Method for producing agent for intracellular delivery of biologically active low-molecular nanoparticle-based compounds

FIELD: medicine.

SUBSTANCE: method is implemented as follows: preparing a mixture 1 by adding 0.5M aqueous solution of selenious acid 250 mcl in PEG 400 8 ml, mixing thoroughly in a magnetic mixture at min. 750 rpm with pH of the given mixture 7.55; that is followed by preparing a mixture 2 by adding 0.5M aqueous solution of hydrazine hydrochloride 250 mcl in PEG 400 8 ml, mixing thoroughly in a magnetic mixture at min. 750 rpm with pH of the given mixture 0.68. The mixture 1 is added to the mixture 2 by mixing thoroughly drop by drop. The prepared solution is dialysed against distilled water with removing PEG 400 and hydrazine hydrochloride; the surplus water is distilled off in a rotary evaporator at 60 rpm and 70°C. The prepared solution is added with a low-molecular compound specified in a group of: gentamicin, hexamethylene tetramine, methionine, cephalexin, indole-3-carbinol; pH is reduced to 7.2-7.4. The components are mixed in an amount to provide their content in the agent, wt %: biologically active low-molecular compound 0.001-5.0; selenium 0.0001-1.0; water up to 100.

EFFECT: simplifying the technology.

2 tbl, 3 ex, 1 dwg

 

The invention relates to the field of pharmacology and medicine, including veterinary medicine, and can be used as an efficient tool for intracellular delivery of low molecular drugs and biologically active molecules for treatment of diseases of various origins.

Intracellular delivery of drugs and biologically active molecules is an important problem in biology, medicine and veterinary medicine. Direct delivery of drugs and biomolecules to the organs-targets, ineffective, due to enzymatic degradation of the active substances, by neutralization of the liver and excretion by the kidneys. Therefore, the search for effective and safe vectors to genes, cells and organs-targets is an important area of research.

As particulate carriers for biologically active substances used structures, such as fullerenes, dendrimers, liposomes, polyacrylate particles, micelles, colloidal gold, and other.

The most promising developments in this area are lipidsoderžaŝie (liposomal) nanosystems, polymeric nanoparticles, colloidal particles, and the nanocarriers from porous silicon.

These transport systems are imperfect and have a number of drawbacks (in particular, the lack of stability and high complexity technical�ogy of production and, accordingly, the extremely high cost), which explains the lack of "targeted" drugs in the practice of medicine.

In particular, liposomal nanosystems in most cases are offered in the form of solutions containing particles, comprising more than 100 nm. Liposomes are used as a container for drug delivery to the target organs.

Particles of this size are typically unstable and, therefore, not possible to control the particle size during formation of the system. In addition, when administered liposomes are rapidly cleared from the bloodstream of the reticulo-endothelial system, resulting in reduced efficacy of the drugs.

Similar drawbacks are inherent to the next development - "Immunoliposomes nanosystem targeted delivery to connexin-43-positive tumor cells" (patent RF 2422154 on CL IPC AC 39/00, pub. 27.02.2011). The system includes biotinylated monoclonal antibody to extracticona fragment of connexin-43 (first component) and Paglierani liposomal containers with a diameter of 70-100 nm, covalently linked to streptavidin (second component). Serial introduction to living system of the first and second components of the binary system leads to the selective delivery of liposomes to the CX-43-positive cells by specific�die of binding of biotinylated antibodies with extraclean fragment of CX-43 in titlemma glioma cells and the subsequent formation of the streptavidin-bioteknologi complex.

However, this nanosystem has a specific action - is intended only for the targeted delivery of liposomes to tumor cells. In addition, a significant drawback of this system is the low stability of liposomes during storage.

Also known dosage form of delivery of water insoluble and poorly soluble drugs in the form of nanoparticles and a method of producing a delivery system dosage form (see application No. 2006123043 on CL IPC AC 31/00, pub. 20.01.2008). The method comprises freeze-drying a water-insoluble and poorly soluble biologically active substances with the formation as a result of particles in the form of spherical nanosized structures. The lyophilization is carried out with liposomes composed of lipids that form the Association vector environment around the nucleus - liposomal membrane. The nanoparticles have a size from 5 to 1500 nm. As poorly soluble and water-insoluble biologically active substances the system contains hypnotics, sedatives, anti-convulsants, tranquilizers, analgesic, anti-inflammatory, cardiovascular, hormones, vitamins, enzyme and stimulating metabolic processes, tools, antifungal, antitumor, biologically active peptides and proteins. However, a significant disadvantage Dunn�th system is the low storage stability and the inability to control the particle size during formation of the system.

The most stable is highly dispersed nanosystem proposed in 2011, research Institute of biomedical chemistry named after V. N. Full member (IBMC RAMS). This system obtained on the basis of soybean phospholipid and is a system for transport as fat-soluble (hydrophobic) and water-soluble (hydrophilic) of biologically active compounds. The authors develop on the basis of screening of drugs of different spectrum was analyzed by their ability to be embedded in phospholipid transport nanostructure, as well as conducted preliminary tests in vitro and in vivo. The results of these tests as the most promising for industrial production were selected drugs containing nizkomolekulyarnye compound drugs doxorubicin (drug "Localip") and indomethacin (drug "Indole") with phospholipid transport nanoscale. Conducted preclinical studies of these nanolasers developed a full set of normative-technical documentation, required for their production, prepared documents for submission to the Ministry of Health for permission to conduct clinical trials.

The proposed technology allows to obtain nonrecursive in the form of lyophilized powder, which increases their with�OK storage of up to 5 years, protects phospholipids from aggregation. However, a process that requires unique equipment, it is highly complex and time-consuming. It includes obtaining a stable phospholipid nano-emulsion particles with an active drug substance (high pressure homogenization) and subsequent freeze drying of the resulting emulsions. This factor has a negative impact on production costs.

The nanocarriers from porous silicon is represented by the following developments:

- "Method of producing nano-sized drug delivery system based on silicon dioxide" (patent RF №2372890 on CL IPC 61J 3/00, AK 47/04, AK 38/33, pub. 20.11.2009),

- "Pharmaceutical nanocarriers with. The production of pharmaceuticals with the nanocarriers from porous silicon" (developers - Nanolek Holding Limited (Cyprus) LTD. "Nanosec" Russia).

The closest of them to the claimed method is a method of producing nano-sized drug delivery system on the basis of silicon dioxide (see patent RF №2372890 on CL IPC A61J 3/00, AK 47/04, AK 38/33, pub. 20.11.2009) - met-enkephalin on the Hydrosol nanoparticles of SiO2. The method involves mixing distilled water, hydrochloric acid and tetraethoxysilane, the addition of the solution prepared in NaOH, evaporation and filtration with obtaining Hydrosol SiOsub> 2, ultrasonic treatment of the obtained Hydrosol SiO2the addition of met-enkephalin and surfactant solution in an amount of 0.5-2% of the total volume of the resulting system. The delivery system met-enkephalin is able to cross the blood brain barrier and deliver a drug to the brain cells. The method allows to create nanoparticles with diameter of 6-10 nm. This drug and the system of delivery have focused the action is intended only to overcome the blood-brain barrier.

As follows from the description of the project "Pharmaceutical nanocarriers with. The production of pharmaceuticals with the nanocarriers from porous silicon" (developers - Nanolek Holding Limited (Cyprus) LTD. "Nanosec" Russia technology for targeted delivery of nanocarriers from porous silicon involves the prolongation of biologically active substances due to sorbirovaniya it on porous silicon nanoparticles that perform the transport of active substances, providing a gradual release from the pores size of about 10 nm. On this principle it is planned to develop the group of solid dosage forms containing drugs for the treatment of cardiovascular diseases, anticancer and antiviral drugs. The action of already known active substances in these drugs is prolonged at the expense of their sorbirovaniya on Nan�particles of porous silicon.

As follows from the description, this nanosystem does not imply targeted delivery, but only the prolongation of the action of active substances. Unclear remains the question of the release rate of the substance from the pores of the nanoparticles. Also dubious about the possibility of further utilization (biodegradation) of sufficiently large silicon particles (more than 150 nm) in the bloodstream. It is not excluded that these nanoparticles can have an irritating effect on the immune system, which may subsequently lead to allergic and autoimmune diseases.

It should be noted that the main disadvantage of the currently used nanoparticles is their high toxicity compared to the particulates. They are able to pass unchanged through cellular barriers, the blood-brain barrier into the Central nervous system, circulate and accumulate in organs and tissues, causing more severe pathological lesions of internal organs (such as the formation of granulomas in the lungs, liver cirrhosis, glomerulonephritis), as well as having a long half-life.

Toxicity of nanoparticles is determined by their shape and size, with the smallest nanoparticles fusiform shape cause more devastating effects in the body than similar particles of spherical shape Under the influence of the nanoparticles on the body is clearly apparent dependence "dose-effect". Clinical manifestations depend on the content of a given chemical element in the composition of each specific nanoparticles, however, we have observed a significant increase of toxic effect.

In addition, the nanoparticles can have an irritating effect on the immune system. Since the removal of nanoparticles from the body is a long time, constant migration of nanostructures on the body in small amounts can lead to destabilization of the immune system and allergic and autoimmune diseases.

The invention is aimed at solving the problem of creating non-toxic and effective means of intracellular delivery of biologically active low-molecular compounds based on nanoparticles of selenium, namely to a method of its receipt.

To solve this problem the method is as follows

First, prepare mixture 1 by adding 250 ál of 0.5 M aqueous solution se acid in 8 ml of PEG 400, intensive mixing on a magnetic stirrer at 750 rpm, pH of this mixture and 7.55, then cook the mixture 2 by adding 250 µl of 0.5 M aqueous solution of hydrochloric acid hydrazine in 8 ml of PEG 400, intensive mixing on a magnetic stirrer at 750 rpm, pH of this mixture to 0.68, with vigorous stirring is introduced into the mixture of 1 CME�y 2 dropwise, the resulting solution was put on dialysis against distilled water, removing the PEG 400 and hydrazine hydrochloric acid, the excess water is distilled off on a rotary evaporator, the resulting solution is brought low molecular weight compound selected from the group gentamicin, hexamethylenetetramine, methionine, cephalexin, indole-3-carbinol. The pH was adjusted to 7.2 to 7.4.

As a result of the claimed method provides a medium of the following composition, in wt. %:

Biologically active low-molecular compoundOf 0.001 to 5.0
SeleniumIs 0.0001 to 1.0
Waterelse

As a low-molecular biologically active substances compound may contain gentamicin, hexamethylenetetramine, methionine, cephalexin, indole-3-carbinol.

As a selenium-containing substance use se acid.

In the known authors of the sources of patent and scientific and technical information not described non-toxic and effective means of intracellular delivery of low-molecular biologically active substances and methods for its preparation, wherein the recording medium (nonplatform) use colloidal selenium, made by conjugation of p�Yes biologically active molecules.

It is known the use of selenium as a therapeutic agent, i.e. drug, capable of participating in redox processes of the cells of the body (see, for example, patent RF №2394583, No. 2426444, sites http://fitoapteka.com/read/ru: http://www.naturoprof.ru, http://bezvreda.com/selen-v-pitanii/). Selenium normalizes the metabolism of proteins and nucleic acids; regulates specific and non-specific immunity through activation of the functions of neutrophils, proliferation of T-, b-lymphocytes, generate production of antibodies, lymphokines, improves the adaptation of the organism; reduces toxic effect of substances, salts of heavy metals, drugs, various antibiotics; prevents the development of oxidation, free radical diseases, including atherosclerosis and its complications, liver necrosis, pancreatitis, multiple sclerosis, Parkinson's disease, other diseases.

It is generally accepted that the trace element selenium (Se) is a necessary nutrient for the normal functioning of the human body and animals, as it is part of the majority of hormones and enzymes involved in metabolism. He performs in the body of catalytic, structural and regulatory function; interacts with vitamins, enzymes and biological membranes; involved in oxidation-reduction process, cellular respiration, metabolism fat�in, proteins and carbohydrates. The role of selenium in the body is largely determined by its inclusion in the composition of one of the most important enzymes - glutathione peroxidase, which protects cells against peroxidation products. Thus, selenium and its compounds exhibit significant antioxidant activity. This element is included and other enzymes involved in the detoxification of xenobiotics, regulates the thyroid and pancreas, exhibits a hepatoprotective effect, stimulates antitoxic protection body, has a positive effect on the reproduction system, has radioprotective action.

The use of colloidal selenium as nanooxides for delivery of low-molecular biologically active substances hitherto unknown.

The advantage of the inventive method of delivery of low-molecular biologically active substances with nanoparticle-based colloidal selenium is the ability of intracellular penetration of the nanoparticle colloidal selenium conjugated with it medicinal substances of low molecular weight compounds. The biodynamics of drugs and biologically active molecules connected with colloidal selenium occurs by lymphogenous and to a lesser extent subjected to enzymatic degradation and neutralization of the liver.

Also of particular advantage is the fact that colloidal selenium is part of the metabolic chain of the body and are capable of binding to intracellular space. Thereby eliminated unwanted effects related to "dispose of" the body of the vehicle.

This allows us to conclude that the presence of the claimed invention, the criterion of "inventive step".

The means obtained by the proposed method, is a colloidal solution of selenium nanoparticles with adsorbed on its surface particles of low molecular weight compounds. It is a clear liquid, the color of which varies from brick-red to orange. Store at temperature from +2 to +20°C in a dark place. Shelf life is 1 year.

To obtain the intracellular delivery of biologically active low-molecular compounds, prepare a mixture of 1 by adding 250 ál of 0.5 M aqueous solution se acid in 8 ml of PEG 400, intensive mixing on a magnetic stirrer at 750 rpm, pH of this mixture and 7.55, then cook the mixture 2 by adding 250 µl of 0.5 M aqueous solution of hydrochloric acid hydrazine in 8 ml of PEG 400, intensive mixing on a magnetic stirrer at 750 rpm, pH of this mixture to 0.68, with vigorous stirring is introduced into the mixture 1 mixture 2 p� drops, the resulting solution was put on dialysis against distilled water, removing the PEG 400 and hydrazine hydrochloric acid, the excess water is distilled off on a rotary evaporator at 60 rpm and 70°C, the resulting solution is brought low molecular weight compound selected from the group gentamicin, hexamethylenetetramine, methionine, cephalexin, indole-3-carbinol, at a concentration of 0.001 to 5.0% by mass, the pH was adjusted to 7.2 to 7.4.

Bring the pH to 7.2 to 7.4 due to the need to create a stable system because of the acidity values above or below the specified limits will result in the destruction of the colloidal solution.

The choice of the quantitative content of selenium in the medium is 0.0001 to 1.0 wt.%, as well as biologically active low-molecular compounds is 0.001-5% wt., due to the necessity of matching the task to be solved. The concentrations of the components ensures the homogeneity and stability of the funds.

The preferred route of administration tools for intracellular delivery of parenteral. The dose is calculated based on biologically active substances used in the system, the rate of 0.1-7 mg per kilogram of body weight biological organism.

Example 1

Prepare mixture 1 by adding 250 ál of 0.5 M aqueous solution se acid in 8 ml of PEG 400, intensive mixing on a magnetic stirrer at 750 rpm, pH of this �Mesa 7,55, then cook the mixture 2 by adding 250 µl of 0.5 M aqueous solution of hydrochloric acid hydrazine in 8 ml of PEG 400, intensive mixing on a magnetic stirrer at 750 rpm, pH of this mixture to 0.68, with vigorous stirring is introduced into the mixture 1 mixture 2 dropwise, the resulting solution was put on dialysis against distilled water, removing the PEG 400 and hydrazine hydrochloric acid, the excess water is distilled off on a rotary evaporator at 60 rpm at 70°, the resulting solution is brought methionine to a final concentration of 1.7 wt. %, the pH was adjusted to 7.2 to 7.4.

So we get:

Low molecular weight biologically active substance methionine0,017 g/ml
Selenium0,00062 g/ml
Waterelse

As a percentage of the total volume of the composition of funds will be as follows, in mass. %:

Low molecular weight biologically active substance methionine1,7
Selenium0,062
Waterto 100

Example 2

Prepare mixture 1 by vnesti� 250 µl of 0.5 M aqueous solution se acid in 8 ml of PEG 400, intensive mixing on a magnetic stirrer at 750 rpm, pH of this mixture and 7.55, then cook the mixture 2 by adding 250 µl of 0.5 M aqueous solution of hydrochloric acid hydrazine in 8 ml of PEG 400, intensive mixing on a magnetic stirrer at 750 rpm, pH of this mixture to 0.68, with vigorous stirring is introduced into the mixture 1 mixture 2 dropwise, the resulting solution was put on dialysis against distilled water, removing the PEG 400 and hydrochloric acid hydrazine, the excess water is distilled off on a rotary evaporator at 60 rpm at 70°, the resulting solution is brought doxycycline or cephalexin to final concentration of 1 mass. %, the pH was adjusted to 7.2 to 7.4.

So we get:

Low molecular weight biologically active substance doxycycline or cephalexin0,095 g
Selenium0,0098 g

As a percentage of the total volume of the composition of funds will be as follows, in mass. %:

Low molecular weight biologically active substance doxycycline or cephalexin1
Colloidal selenium0,2
Water to 100

Example 3

Prepare mixture 1 by adding 250 ál of 0.5 M aqueous solution se acid in 8 ml of PEG 400, intensive mixing on a magnetic stirrer at 750 rpm, pH of this mixture and 7.55, then cook the mixture 2 by adding 250 µl of 0.5 M aqueous solution of hydrochloric acid hydrazine in 8 ml of PEG 400, intensive mixing on a magnetic stirrer at 750 rpm, pH of this mixture to 0.68, with vigorous stirring is introduced into the mixture 1 mixture 2 dropwise, the resulting solution was put on dialysis against distilled water, removing the PEG 400 and hydrazine hydrochloric acid, the excess water is distilled off on a rotary evaporator at 60 rpm at 70°, the resulting solution is brought hexamethylenetetramine to a final concentration of 5 mass. %, the pH was adjusted to 7.2 to 7.4.

So we get:

Low molecular weight biologically active substance hexamethylenetetramine0.05 g/ml
Selenium0,00062 g/ml

As a percentage of the total volume of the composition of funds will be as follows, in mass. %:

Low molecular weight biologically active substance hexamethylenetetramine5
Selenium0,062
Waterto 100

The particle size of the obtained means were studied by electron microscopy, the results of which showed that the means obtained by the proposed method, contains particles of selenium from 40 to 100 nm (see Fig. 1). Figure visible nanoparticles with size from 40 to 100 nm.

The influence of colloidal selenium on the intracellular penetration of methionine was determined by its accumulation in the extracellular space. Measurement of methionine was determined chromatographically by the following method:

Determination of the concentration of methionine was carried out by high performance liquid chromatography. Measurement of the concentration of methionine in cells was determined by liquid chromatograph type "the Stayer", the analysis was carried out at a wavelength of 288 nm, flow rate - 0.9 cm3/min, the amount of insertion of the sample was 20 µl, temperature analysis-30-35°C, eluent - acetonitrile for liquid chromatography" - 1% solution of acetic acid in the ratio 2:3 by volume. In these conditions chromatographic standard solutions twice each. Measure the peak area at the retention time of 3.9 to 4.3 min (depends on the efficiency of the column). On average parallel measurements, the discrepancy between them should not exceed 5%(Rel.), build a calibration curve in the coordinates: the peak area - concentration mg/ml. Chromatography of the analyzed samples was carried out analogously. The obtained average value of two parallel measurements (relative standard deviation of which should not exceed 5%) of the calibration curve to find the concentration of methionine in the sample solution.

The accumulation of methionine was observed in peritoneal cells of rats and lymphocytes.

Obtaining peritoneal macrophages

Peritoneal macrophages obtained from the peritoneal cavity of rats. Sleeping chloroform to rats intraperitoneally administered 10 ml of medium for the isolation of cells from the peritoneal cavity (medium 199+2% horse serum + 100 units of heparin + 100 units of penicillin) and stood for 1 min, massaging the abdomen. Then the rat was dissected and collected entered Wednesday with washed away by macrophages. After the cells were precipitated at 2500 rpm for 30 min and washed 3 times with medium to launder cells (medium 199+2% horse serum + 100 units of penicillin). Next, the cells were resuspended in the medium for cultivation (medium 199+10% horse serum + 100 units of penicillin). The content and the leukogram of the selected cells was determined on generalizatio "Arkus" (Austria).

Obtaining lymphocytes

The lymphoid cells were obtained from peripheral blood of calves. Peripheral blood was centrifuged at 1500 �b/min for 30 min, then selected the plasma. The resulting plasma was diluted with Hanks solution 3:1. Diluted plasma was layered on ficol-verografin without violating the boundary plasma/fecal and centrifuged at 1500 rpm for 30 min.

After centrifugation were collected formed the ring of mononuclear cells in a separate tube, diluted with Hanks solution 1:1 and precipitated cell fraction at 3000 rpm for 10 min. the Precipitate was washed with Hanks solution 1:7 from fecal-verografin, precipitating the cellular fraction as well. The residue with the remainder of the supernatant to a total volume of 1 ml were diluted with medium for the production of lymphocytes (medium 199+5% horse serum + 0.1% of gelatin) of 1:1 was precipitated at 2000 rpm for 10 min and the resulting pellet was resuspended in the same environment. The content and the leukogram of the selected cells was determined on generalizatio "Arkus" (Austria).

Incubation of the particles of colloidal selenium conjugated with methionine with peritoneal macrophages and lymphocytes

The investigated colloidal particles were incubated with peritoneal macrophages and lymphocytes within 3 hours in a thermostat at 37°C.

After the incubation the cells were washed thrice with cold physiological saline to remove unabsorbed particles. After washing the cells suspended in 200 µl of saline. In the obtained cell susp�nsii measured the amount accumulated in the cells of methionine by HPLC, and prepared smears.

As a result of the experiments the following data were obtained. When cultured methionine, conjugated with colloidal selenium as a carrier, the highest concentration of drug was determined using as the object of study of peritoneal macrophages of rats. When using whole blood lymphocytes of calves as the object of penetration, we observed a reduction in the amount of methionine in 2 times a cell population (table. 1). The data obtained indicate the ability of colloidal selenium methionine to transfer into the intracellular space.

Table 1
The concentration of methionine in cells using colloidal selenium as media
Type of conjugate/type of cellsThe introduced methionine, ugFound methionine, µg ×108/Li
conjugate methionine/selenium/peritoneal macrophages30,4529,13
conjugate methionine/selenium/lymphocytes whole blood30,4515,63
methionine/lymphocytes whole blood30,40,06
methionine/peritoneal macrophages30,42,4

Similar results are obtained when using other low molecular weight substances, gentamicin, hexamethylenetetramine, cefalexina, indole-3 carbinol.

Studied biodynamics means of intracellular delivery of biologically active substances by assessing the penetration of colloidal selenium particles into the cell.

As a means of intracellular delivery of the product obtained in accordance with example 1 or 2.

Carried out the labeling of the drug fluorescent dye (FITZ) using the methodology described, for example, in the book: Immunologische methods / edited by G. Frimel, translated from the German by A. P. Tarasova. - M.: Medicine, 1987, p. 130.

The object of studies were white mice weighing 20 grams.

All animals were divided into groups: the first group of mice (n=3) the drug is labeled FITZ, was injected intramuscularly at a dose of 0.1 ml; the second intraperitoneally at a dose of 0.2 ml, the third - orally at a dose of 0.2 ml. One mouse was the control.

After 2, 6 and 24 hours postmortal fingerprints were obtained for liver, kidney, spleen, bone marrow and whole blood smear.

p> The results are presented in table 2.

Table 2
№p/pBodiesIntracellular fluorescence
2 hours6 hours24 hours
ORAL INTRODUCTION
1.Blood+
2.Liver++
3.Kidney+
4.Spleen+++
5.Bone marrow++
INTRAPERITONEAL administration
1.Blood++
2.Liver++
3.Kidney+
4.Spleen++
5.Bone marrow++
INTRAMUSCULAR TO the JURISDICTION of
1.Blood++
2.Liver++
3.Kidney+
4.Spleen++
5.Bone marrow++

As can be seen from table 2, the complex on the basis of colloidal selenium methionine or gentamicin in the animals body is inside the structural elements, having a shell, i.e. inside immune cells. Wherein the means often gets in the spleen cells. The drug is quickly absorbed in the gastrointestinal tract.

However, oral administration of the drug in the cellular elements of the blood, he falls much later than when administered parenterally. With this in injecting the drug reaches the liver and spleen later than during oral.

The data presented indicate the penetration of the drug in the cellular elements of the immune organs, where it metabolizing. The absence of the drug in the intercellular space indicates its complete biodegradation of the cellular elements.

Thus, the tool is not subjected to enzymatic degradation and neutralization of the liver, i.e., it fully penetrates into nutrici�full-time space.

Himself colloidal selenium, as part of the metabolic chain of the body, is absorbed in the extracellular space, i.e., in the present invention solved the task of "recycling" in the body of the vehicle.

Determined the acute toxicity of the preparation method of Kerber.

This used albino mice in the amount of 10 goals a body weight of 20-25 g.

Formulated with methionine or gentamicin was administered to mice intraperitoneally at the highest possible dose of 0.5 ml. the Tool was administered during the day every 3-4 hours.

During the first day the mice were closely monitored, noting the behavior, consumption of food, water, and General condition.

All observations were within 2 weeks of death of mice was observed. Thus, we can conclude that the drug belongs to the group of non-toxic compounds.

Similar results are obtained when using other low molecular substances of hexamethylenetetramine, cefalexina, indole-3-carbinol.

The above experiments demonstrate that the remedy obtained by the developed method has low toxicity and high bioavailability.

In addition, biodynamics biologically active low-molecular compounds, conjugated with colloidal selenium occurs by lymphogenous and to a lesser extent is exposed to a�Xia enzymatic degradation and neutralization of the liver, thus biologically active low-molecular substances exert their action directly in the pathological focus.

A method of obtaining a means of intracellular delivery of biologically active low-molecular compounds, namely, that prepare a mixture of 1 by adding 250 ál of 0.5 M aqueous solution se acid in 8 ml of PEG 400, intensive mixing on a magnetic stirrer at 750 rpm, pH of this mixture and 7.55, then cook the mixture 2 by adding 250 µl of 0.5 M aqueous solution of hydrochloric acid hydrazine in 8 ml of PEG 400, intensive mixing on a magnetic stirrer at 750 rpm, pH of this mixture to 0.68, with vigorous stirring is introduced into the mixture 1 mixture 2 dropwise, the resulting solution was put on dialysis against distilled water, removing the PEG 400 and hydrazine hydrochloric acid, the excess water is distilled off on a rotary evaporator at 60 rpm and 70°C, the resulting solution is brought low molecular weight compound selected from the group gentamicin, hexamethylenetetramine, methionine, cephalexin, indole-3-carbinol, the pH was adjusted to 7.2 to 7.4, while the components are mixed in quantities to ensure the content of their means, in the mass. %:

Biologically active low-molecular compoundOf 0.001 to 5.0
SeleniumIs 0.0001 to 1.0
Waterto 100



 

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FIELD: chemistry.

SUBSTANCE: ceramic membrane, applied for the separation of gas mixtures, has the following composition, wt %: aluminium oxide 30-54; sodium silicate 42-68; carbon nanotubes CNT with an external diameter of 1-5 nm with a three-layer structure and a specific surface of 350-1000 m2/g 1-4. The method of preparing the ceramic membrane for the separation of gas mixtures includes mixing of thermoactivated gibbsite - Al(OH)3 with sodium silicate and the carbon nanotubes CNT with an external diameter of 1-5 nm with the three-layer structure and the specific surface of 350-1000 m2/g, following addition of a nitric acid solution. The obtained mass is thoroughly mixed and an excess of moisture is removed until powder has a half-dry condition. The obtained powder is pressed, the pressed tablets are subjected to thermal-processing - first, they are exposed at a temperature of not higher than 150°C, then at a temperature of not higher than 400°C. The obtained membrane in the form of a tablet is annealed without air access at 850-1100°C.

EFFECT: increased factor of the gas mixture separation.

2 cl, 3 dwg, 2 tbl, 5 ex

FIELD: chemistry.

SUBSTANCE: invention relates to electronics and is intended to design devices which convert the chemical reaction of adsorbed molecules of a fuel gas (vapour) and oxygen (or air) into an electrical signal. The invention can be used to design compact batteries for electronic equipment in the form of single-chamber fuel cells, which consist of a working chamber having a fuel-gas mixture inlet and a gas outlet, inside of which there is a composite film with electrical contacts connected to an external load, the space between which is filled with a conducting material. The conducting material used is a nanocomposite material which consists of a non-conducting polymer film of polypropylene and conducting filler in the form of carbon nanotubes. Concentration of the carbon nanotubes with p-type conductivity is about 0.5-5% near the percolation threshold. The nanocomposite material may contain catalytic nanoparticles of Pt or Pd, or Rh, or Ru. Also disclosed is a method of producing a conducting nanocomposite material, which includes mixing carbon nanotubes and polymer material and then holding the nanocomposite material at external voltage of 4-10 V for 2-30 min in an atmosphere of saturated acetone vapour.

EFFECT: higher current density in the active layer.

7 cl, 3 dwg

FIELD: medicine.

SUBSTANCE: invention refers to medicine and describes a method for producing chondroitin sulphate nanocapsules by non-solvent addition, characterised by the fact that chondroitin sulphate is added in small portions into xanthane gum suspension used as a nanosuspension shell, in butanol containing 0.01 g of the preparation E472 as a surfactant; the produced mixture is stirred and added with the nonsolvent hexane 6ml, filtered, washed in hexane and dried.

EFFECT: invention provides simplifying and accelerating the process of nanoencapsulation in xanthane gum and higher weight yield.

2 ex

FIELD: medicine.

SUBSTANCE: invention represents a method for drug encapsulation by non-solvent addition, wherein according to the invention cores of nanocapsules are vitamins, whereas a shell is sodium carboxymethyl cellulose precipitated from isopropyl alcohol suspension by adding chloroform as a non-solvent and dried at room temperature.

EFFECT: simplifying and accelerating the process of nanoencapsulation, reducing accompanying loss.

7 ex

FIELD: veterinary medicine.

SUBSTANCE: method comprises subcutaneous administration of antibiotic preparation enroxyl 5% at a dose of 0.1 ml/kg daily one time a day for 7 days and intramuscular administration of homeopathic preparation ovarinin at a dose of 1 ml/kg one time for 4 days, 4-fold.

EFFECT: use of the invention enables to increase the efficiency of treatment, to reduce treatment time and to restore reproductive function of dogs.

2 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: group of inventions refers to veterinary science and is applicable for treating bovine mastitis. What is declared is a nosode for producing a preparation for treating bovine mastitis. That involves taking mastitis milk 1 ml with clinical signs of purulent-catarrhal mastitis, filling it 70° alcohol 9 ml to produce a homogenous solution; the produced solution is diluted to "Д6" with 70° alcohol. What is also declared is a preparation for treating bovine mastitis containing an aqueous-alcoholic solution of components. The components are Belladonna, Sulphur, Apis mellifica, Conium, Phytolacca, Silicea and above nosode in the following proportions, wt %: Belladonna - 5, Sulphur - 20, Apis mellifica - 20, Conium - 10, Phytolacca - 10, Silicea - 5, nosode - 15, aqueous-alcoholic solution up to 100%. What is also declared is a method of treating bovine mastitis involving the intracisternal administration of the preparation. The preparation is administered in a dose of 5-10 ml per 1 animal once a day.

EFFECT: declared group of inventions is highly effective in treating bovine mastitis.

3 cl, 5 tbl, 1 ex

FIELD: veterinary medicine.

SUBSTANCE: method comprises administering the preparation in a dose of 0.5 ml/25 kg live weight. The preparation is administered two times intramuscularly with an interval of 35 days. The first administration of the preparation is carried out 35 days before weaning, and the second - at the time of weaning the lambs. The preparation is used as a vitamin and mineral complex containing selenium taken in nanoscale state and zero valence of 0.125%, fat-soluble vitamin E 3.8%, water-soluble vitamin B1 0.1%, water-soluble vitamin B2 0.1%, water-soluble vitamin B5 0.1%, water-soluble vitamin B6 0.1%, water-soluble vitamin B12 0.1%, vitamin K3 - 0.1%, solubiliser Solutol HS 15 1.0-20.0%, and water.

EFFECT: use of the method enables to reduce the negative effect of stress on animal body and to reduce the loss of live weight.

7 dwg, 2 ex

FIELD: chemistry.

SUBSTANCE: invention relates to encapsulation, particularly a method of producing microcapsules of a mixture of Vetom 1.1 and Sel-Plex in a carrageenan envelope. According to the disclosed method, Vetom 1.1 and Sel-Plex, taken in weight ratio of 60:40, are dissolved in dimethyl sulphoxide or dimethyl formamide. The obtained mixture is dispersed in carrageenan solution in benzene in the presence of an E472c preparation while mixing at 1000 rps. The mixture of the preparations and carrageenan are taken in weight ratio of 1:1 to 1:5. Butanol and distilled water, taken in ratio of 5:1 vol/vol, are then added. The obtained suspension of microcapsules is filtered out and dried. The process of producing the microcapsules is carried out at 25°C for 20 minutes.

EFFECT: invention simplifies and speeds up the process of producing microcapsules, reduces losses during production thereof (high mass output).

6 ex, 10 dwg

FIELD: medicine.

SUBSTANCE: method for producing an agent for stimulating body cells involving preparing a mixture of aqueous solution of selenious acid and PEG 400; that is followed by preparing a mixture of hydrazine hydrochloride and PEG 400; the prepared mixtures are combined; the solution is put to dialyse against distilled water; surplus of water is driven off; the produced solution is added with hexamethylene tetramine; pH is reduced to 7.2-7.4; the method is implemented in certain circumstances.

EFFECT: producing high-effective, ecologically safe agent by the synergism of colloidal selenium and hexamethylene tetramine on body cell stimulation.

1 dwg, 2 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: method for producing an agent inhibitory the tumour cell growth, involving preparing a mixture of aqueous solution of selenious acid and PEG 400; that is followed by preparing a mixture of aqueous solution of hydrazine hydrochloride and PEG 400; the produced mixtures are combined; the solution is put to dialyse against distilled water; surplus of water is driven off in a rotary evaporator; the produced solution is added with silymarin dissolved in Solufor with dialysis against distilled water; pH is reduced to 7.2-7.4; the method is implemented in certain circumstances.

EFFECT: agent produced by the given method possesses high inhibitory action on the tumour cell growth.

4 dwg, 2 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: wound surface is treated with 3.0-1.5% hydrogen peroxide; then keeping the wound wet, Ichthyosin dressing prepared by streptocide powder 0.5-2.5g and Ichthyol ointment 5.0-10.0g dissolved in castor oil 94.5-87.5ml is applied. The agent is applied once a day; the therapeutic course makes 16 days. That is combined with performing an integrated treatment.

EFFECT: invention provides both the antibacterial and anti-infectious, and local anti-inflammatory, local anaesthetic, wound-healing effects, improves the blood supply, stimulates the epidermis regeneration, intensifies the keratosis processes that enables accelerating the wound cleansing from the purulonecrosis process, and a length of preparation for aurografting.

1 tbl, 2 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to the pharmaceutical industry, particularly to a composition for treating or preventing the human immunodeficiency virus (HIV) or hepatitis C. The herbal composition used for treating or preventing the patients infected by the HIV or hepatitis C viruses and containing a herbal ingredient with tannin agents and catechin, and a pharmaceutically acceptable carrier; the herbal ingredient is Agrimonia Eupatoria (GAFT) and/or gambier (Uncaria gambir). A method for preparing the herbal composition for treating or preventing the patients infected by the HIV or hepatitis C viruses.

EFFECT: composition is effective for treating or preventing the patients infected by the HIV or hepatitis C viruses.

12 cl, 6 dwg, 2 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to veterinary science and aims at treating and preventing alimentary anaemia in young pigs. A preparation contains an iron dextran complex, nanosized and zero-valent selenium (Se0), vitamin E, vitamin B12 and water in the following proportions, wt %: iron dextran complex 0.0001-80.0, selenium (Se0) 0.0001-5.0, vitamin E 0.0001-20.0, vitamin B12 0.0001-10.0, water for injections - the rest.

EFFECT: using the declared method provides compensating iron deficiency in young pigs, improving animal's growth and development, increasing total immunity and rapid adaptation to the varying ambient environment.

4 tbl, 4 ex, 4 dwg

FIELD: veterinary medicine.

SUBSTANCE: preparation for normalisation of lipid peroxidation processes in animals comprises, wt %: 2-ethyl-6-methyl-3-hydroxypyridine succinate 20.0-30.0, ascorbic acid 5.0-7.0, selenium (Se°) 0.3-0.5, polyvinylpyrrolidone 3.0-5.0, water for injection - the rest.

EFFECT: antioxidant effect, prevention of excessive formation of lipid peroxidation products, high efficiency in normalisation of functioning of the system of antioxidant protection of animals and the process of lipid peroxidation, low toxicity and ease of administration.

6 dwg, 5 ex

FIELD: medicine.

SUBSTANCE: the present innovation includes synthetic zeolite Na-A, sulfur and flavoring at the following ratio of components, weight%: sulfur 0.03-0.08, flavoring 1.0-1.5, synthetic zeolite Na-A - the rest. As flavoring one should apply food flavorings with the odor of vanillin, lemon, prune, almond, coffee. Organoleptic evaluation of preparation's properties has shown that it causes no irritation of human hand skin: hands become dry and demonstrate pleasant odor. Due to supplementing sulfur the suggested preparation provides elimination of harmful microflora, protects hand skin against infection. Preparation is simple to be manufactured and applied along with its low price and ecological safety.

EFFECT: higher efficiency.

1 cl, 1 tbl

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