmosphere and washing in water from the residual monomer at room temperature for 0.5-2 hours.

The proposed carbon sorbent with antibacterial properties is round-shaped granules, contains 4.5-5.5% polyvinylpyrrolidone, characterized by a specific adsorption surface of less than 50 m ² / g and a total pore volume of less than 0.30 cm ³ / g.

Distinctive features of this invention are:

- the use of carbon hemosorbent as a starting material with a developed specific adsorption surface of 300-400 m ² / g and a total pore volume of at least 0.4 cm ³ / g;

- surface modification of carbon hemosorbent with a solution of initiator in N-vinylpyrrolidone with subsequent polymerization of the deposited substance.

The modification process is carried out in several stages:

- impregnation of granules of carbon hemosorbent with a solution of initiator in N-vinylpyrrolidone with a concentration of 0.2-1.0% at a pH of 7.0-7.5, a residual pressure of 15-20 mm Hg and the ratio of hemosorbent: initiator solution in N-vinylpyrrolidone 1: 1.4-2.0 for 15-30 minutes;

- polymerization of the applied N-vinylpyrrolidone, for which the temperature is raised to 65-75 ° C and the granules are kept at this temperature for 0.5-8 hours in an inert atmosphere;

- washing the granules from the residual monomer in water at room temperature for 0.5-2 hours;

- drying the washed product at room temperature for 18-24 hours to obtain a carbon sorbent with antibacterial properties.

Modification of the carbon hemosorbent according to the proposed method with a modifier — monomer N-vinylpyrrolidone — with its subsequent polymerization changes both the chemical nature of the surface of the carbon matrix and its porous structure. The results illustrating changes in the parameters of the porous structure and chemical composition of carbon sorbent samples obtained by the prototype and when modified with N-vinylpyrrolidone followed by polymerization, in accordance with the examples of the present invention, are shown in table 1.

The physicochemical characteristics of the samples of the initial and modified carbon sorbent were determined by standard methods used in the study of porous materials: the main texture characteristics — specific adsorption surface, total pore volume, and pore size distribution were determined by nitrogen adsorption-desorption isotherms obtained on a Gemini device 2380 "(USA). The calculation of the adsorption specific surface area was carried out according to the BET equation. The relief and surface morphology of the studied carbon sorbent samples were studied by scanning electron microscopy using a JSM - 6460 LV electron microscope (JEOL, Japan). X-ray microanalysis of the surface of the studied samples was carried out on an EDAX energy dispersive spectrometer (EDAX, Japan) equipped with a Si (Li) detector with an energy resolution of 130 eV. IR spectra were recorded on a NICOLET 5700 spectrometer from Thermo Fisher Scientific with a resolution of 4 cm ^-1 and a spectral accumulation number of 32. The spectra are presented after processing in the ORIGIN software package (baseline correction and smoothing). Measurements by X-ray photoelectron spectroscopy were carried out on an ES-300 Kratos Analytical instrument using a Mg anode (hv = 1253.6 eV). The amount of PVP polymer deposited on the hemosorbent surface was determined by differential thermal analysis by mass loss, which was recorded by thermograms in the temperature range 330-340 ° C.

Figure 1 presents the data of thermogravimetric (TGA) and differential thermal (DTA) analyzes of the samples of the prototype (a) and carbon sorbents according to example 3 (b) and example 7 (c).

The studies were carried out on a DTG-60H device from SHIMADZU (Japan), which simultaneously records the thermal curves: temperature - T and DTA, TG. The accuracy of determining the temperature was - 1 ° C, and the change in weight was 0.1%. The shooting was carried out in an atmosphere of air, in the temperature range from room temperature to 1000 ° C, the sample was 10 mg, the temperature rise rate was 10 ° / min.

The presence of a monomer (N-vinylpyrrolidone) was determined by analyzing the composition of the wash water of the modified sorbents by C ¹³ NMR on an Advance-400 spectrometer. In the case of the presence of VP in the composition of the wash water, peaks characteristic of the VP were recorded on the NMR spectra.

To study the antibacterial properties of modified samples of carbon hemosorbent in the Central Research Laboratory of the Omsk State Medical Academy conducted biomedical research (bench tests).

We used gram-positive test microorganisms (Staphylococcus aureus, Staphylococcus aureus) and gram-negative test microorganisms (Pseudomonas aeruginosa, Pseudomonas aeruginosa; Klebsiella pneumonia, Klebsiella pneumoniae, Escherichia coli, Escherichia coli). In addition, we studied a mixture of test cultures, which was prepared by mixing equal volumes of prepared working concentrations of microbial cells. The initial dilution with a concentration of 0.5 units according to Mac Farland using a densitometer, mixing cultures, suspensions of the sorbent with the culture was carried out on a Vortex shaker. Working cultivation of crops for the experiment - 1 × 10 ³ CFU in 1 ml. Cell sorption is carried out from physiological saline with a concentration of microbial cells of 1 × 10 ³ CFU / ml.

To assess the antibacterial effect of carbon sorbents, the direct inoculation method on nutrient media was used. As a nutrient medium, meat-peptone agar was selected.

Microorganisms were quantified by counting bacterial colonies on two Petri dishes with a diameter of 90 mm, the average value was found, and multiplying by the dilution index, the number of bacteria in 1 g per 1 ml of the sample was calculated. The control was the crops of working dilutions of the tested cultures.

Sterilization of sorbents before conducting biomedical biological tests was carried out with saturated steam at an overpressure of 0.11 ± 0.02 MPa and a temperature of (12 ± 1) ° C in an autoclave.

The following examples are provided to illustrate the invention.

Example 1 (prototype)

To the hemosorbent (in a volume of 0.5 ml) add 1 ml of microbial suspension (test microorganisms) and incubated for 24 hours with periodic manual shaking for the first 30 minutes of contact (to remove air bubbles). Three parallel tests are carried out for each type of bacterial cell and for a mixture of microorganisms.

The supernatant in an amount of 100 μl is seeded on agar plates with a nutrient medium after 24 hours of contact with the sorbent. Inoculated Petri dishes are incubated for 18-20 hours at 37 ° C in a thermostat. Count the grown colonies by stencil visually in 5 fields of 1 cm ² and count on the area of the cup. The result is evaluated by the number of colony forming units on the surface of the agar in comparison with the control.

Carbon hemosorbent showed antibacterial activity against test microorganisms Staphylococcus aureus: the number of bacterial cells after contact with hemosorbent decreased from 1x10 ³ to 30 CFU / ml (from 300 to 11 colonies per cup). The concentration of gram-negative bacteria after contact with carbon hemosorbent is comparable to the concentration in control samples.

Example 2

A portion of hemosorbent (1.0 g) is poured into a three-necked flask, hermetically sealed and connected to a vacuum pump with a vacuum of at least 15 mm Hg. and incubated at room temperature for 1 hour. Next, a pre-prepared 0.5% initiator solution (dinitrile azobisisobutyric acid - DYNIZ) in VP is impregnated with a vacuum sample for 15 minutes at a ratio of hemosorbent: initiator solution in N-vinylpyrrolidone 1: 1.4. Then the vacuum pump is turned off, an inert gas (argon) is supplied to the flask, the temperature is raised to 75 ° C and held for 0.3 hours. Upon completion of the modification, the sample of the sorbent is removed from the flask and washing is carried out with distilled water at room temperature for 0.5 hours, after which the sample is dried at room temperature for 24 hours.

Example 3

A sample of hemosorbent is impregnated with a 1% initiator solution in VP at a hemosorbent: solution ratio of 1: 1.5 and polymerization is carried out at a temperature of 68 ° C for 2 hours, followed by washing and drying.

The bacterial cells are adsorbed according to Example 1.

It was found that the carbon sorbent has antibacterial activity against the test microorganisms under study - after the interaction of the modified sorbent with pathogenic microflora, microorganisms did not grow on Petri dishes.

Example 4

A sample of hemosorbent is impregnated with a 0.1% initiator solution in VP at a hemosorbent: solution ratio of 1: 1.2 and polymerization is carried out at a temperature of 85 ° C for 4 hours, followed by washing and drying.

Example 5

A sample of hemosorbent is impregnated with a 1.5% initiator solution in VP at a hemosorbent: solution ratio of 1: 1.6 and polymerization is carried out at a temperature of 50 ° C for 8 hours, followed by washing and drying.

Example 6

A sample of hemosorbent is impregnated with a 0.7% initiator solution in VP at a hemosorbent: solution ratio of 1: 1.4 and polymerization is carried out at a temperature of 68 ° C for 2 hours, followed by washing and drying.

Example 7

A sample of hemosorbent is impregnated with a 0.8% initiator solution in VP at a hemosorbent: solution ratio of 1: 2 and polymerization is carried out at a temperature of 70 ° C for 6 hours, followed by washing and drying.

An analysis of the physicochemical characteristics of the carbon sorbent samples made it possible to establish the effect of the modification on the parameters of the porous texture and on the chemical composition (table 1). In the process of modification, micropores are completely closed; the volume of mesopores decreases by a factor of ~ 2. Screening the carbon surface with a PVP polymer film reduces its value by a factor of ~ 2–3 (from 394 to 32 m ² / g), and the total pore volume decreases from 0.630 cm ³ / g to 0.208 cm ³ / t. Moreover, the larger the amount of the introduced modifier in the porous structure, the lower the specific surface area and the total pore volume. The nitrogen content in the sorbent increases from 0 to 3.52% for modified samples.

As follows from the examples and table 1, a decrease in the amount of modifying solution leads to a decrease in the content of PVP sorbed on the surface of the hemosorbent and, accordingly, to smaller changes in texture: pore volume and specific surface. A decrease in the initiator concentration in the modifying formulation causes incomplete polymerization of the supported monomer and the presence of VP on the surface of the modified hemosorbent even with a polymerization time of 4 hours and subsequent washing. An increase in the initiator content to 1.5% also reduces the amount of polymer on the surface of the hemosorbent. Lowering the polymerization temperature to 50 ° C practically does not lead to the polymerization of VP, the texture parameters are practically not reduced, and the amount of PVP at a polymerization time of 8 hours is 1.0%.

To remove PVP, carbon sorbent samples were kept in physiological saline (0.15 M NaCl) under static conditions at room temperature for 24 hours. Then, the samples were dried at room temperature and analyzed: the adsorption properties of nitrogen adsorption were studied with the determination of the specific surface area (S _BET ), the total nitrogen content was determined (table 1). Aqueous solutions after contact with carbon sorbent samples were analyzed by NMR to identify PVP in them.

It was found that after contact with physiological saline for 24 hours, the specific surface area increases, while the nitrogen content naturally decreases. The results obtained indicate the migration of the applied PVP into physiological saline.

The results of the study of the antibacterial properties of the modified samples of carbon hemosorbent are presented in table 2. In the control crops, the concentration of microorganisms was 1 × 10 ³ CFU / ml. After contact with the studied carbon sorbents, it was found that the carbon hemosorbent sample (prototype) exhibits antibacterial properties against gram-positive bacteria - (Staphylococcus aureus, Staphylococcus aureus). A carbon sorbent sample modified by VP with subsequent polymerization, according to medical bench tests, exhibits antibacterial properties both in relation to gram-positive and in relation to gram-negative bacteria. After the contact of this sorbent with pathogenic microflora, the growth of bacterial cell colonies on Petri dishes was absent.

Thus, a set of physicochemical methods has established that the process of modifying the carbon hemosorbent with N-vinylpyrrolidone proceeds with the formation of a polymer poly-N-vinylpyrrolidone, which is capable of migration into the biological medium (physiological saline), causing the manifestation of the antibacterial properties of the obtained carbon sorbent.

The proposed carbon sorbent with antibacterial properties is a promising material for vulnerosorption (application medicine).

table 2 No. p / p The culture The control Samples Example 1 (prototype) Example 3 Concentration, CFU / ml Cup growth CFU / ml concentration Cup growth Concentration, CFU / ml Cup growth one Pseudomonas aeruginosa 1 · 10 ³ drain growth, 300 colonies 1 · 10 ³ comparable to control 0 no growth 2 Staphylococcus aureus 1 · 10 ³ drain growth, 300 colonies ~ 30 11 colonies 0 no growth 3 Escherichia coli 1 · 10 ³ drain growth, 300 colonies 1 · 10 ³ comparable to control 0 no growth four Klebsiella pneumoniae 1 · 10 ³ drain growth, 300 colonies 1 · 10 ³ comparable to control 0 no growth 5 Mix of cultures 1 · 10 ³ drain growth, 300 colonies 1 · 10 ³ comparable to control 0 no growth

Claims (2)

1. A method of producing a carbon sorbent with antibacterial properties, comprising impregnating granules of carbon hemosorbent with a modifier solution and drying the product, characterized in that the granules are impregnated with a 0.2-1.0% initiator solution in N-vinylpyrrolidone at pH 7.0-7, 5, the residual pressure of 15-20 mm RT.article and the ratio of hemosorbent: initiator solution in N-vinylpyrrolidone 1: 1.4-2.0 for 15-30 minutes, followed by a rise in temperature to 65-75 ° C, holding at this temperature for 0.5-8 hours in an inert atmosphere and washing in water from the residual monomer at room temperature for 0.5-2 hours 2. A carbon sorbent with antibacterial properties in the form of round granules, characterized in that it is obtained by the method according to claim 1, contains polyvinylpyrrolidone in an amount of 4.5-5.5%, characterized by a specific adsorption surface of less than 50 m ² / g and a total volume then less than 0.30 cm ³ / g

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