RU2801869C2 - Wound healing gel with liposomes containing a compound of iodine with polyvinyl alcohol, and a method of its preparation - Google Patents

Abstract

FIELD: veterinary medicine; physiology; biochemistry; pharmacology.

SUBSTANCE: group of inventions relates to a method of producing a wound healing gel with liposomes and the wound healing gel itself. The method of producing a wound healing gel with liposomes involves mixing the components of the gel base and adding liposomes to it. 0.2 g of iodine, 0.6 g of potassium iodide, 1.8 g of polyvinyl alcohol and 0.4 g of carbomer-940 are dissolved in 200 ml of distilled sterile water, the mixture is stirred on a magnetic stirrer until a homogeneous gel is obtained, and liposomes prepared by dissolution are added 4.0 g of phosphatidylcholine in 50 ml of ethanol, adding 8 mg to the solution α-tocopherol, 0.06 g of solid fat, 0.04 g of soybean oil and 0.02 g of linseed oil, by dissolving 10 g of potassium orotate in 50 ml of sterile distilled water, followed by injection of an alcoholic solution of lipids into an aqueous solution of potassium orotate 300–400 times with a needleless injector BI-1 to obtain liposomes with encapsulated potassium orotate, which are then separated from the solution by centrifugation for 1 hour at 15,000 rpm, added to the gel base and the resulting mixture is stirred until a homogeneous state. The wound healing gel with liposomes obtained by the above method contains a gel base — iodine and potassium iodide in combination with polyvinyl alcohol, gelling agent carbomer-940, liposomes containing phosphatidylcholine, α-tocopherol, potassium orotate, tallow, soybean oil and linseed oil.

EFFECT: group of inventions described above makes it possible to develop an effective and easy-to-manufacture wound healing gel.

2 cl, 7 dwg

Description

SUBSTANCE: group of inventions relates to the field of veterinary medicine, physiology, biochemistry and pharmacology, in particular to methods for activating wound healing and tissue regeneration processes.

Medicines containing iodine and iodine compounds as an active ingredient have a good antiseptic and wound-healing effect.

Known remedy for the treatment of various skin diseases, bruises, stab, incised and purulent wounds, burns and frostbite, which has an antiseptic, anti-inflammatory and wound healing effect (RF Patent No. 2327473, 2008). This ointment contains iodophor as an antiseptic component - a compound of iodine with polyethylene glycol. Iodophors are complex compounds of molecular iodine with nonionic surfactants that have bactericidal and virucidal properties. An important feature of iodophors is their good solubility in water.

In terms of their biological activity, iodophors are significantly superior to tincture of iodine or Lugol's solution. Compared to the latter, they are less toxic. Upon contact with the skin or mucous membranes, active iodine, which is part of the iodophor, is gradually released from the complex, exerting its antiseptic effect.

This ointment, however, does not contain components that enhance tissue regeneration, in particular, liposomes.

The therapeutic effect of many medicinal compounds is limited by the low level of its entry into target cells. To eliminate this, various delivery vehicles are used, for example, liposomes.

For example, a wound healing gel with liposomes is known (Patent 2697669, 2019), containing metronidazole as an antibacterial substance, local anesthetic lidocaine, gelatin, an amino acid donor for tissue regeneration, hyaluronic acid, which is one of the components of the extracellular matrix, and the gel-forming substance Carbomer 940. Liposomes consist of the phospholipid lecithin, which forms a bilayer of liposomes, α-tocopherol (vitamin E), an antioxidant that prevents lipid peroxidation of liposomes, and potassium orotate as a stimulator of regeneration processes. However, this gel does not contain iodine and is not effective enough in the treatment of wounds in animals in livestock farms.

The objective of the present invention is to develop an effective, cheap, easy-to-manufacture wound healing gel. To solve the problem of the present invention, a gel with liposomes containing an iodine compound with polyvinyl alcohol, called by the authors "Liposomide", is proposed.

For targeted delivery of the drug to the cells of the wound tissue, it was proposed to use liposomes. However, to prevent the destruction of liposomes by an antiseptic (compound of iodine with polyvinyl alcohol), it was proposed to modify their membrane by introducing solid fat, soybean oil and linseed oil into its composition, as well as to increase the amount of vitamin E.

The gel has the following composition:

Gel base

1. Iodine (0.1%) - a broad-spectrum antiseptic.

2. Potassium iodide (0.3%) - excipient.

3. Polyvinyl alcohol (9%) - excipient.

4. Carbomer 940 is a gelling agent widely used in medicine.

Liposomes

Liposomes (content to the volume of the gel 0.5-1.0%) - nanocontainers for targeted delivery of the drug to enhance regeneration in tissue cells.

1. Phosphatidylcholine (lecithin group) 500 mg - a phospholipid that forms a bilayer of liposomes;

2. α-tocopherol (vitamin E) 200 IU - antioxidant agent; preventing peroxidation of lipids that make up liposomes;

3. Solid fat 0.06 g;

5. Soybean oil 0.04 g;

6. Linseed oil 0.002 g;

7. Potassium orotate (potassium salt of vitamin B ₁₃ ) - a substance that enhances regeneration.

The novelty of the group of inventions is the modification of the composition of liposome membranes and the use of a solution of an iodine compound with polyvinyl alcohol as an antiseptic.

A method for obtaining a wound healing gel with liposomes.

1. Getting a gel base.

In 200 ml of distilled sterile water dissolve 0.2 g of iodine, 0.6 g of potassium iodide and 1.8 g of polyvinyl alcohol and 0.4 g of carbomer-940; the mixture is stirred on a magnetic stirrer until a homogeneous gel is obtained.

2. Obtaining liposomes with encapsulated potassium orotate.

To obtain liposomes, an injection method is used using a BI-1 needleless injector. A portion of 4.0 g of phosphatidylcholine is dissolved in 50 ml of ethyl alcohol, 8 mg of α-tocopherol, 0.06 g of solid fat, 0.04 g of soybean oil, 0.02 g of linseed oil are added to the solution. A portion of potassium orotate 10 g is dissolved in 50 ml of sterile distilled water. An alcoholic solution of lipids is injected into an aqueous solution of potassium orotate 300-400 times using a BI-1 needleless injector to obtain liposomes with encapsulated potassium orotate.

As a result, an emulsion of 4-10 μm encapsulated potassium orotate liposomes was obtained in a solution of potassium orotate.

To separate the liposomes from the solution, centrifugation is used for 1 hour at 15,000 rpm.

3. Obtaining a gel with liposomes.

Liposomes with encapsulated potassium orotate are added to the resulting gel base, the mixture is stirred until a homogeneous state.

The group of inventions is illustrated by the following figures. Fig. 1-Dynamics of changes in the area of wounds in experimental and control rats, Fig. 2 - Morphological parameters of the blood of experimental rats: 1 - leukocytes, 10 ⁹ ; 2 - lymphocytes, 10 ⁹ ; 3 - monocytes, 10 ⁸ ; 4 - granulocytes, 10 ⁹ ; 5 - erythrocytes, 10 ¹² ; 6 - platelets, 10 ¹¹ , fig. 3 - Hematological parameters of rats: 1 - hemoglobin, g/l, 2 - hematocrit, 3 - average hemoglobin content in an erythrocyte; 4 - the average concentration of hemoglobin in the erythrocyte g/l; 5 - distribution width of erythrocytes, %; 6 - average platelet volume, fl; 7 - platelet distribution width; 8 - average volume of erythrocyte, fl; 9 - thrombocrit, fig. 4 - Micrographs of the scar zone in rats of the control group (Monklavit-1), fig. 5 - Micrographs of the scar zone in rats (gel with liposomes), fig. 6 - Healing of post-castration wounds in piglets, FIG. 7 - Dynamics of changes in the area of wounds in piglets of the experimental and control groups.

Evaluation of the effectiveness of a wound healing gel with liposomes The experiments were carried out in two stages, following the ethical principles of animal experiments in accordance with the provisions of the European Convention for the Protection of Vertebrate Animals used for experiments.

Stage 1. Model experiments were carried out on white rats in the vivarium of the Faculty of Veterinary Medicine of the Kursk State Agricultural Academy (body weight - 200-300 g). Animals for participation in the experiment were selected taking into account the general condition. The rats had an active behavior, smooth shiny coat and clean skin; there were no external signs of disease. Based on the selection results, the animals were randomized and the experimental and control groups were formed from them. In the control group, Monklavit-1, which is widely used in the clinic, was used, and in the experimental group, Liposomide gel, developed at the Department of Physiology and Chemistry, Faculty of Veterinary Medicine, Kursk State Agricultural Academy, was used.

For comparison, Monclavit-1 was chosen, which is an aqueous solution of iodine and potassium iodide in combination with a nonionic polymer polyvinylpyrrolidone (polyvidone), which has a prolonged antiseptic and disinfectant effect.

To create a model of a castration wound on the skin of the back in the interscapular region after depilation under anesthesia, a 1.5-2 cm incision was made.

The wound remained open throughout the experiment. To exclude wound licking, the rats were kept in individual cages. Application of drugs was carried out once.

To assess the effectiveness of the rate of regeneration, planimetric measurements of the wound were regularly carried out, taking into account the timing of the completion of epithelialization. At the end of the experiment, under chloral hydrate anesthesia (700 mg/kg intraperitoneally), a full-thickness area of skin with a scar was taken for histological analysis.

Blood for the study was taken from the animals at the end of the experiment. Hematological analysis was performed on a PCE90Vet analyzer in the regional veterinary laboratory.

Stage 2. Evaluation of the effectiveness of the gel with liposomes in the castration of piglets was carried out in JSC AF "Opening", Kursk region, Korenevsky district, with. Gracious.

Experiments were carried out on 4-day-old suckling piglets. The number of animals in the experimental and control groups - 5 heads.

The duration of the experiment is 10 days. All animals were kept in the same zoohygienic conditions.

Animals of the control group were treated with Monclavit-1 for the treatment of postoperative wounds, the piglets of the experimental group were treated with a wound healing gel with liposomes containing iodine compounds with polyvinyl alcohol. The drugs were applied once, immediately after the operation.

To assess the effectiveness of the rate of regeneration, planimetric measurements of the wound were regularly carried out, and the timing of the completion of epithelialization was taken into account. The results obtained were subjected to statistical processing using computer programs Microsoft Excel, ImageJ, Statistica. The significance of differences between the compared values was determined using Student's t-test. Significant differences were considered at P<0.05.

The results of experiments to evaluate the effectiveness of the gel Stage 1

The results of the study showed that the use of a wound healing gel with liposomes affects the dynamics of wound healing in rats (Fig. 1). Thus, complete healing of wounds in rats of the experimental group occurred on the 4th day. experiment, and in the control group - for 5 days.

In rats, when using the gel with liposomes, the content of leukocytes, lymphocytes, monocytes and platelets, granulocytes was significantly higher compared to the control group (Fig. 2, 4).

There were no significant differences in hematological parameters in rats by experimental groups (Fig. 3).

In the micrograph of the scar zone in rats of the control group (Fig. 4), the epithelium was completely regenerated. The underlying collagen fibers with a moderately pronounced lymphocytic infiltration, a few newly formed vertically located vessels are visualized.

On a micrograph of the scar zone in rats of the experimental group (Fig. 5). The epithelium is completely regenerated. The underlying layers of the dermis with slight lymphoid infiltration, newly formed vertically located vessels are visualized.

Thus, when using the gel, it is possible to ascertain the formation of a more complete scar than when using Monclavit-1. When using a gel with liposomes, the inflammatory reaction is less pronounced. The use of the gel with liposomes also led to an enhanced process of angiogenesis (the formation of new blood vessels). The components of the gel base - antiseptics of the Liposomed gel, when applied to wounds, had a disinfecting and antiseptic effect, like the components of the Monclavit-1 solution, however, liposomes with encapsulated potassium orotate, which has a regenerative effect on cells and tissues, could penetrate deeper into the damaged layers and activate regeneration processes throughout the depth of the wound, which was not observed when Monclavit was applied, since the skin is a protective organ of a living organism that prevents the penetration of foreign substances into the deeper layers of the skin and underlying tissues, and liposomes have the ability to penetrate into the cells and intercellular spaces of the skin. This is confirmed by the strengthening of the processes of formation of new vessels, which in turn improved the nutrition and healing of regenerating tissues. This proves the effectiveness of the wound healing gel.

Stage 2

The case, any complications were not revealed in any group of animals (Fig. 6).

Complete healing of wounds in piglets treated with Liposomid occurred on the 9th day after castration, and in those treated with Monclavit-1 - on the 11th day (Fig. 7).

From the graphs shown in figure 3, it can be seen that in piglets of the experimental group (Liposomide) the area of the postoperative wound was less than in the control (Monclavit -1).

The study of the effectiveness of a wound healing gel with liposomes containing iodine compounds with polyvinyl alcohol confirmed its effectiveness in laboratory animals (rats).

The study of the effectiveness of a wound healing gel with liposomes containing iodine compounds with polyvinyl alcohol in experiments on suckling pigs confirmed its effectiveness under production conditions.

The use of the wound healing gel with liposomes Liposomed, containing a compound of iodine with polyvinyl alcohol, led to more effective healing of post-castration wounds compared with the use of Monclavit-1 used in the control group. Purulent-inflammatory processes in both groups of animals were not found.

Thus, a wound healing gel for external use has been obtained, which has antimicrobial and anesthetic effects. It enhances tissue regeneration by enhancing the synthesis of pyrimidine nucleotides (uracil, thymine, cytosine), which are part of nucleic acids, and leads to increased protein synthesis in a rapidly regenerating tissue and, consequently, faster wound healing without complications, which is important in industrial conditions. production, for example, during the mass castration of piglets and other surgical interventions.

Claims (2)

1. A method for producing a wound healing gel with liposomes, including mixing the components of the gel base and adding liposomes to it, characterized in that 0.2 g of iodine, 0.6 g of potassium iodide, 1.8 g of polyvinyl alcohol are dissolved in 200 ml of distilled sterile water and 0.4 g of carbomer-940, the mixture is stirred on a magnetic stirrer until a homogeneous gel is obtained and liposomes prepared by dissolving 4.0 g of phosphatidylcholine in 50 ml of ethyl alcohol are added, adding 8 mg of α-tocopherol, 0.06 g of solid fat to the solution , 0.04 g of soybean oil and 0.02 g of linseed oil, by dissolving 10 g of potassium orotate in 50 ml of sterile distilled water, followed by injecting an alcoholic solution of lipids into an aqueous solution of potassium orotate 300-400 times with a BI-1 needleless injector to obtain liposomes with encapsulated potassium orotate, which are then separated from the solution by centrifugation for 1 hour at 15,000 rpm, are added to the gel base and the resulting mixture is stirred until a homogeneous state. 2. Wound healing gel with liposomes, obtained by the method according to claim 1, containing a gel base - iodine and potassium iodide in combination with polyvinyl alcohol, gelling agent carbomer-940, liposomes containing phosphatidylcholine, α-tocopherol, potassium orotate, solid fat, soybean oil beans and linseed oil.