RU2460532C1 - Preparation fastening wound healing - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine. The preparation represents a solution of low-molecular chitosans in hydrogel containing methyl cellulose (MC-100) and Nipagin. The preparation represents a dispersed suspension of copper nanoparticles, particle size 33.8÷103 nm; oxide film thickness 6÷10 nm and phase composition: crystalline copper 67-96% and copper oxide CuO - 4÷33%, or more oxidised copper nanoparticles having the following characteristics: particle size 77÷119 and phase composition: crystalline copper 0.5÷3.3%, CuO - 27.1÷90.0% and Cu₂O -9.05÷69.50 in hydrogel containing methyl cellulose (MC-100), Tween-80, Nipagin and the solution of solution of low-molecular chitosans, the suspension of chitosan nanoparticles.

EFFECT: invention provides higher wound healing effect and antimicrobial action.

15 cl, 9 tbl, 6 dwg, 10 ex

Description

The invention relates to medicine, in particular to drugs that accelerate wound healing.

The creation of modern wound healing agents is dictated by the high level of injuries in Russia, when every tenth resident of the country is injured every year. The level of victims increases sharply as a result of terrorist attacks, military operations and in the event of natural disasters. In any case, injuries, as a damaging factor, lead to impaired body functions. In addition, the high resistance of microorganisms infecting wounds complicates the healing process. Therefore, the development of new modern drugs with wound healing and antimicrobial effects is relevant.

Wound healing is a complex process, the course of which requires a balance of trace elements, antioxidants, matrix metalloproteinases and other factors. One of the metals, the deficit of which inhibits wound healing, is copper [13]. Recently, preparations based on highly dispersed metal powders have been used as agents for treating skin lesions. So, a bandage based on silver nanoparticles has been developed. Among the drugs of this class, popular, for example, in the USA, are the trademarks Acticoat, Nucryst. According to researchers, these drugs accelerate the healing of burns, wounds, eczema, acne [16, 17]. Domestic preparations based on highly dispersed metal compounds with a therapeutic effect include magnetic suppositories based on highly dispersed barium ferrite [4].

As analogues of the present invention, wound healing preparations with magnesium nanoparticles (NPs) and iron nanoparticles described in patents No. 2296571, No. 2306141 [5, 6] can be cited.

Interest in nano-objects is associated with an understanding of the difference between their physicochemical and biological properties, both from the properties of massive metal objects and the properties of individual atoms. Studies on the influence of metal nanoparticles on biosystems have revealed their unique properties [3, 7]:

1) metal nanoparticles are 7-50 times less toxic than metals in ionic form;

2) nanoparticles have a prolonged effect due to their ability to play the role of depot elements in the body;

3) nanoparticles introduced in biotic doses stimulate metabolic processes in the body;

4) nanoparticles have a multifunctional effect.

In the scientific literature there is much information about the prospects of using chitosans of various structures as wound healing agents [1, 2, 10, 11, 12, 14].

The dependence of the wound healing properties of chitosan, a non-toxic biodegradable polymer of natural origin, on its main characteristics — molecular weight (M. m.) And degree of deacetylation (DM), was established by us earlier [1, 2].

Chitosan nanoparticles (NPs) also have biological activity and are able to influence physiological processes in the body. So, it has been shown that chitosan NPs have the ability to stimulate the proliferation of lymphocytes and, thus, have a positive effect on wound healing [15].

The biological effects of chitosan NPs are not unambiguous. It is known, for example, that some chitosan NPs exhibit a high cytotoxic effect on various tumor cell cultures in in vitro experiments [14].

It is also known that chitosan binds ions of various metals and, in particular, has a high affinity for copper ions. In this case, a change in the properties of chitosan is observed [14].

Therefore, the use of nanoparticles of various chemical nature in the composition of wound healing agents seems to be a promising area of pharmacy and medicine.

The objective of the invention is the creation of modern wound healing preparations of multifunctional action, providing a high level of wound healing and having an antimicrobial effect.

The problem is solved in that pharmaceutical compositions have been developed that accelerate wound healing and have antimicrobial activity based on copper nanoparticles. For the preparation of drugs that accelerate wound healing, copper nanoparticles were used in the concentration range,%: 2 × 10 ^-5 -2 × 10 ^-2 .

Preferably, copper-containing nanoparticles have the following characteristics: copper nanoparticles have a particle size of 33.8 ÷ 103 nm; the thickness of the oxide film is 6 ÷ 10 nm, the phase composition: crystalline copper 67 ÷ 96%, copper oxide CuO - 4 ÷ 33%; the more oxidized copper nanoparticles have a particle size of 77 ÷ 124 nm, phase composition: crystalline copper 0.5 ÷ 3.3 ÷ 23%, CuO - 27.1 ÷ 90% and Cu ₂ O - 9.05 ÷ 69.5%.

The invention also relates to combination preparations, which further comprise chitosan derivatives, which are derivatives of low molecular weight chitosans: O-sulfochitosan and N-sulfosuccinoyl-N-carboxymethylchitosan, or chitosan nanoparticles.

Methylcellulose (MTs-100), as well as Tween-80, glycerol are preferably used as the basis for the developed preparations; nipagin is more preferably used as a preservative. Preferably, the claimed preparations are ointments of the following composition (in g):

1) copper nanoparticles - 0.00002-0.002; liquid paraffin - 10.0-15.0; methyl cellulose (MTs-100) - 3.0-4.0; Twin-80 - 2.0-3.0; nipagin - 0.1-0.3; purified water to 100.0;

2) copper nanoparticles - 0.00002-0.002; chitosan - 0.005-0.05; liquid paraffin - 10.0-15.0; methyl cellulose (MTs-100) - 3.0-4.0; Twin-80 - 2.0-3.0; nipagin - 0.1-0.3; purified water to 100.0;

3) copper nanoparticles - 0.00002-0.002; chitosan nanoparticles - 0.01-0.0002, liquid paraffin - 10.0-15.0; methyl cellulose (MC - 100) - 3.0-4.0; Twin-80 - 2.0-3.0; nipagin - 0.1-0.3; purified water to 100.0.

In another embodiment of the invention, there is provided a pharmaceutical composition accelerating wound healing and comprising an active substance and a base, which as an active substance contains a chitosan derivative, which is selected from the group consisting of low molecular weight derivatives of chitosan or chitosan nanoparticles. Derivatives of low molecular weight chitosans - O-sulfochitosan and N-sulfosuccinoyl-N-carboxymethylchitosan - were synthesized by us from the initial chitosan SD 89% and M.m. 56 kDa and 200 kDa. Chitosan nanoparticles synthesized from M. chitosan 10 kDa, SD 89%. The size of chitosan nanoparticles is 60-106 nm. Chitosan nanoparticles have a zeta potential of 38-42 mV.

In the most preferred embodiments of the invention, the preparations have the following composition, g:

I.

Copper nanoparticles 0.00002-0.002 Vaseline oil 10.0-15.0 MTs-100 3.0-4.0 Twin 80 2.0-3.0 Nipagin 0.1-0.3 Purified water up to 100.0

II.

Derivatives of low molecular weight chitosans 0.005-0.05 MTs-100 3.0-4.0 Nipagin 0.1-0.3 Purified water up to 100.0

III.

Chitosan Nanoparticles 0.01-0.0002 MTs-100 3.0-4.0 Nipagin 0.1-0.3 Purified water up to 100.0

Combined ointments:

IV.

Copper nanoparticles 0.00002-0.002 Derivatives of low molecular weight chitosans 0.005-0.05 Vaseline oil 10.0-15.0 MTs-100 3.0-4.0 Twin 80 2.0-3.0 Nipagin 0.1-0.3 Purified water up to 100.0

V.

Copper nanoparticles 0.00002-0.002 Chitosan Nanoparticles 0.01-0.0002 Vaseline oil 10.0-15.0 MTs-100 3.0-4.0 Twin 80 2.0-3.0 Nipagin 0.1-0.3 Purified water up to 100.0

The following diagrams illustrate methods for implementing the most preferred embodiments of the invention.

I. Composition

Stage 1. Preparation of MC gel

Preparation of the gel on the MC-100.

Nipagin was dissolved in water with heating (80-90 ° C). Then, MC-100 was added to the resulting warm solution (60 ° C) and left to swell for 1 hour.

Step 2. Preparation of a Vaseline Suspension of Copper Nanoparticles

An exact sample of copper nanopowder was placed in liquid paraffin and dispersed on an ultrasonic disperser UZDN-A in 44 kHz, 0.5A mode according to the following scheme: 20 seconds of sounding - 2 minutes break (3 cycles).

Stage 3. Introduction of copper nanoparticles into the base

To the prepared MC gel obtained in the first stage, tween-80 was added and emulsified for 10-15 minutes on a MLW mixer (model ER-10) at a speed of 300 rpm. An equal amount of the obtained gel was added to the vaseline suspension of copper nanoparticles and mixed with an MLW mixer (model ER-10) at a speed of 300 rpm, making sure that no air bubbles appeared in the resulting mass. Then the remaining amount of gel was gradually added, each time thoroughly mixing.

II. Structure

Stage 1. Preparation of MC gel

Nipagin was dissolved in 2/3 of the calculated volume of water purified by heating (80-90 ° C). In the resulting warm solution (60 ° C), MC-100 was added and allowed to swell for 1 hour. In the remaining volume of water (1/3), a weighed portion of derivatives of low molecular weight chitosans was dissolved.

Stage 2. The introduction of low molecular weight chitosans

The resulting solution of low molecular weight chitosans was added to the MC gel and thoroughly mixed using an MLW mixer (model ER-10) at a speed of 300 rpm, avoiding the appearance of air bubbles.

III. Structure

Step 1. Gel Preparation

Nipagin was dissolved in 2/3 of the calculated volume of water purified by heating (80-90 ° C), then MC-100 was added to the resulting warm solution (60 ° C) and allowed to swell for 1 hour.

Stage 2. The introduction of chitosan nanoparticles

In the remaining volume of water (1/3), an aqueous suspension of chitosan nanoparticles was added, added to the prepared gel and gently mixed using an MLW mixer (model ER-10) at a speed of 300 rpm, avoiding the appearance of air bubbles.

IV. Structure

Stage 1. Preparation of MC gel

Nipagin was dissolved in 2/3 of the calculated volume of water purified by heating (80-90 ° C). In the resulting warm solution (60 ° C), MC-100 was added and allowed to swell for 1 hour. In the remaining volume of water (1/3), a weighed portion of derivatives of low molecular weight chitosans was dissolved.

Stage 2. The introduction of low molecular weight chitosans

The resulting solution of low molecular weight chitosans was added to the MC gel and thoroughly mixed using an MLW mixer (model ER-10) at a speed of 300 rpm, avoiding the appearance of air bubbles.

Step 3. Preparation of a Vaseline Suspension of Copper Nanoparticles

An exact sample of copper nanopowder was placed in liquid paraffin and dispersed on an ultrasonic disperser UZDN-A in 44 kHz, 0.5A mode according to the following scheme: 20 seconds of sounding - 2 minutes break (3 cycles).

Stage 4. The introduction of a vaseline suspension of copper nanoparticles

The gel obtained in the second stage was added to the vaseline suspension of copper nanoparticles and mixed using an MLW mixer (model ER-10) at a speed of 300 rpm, making sure that no air bubbles appeared in the resulting mass.

V. Composition

Step 1. Gel Preparation

Nipagin was dissolved in 2/3 of the calculated volume of water purified by heating (80-90 ° C). Then, MC-100 was added to the resulting warm solution (60 ° C) and left to swell for 1 hour.

Stage 2. The introduction of chitosan nanoparticles

An aqueous suspension of chitosan nanoparticles was introduced into the remaining volume of water (1/3), added to the prepared gel and carefully mixed using an MLW mixer (model ER-10) at a speed of 300 rpm, avoiding the appearance of air bubbles.

Step 3. Preparation of a Vaseline Suspension of Copper Nanoparticles

An exact sample of copper nanopowder was placed in liquid paraffin and dispersed on an ultrasonic disperser UZDN-A in 44 kHz, 0.5A mode according to the following scheme: 20 seconds of sounding - 2 minutes break (3 cycles).

Stage 4. Introduction of copper nanoparticles into the base

The gel obtained in the second stage was added to the vaseline suspension of copper nanoparticles and mixed using an MLW mixer (model ER-10) at a speed of 300 rpm, making sure that no air bubbles appeared in the resulting mass.

For composition I, IV, V, the resulting product is a white ointment, uniform in composition, without mechanical impurities, with a very weak specific odor.

For composition II, III, the resulting product is a colorless, transparent gel, uniform in composition, without mechanical impurities, odorless. Derivatives of low molecular weight chitosans - O-sulfochitosan and N-sulfosuccinoyl-N-carboxymethylchitosan - were synthesized by us from the initial chitosan SD 89% and M.m. 56 kDa and 200 kDa. Chitosan nanoparticles synthesized from M. chitosan 10 kDa, SD 89%. The size of chitosan nanoparticles is 60-106 nm. Chitosan nanoparticles have a zeta potential of 38-42 mV.

I. Physico-chemical characteristics of copper nanoparticles

Nanoparticles of one of the copper samples, which differ in size, phase composition, and oxide film thickness on the surface of the nanoparticles, were introduced into the ointment (table 1). Figure 1 (A and B) presents photographs of copper nanoparticles of sample No. 2, obtained by electron scanning microscopy (A) and transmission electron microscopy (B). It is seen that the copper nanoparticles of sample No. 2 are single-crystal structures of a circular regular shape, covered with a translucent film of copper oxide (B).

The main physicochemical characteristics of copper nanoparticles are presented in table 1 [8].

According to the particle size distribution diagrams and frequency accumulation curves, it was found that the average size of copper nanoparticles is in the range from 33.8 nm (sample No. 6) to 103.8 nm (sample No. 2). The size of copper nanoparticles of sample No. 3 is 77.3 nm, and of sample No. 7 is 119.0 nm. Copper nanoparticles differ in phase composition. So, the content of crystalline copper in the core of copper nanoparticles of sample No. 6 is 67%, samples No. 5 and No. 2 94% and 96%, respectively.

In copper nanoparticles of copper samples No. 3 and No. 7, the content of crystalline copper is small and is 3.3% and 0.5%, respectively.

The thickness of the oxide film on the surface of copper nanoparticles varies between 6-10 nm. When storing nanoparticles in a sealed container filled with an inert gas, the size of the oxide film remains almost constant. When stored under normal conditions, i.e. in the absence of an inert gas, the degree of oxidation of the particles changes. Therefore, usually before the preparation of ointments, a repeated determination of the degree of oxidation of the nanoparticles was carried out.

The composition of the oxide film on the surface of copper nanoparticles of samples No. 1, No. 2, No. 4, No. 5, No. 6 is the same and is represented by CuO. At the same time, the composition of the oxide film of copper nanoparticles of samples No. 3 and No. 7, in addition to Cu (II) oxide, includes Cu (I) oxide.

Therefore, copper nanoparticles differ in size, phase composition, and oxide film thickness.

II. Wound healing activity

II.1. Wound healing effect of copper nanoparticles with different physicochemical characteristics in the composition of the ointment

Ointments developed by us with copper nanoparticles having the physicochemical characteristics described in Table 1 were daily applied to the surface of full-layer wounds with an initial area of 60 mm ² in an amount of 0.2 g. Factory ointments Solcoseryl and Levomekol were used as a reference preparation, which applied to wounds in the same way, in the same amount and according to the same treatment regimen. In all experimental variants, the change in the area of the wounds was recorded daily and the rate of wound tension was calculated from the kinetic curves of the changes in the area of the wounds and the time for complete healing of the wounds was estimated. The effect of the preparations was studied on female white mice weighing 18–20 g of the SHK line, which are on the general feeding diet [1]. Experimental animal studies were carried out in accordance with the guidelines recommended by our institute, 1987 and The Guide for the Care and Use of Laboratory Animals (National Academy Press Washington, DC 1996).

Example 1. The effect of ointments with copper nanoparticles of sample No. 5 and sample No. 7 on the kinetics of wound healing

Table 2 presents data on the change in the area of wounds during their healing: untreated animals (control 1), treated with an ointment base without copper nanoparticles (control 2), treated with ointments with copper nanoparticles of sample No. 5, sample No. 7, and also treated with factory ointments “Solcoseryl” and “Levomekol” taken as a standard of comparison.

A study of wound healing showed that experimental full-layer wounds in two control groups and groups treated with ointments-standards “Solcoseryl” and “Levomekol”, 14 days after wounding, had an area of 0.3-1.6% of the initial area of the wound. At the same time, when treating full-layer wounds with ointments with copper nanoparticles, the wounds completely heal 14 days after their application.

Therefore, according to the effect on the time of complete healing, the ointments developed by us have an advantage in comparison with the standard ointments “Solcoseryl” and “Levomekol”.

Figure 2 presents the kinetic curves of the change in the area of experimental full-layer wounds under the action of an ointment base without nanoparticles (curve 1), under the action of an ointment with copper nanoparticles of sample No. 7 (curve 2). The kinetics of changes in the area of wounds during treatment with ointments with copper nanoparticles indicates the effective effect of ointments on the course of the wound process.

Example 2. The effect of ointments with copper nanoparticles on the specific speed of the primary tension of experimental full-layer wounds

To assess the effectiveness of the ointments we developed with copper nanoparticles on the course of the wound healing process, we calculated the specific rates of primary wound tension: [Vsp = (So-S ₁ ) / t], where So is the area of the initial wound, S ₁ is the area of the wound in 24 hours after surgery, t is the time between area measurements (in days). The data on the change in the speed of primary wound tension during treatment with ointments with copper nanoparticles are presented in table 3. The results of the study show that ointments containing copper nanoparticles of sample No. 5 and sample No. 7 have different effects on the specific speed of wound tension. Ointments with copper nanoparticles of sample No. 5 and sample No. 7 increase the initial wound tension rate by 23.1 and 31.4%, respectively, compared to the initial wound tension rate of untreated animals and 2.0 times compared to the wound tension rate of animals treated with ointment base. Ointments with copper nanoparticles of sample No. 1 and sample No. 3 do not change the rate of specific wound tension compared to untreated animals and increase the specific rate of wound tension by 57% compared to the rate of wound tension of animals treated with ointment base. The ointment standard Solkoseril practically does not change the specific wound tension rate compared to untreated animals, the ointment standard Levomekol accelerates wound healing by 19.2%. Therefore, ointments with copper nanoparticles have an advantage over the effectiveness of the effect on the speed of the initial wound tension compared with the standard ointments Solkoseril and Levomekol.

Example 3. The effect of ointments on the kinetics of changes in the area of wounds in the healing process, depending on the concentration of copper nanoparticles of sample No. 2 in the preparation

To establish the concentration dependence of the effect of copper nanoparticles in the composition of the ointment on the kinetics of wound healing, we used copper nanoparticles of sample No. 2 in the following concentrations, in%: 2 × 10 ^-5 , 2 × 10 ^-4 , 2 × 10 ^-3 . The research results are presented in table 4. The results show that ointments with copper nanoparticles of sample No. 2 in the studied dose range have wound healing activity, accelerating wound healing for almost the entire period of wound healing. Comparison of the effectiveness of the ointments we developed with ointments-standards for changing the area of wounds during the healing process showed that when treating with ointments with copper nanoparticles, complete wound healing is observed after 14 days, while when treating with factory ointment Solcoseryl and factory ointment Levomekol the sizes of unhealed wounds after 14 days are 1.6 and 0.7% (compared with the initial wounds), respectively. It should also be noted that in the studied concentration range, an ointment with a lower concentration of nanoparticles has the greatest effect.

Example 4. The specific speed of the primary tension of the experimental full-layer wounds in the treatment of ointments with copper nanoparticles of sample No. 2 in different concentrations

Taking into account the effective effect of the ointments developed by us with copper nanoparticles on the course of the wound healing process, we calculated the specific rate of primary wound tension during treatment with ointments containing copper nanoparticles in different concentrations. The results are presented in table 5. We found that at concentrations of copper nanoparticles of sample No. 2 in the composition of the ointment, in%: 2 × 10 ^-5 , 2 × 10 ^-4 , 2 × 10 ^-3 , an increase in the rate of primary wound tension is observed by 55.1% , by 20.1%, by 15.6% compared with the control (untreated animals), respectively, and by 146.5%, 90.9% and 83.6% compared with the rate of wound tension in animals treated with ointment base. When comparing the effectiveness of the action of copper nanoparticles with ointments-standards “Solcoseryl” and “Levomekol” it was found that ointment “Levomekol” accelerates wound healing by 19.2% compared with untreated control, and ointment “Solkoseril” reduces the specific speed of primary tension of wounds by 4.1% .

Therefore, ointments with copper nanoparticles of sample No. 2 in concentrations, in%: 2 × 10 ^-5 , 2 × 10 ^-4 , 2 × 10 ^-3 increase the speed of primary wound tension. In this case, an ointment with copper nanoparticles in a concentration of 2 × 10 ^-5 % shows a higher wound healing activity than ointments with copper nanoparticles in concentrations, in%: 2 × 10 ^-4 and 2 × 10 ^-3 .

II.2. Wound-healing combined effect of copper nanoparticles with different physicochemical characteristics and derivatives of low molecular weight chitosans, as well as with chitosan nanoparticles in the composition of the ointment

Our combined ointments with copper nanoparticles of samples No. 1 and No. 2, having the physicochemical characteristics described in Table 1, with derivatives of low molecular weight chitosans, as well as with chitosan nanoparticles, were daily applied to the surface of full-layer wounds with an initial area of 60 mm ² in an amount of 0.2 d. Factory ointments: Solcoseryl and Levomekol, which were applied to wounds in the same way, in the same amount and according to the same treatment regimen as the combined ointments we developed, were used as a reference preparation. and. In all experimental variants, the change in the area of the wounds was recorded daily and the rate of wound tension was calculated from the kinetic curves of the changes in the area of the wounds and the time for complete healing of the wounds was estimated. The effect of the preparations was studied on female white mice weighing 18–20 g of the SHK line, which are on the general feeding diet [1].

Example 5. The effect of ointments with derivatives of low molecular weight chitosans on the specific speed of the primary tension of full-layer experimental wounds

For the study, we selected derivatives of low molecular weight chitosans: O-sulfochitosan and N-sulfosuccinoyl-N-carboxymethylchitosan, synthesized by us from the initial chitosan SD 89% M. m. 56 kDa and 200 kDa. The research results are presented in table 6.

O-sulfochitosan in the composition of the ointment at a concentration of 0.05% reduces the specific rate of primary wound tension by 8.2% compared with the control (untreated animals) and increases the speed by 45.8% compared with the results of treating animals with an ointment base.

N-sulfosuccinoyl-N-carboxymethylchitosan in the composition of the ointment at a concentration of 0.05% increases the specific healing rate by 17.6% compared with the control (untreated animals) and significantly increases the specific healing rate by 86.9% compared with animals treated with ointment base.

N-sulfosuccinoyl-N-carboxymethylchitosan in the composition of the ointment at a concentration of 0.005% promotes an increase in the specific rate of wound healing by 69.8% compared with animals treated with ointment base, and by 6.9% compared with control (untreated animals).

Example 6. The effect of ointments with chitosan nanoparticles on the specific rate of primary tension of experimental full-layer wounds

Table 7 presents data on the effect of chitosan nanoparticles in the composition of the ointment on the specific speed of the primary tension of experimental full-layer wounds. Chitosan nanoparticles synthesized from low molecular weight chitosan M.m. 200 kDa introduced into the ointment in concentrations, in%: 0.01 and 0.002, as well as chitosan nanoparticles synthesized from low molecular weight chitosan M.m. 10 kDa, introduced into the ointment in concentrations, in%: 0.002 and 0.0002.

It is seen that in the treatment of wounds with ointment with chitosan nanoparticles, the specific rate of wound healing changes. Ointment treatment with chitosan nanoparticles synthesized from chitosan M.m. 200 kDa, at a concentration of 0.01% in the composition of the ointment, significantly increases the specific rate of wound healing by 25.2% compared with the control (untreated animals) and by 98.9% compared with animals treated with ointment base. A decrease in the concentration of chitosan nanoparticles in the ointment to a concentration of 0.002% slows down wound healing, reducing the specific rate of wound healing by 39.7% compared with control (untreated animals) and 2.5% compared with animals treated with ointment base. Ointment with chitosan nanoparticles synthesized from chitosan M.m. 10 kDa, at a concentration of 0.002%, increases the specific healing rate by 39.4% compared with the control (untreated animals) and a significant increase in speed by 2.2 times compared with animals treated with ointment base. Wound treatment with ointment with chitosan nanoparticles synthesized from chitosan M.m. 10 kDa, at a concentration of 0.0002% in the composition of the ointment, leads to a decrease in the specific rate of wound healing by 22.4% compared with the control (untreated animals) and an increase in the specific rate of wound healing by 23.3% compared with animals treated with an ointment base.

Example 7. The effect of a combined ointment with copper nanoparticles and derivatives of low molecular weight chitosans on the specific speed of the primary tension of experimental full-layer wounds

Considering the wound healing effect of ointments containing modified copper nanoparticles, ointments containing chitosan derivatives, we developed ointments of combined composition, including both copper nanoparticles and derivatives of low molecular weight chitosans. The results of studies on the effect of combined ointments on the specific speed of the initial tension of the wounds are presented in FIG.4 and FIG.5.

Figure 4 presents data on the change in the specific rate of wound healing during treatment with a combination ointment containing copper nanoparticles of sample No. 1 at a concentration of 0.002% and a derivative of low molecular weight chitosans - O-sulfochitosan at a concentration of 0.05%. Combined ointment treatment leads to a significant increase in the specific rate of wound healing by 1.5 times compared with the group treated with ointment with O-sulfochitosan, 1.4 times compared with the group treated with ointment with copper nanoparticles of sample No. 1 and 1.4 times compared with the control group (untreated animals)

In the treatment with a combined ointment containing copper nanoparticles of sample No. 1 at a concentration of 0.002% and a derivative of low molecular weight chitosans - O-sulfochitosan at a concentration of 0.05%, the wound healing effect is enhanced due to the synergistic action of the components of an organic (O-sulfochitosan) and inorganic (copper nanoparticles) nature , contributing to an increase in the specific rate of wound healing.

Figure 5 presents data on the change in the specific rate of wound healing during treatment with a combination ointment containing copper nanoparticles of sample No. 2 at a concentration of 0.002% and a derivative of low molecular weight chitosans - N-sulfosuccinoyl-N-carboxymethylchitosan at a concentration of 0.05%. Treatment with a combined ointment leads to an increase in the specific rate of wound healing by 26% compared with an ointment containing N-sulfosuccinoyl-N-carboxymethylchitosan at a concentration of 0.05%, by 16.5% compared with an ointment containing copper nanoparticles of sample No. 2 and by 48.5% compared with a control group (untreated animals).

In the treatment with a combined ointment containing copper nanoparticles of sample No. 2 at a concentration of 0.002% and a derivative of low molecular weight chitosans - N-sulfosuccinoyl-N-carboxymethylchitosan, the wound healing effect is enhanced due to the synergistic action of the components of organic (N-sulfosuccinoyl-N-carboxymethylchitosan) and inorganic copper nanoparticles) of nature, contributing to an increase in the rate of wound healing.

Considering the wound healing effect of ointments containing modified copper nanoparticles, ointments containing chitosan nanoparticles, we developed ointments of a combined composition, including both copper nanoparticles and low molecular weight chitosans nanoparticles. The results of studies on the effect of combined ointments on the specific speed of the primary wound tension are presented in FIG. 6.

Figure 6 presents data on the change in the specific rate of wound healing during treatment with a combination ointment containing copper nanoparticles of sample No. 2 at a concentration of 0.002% and nanoparticles of chitosans synthesized from chitosan SD 89% and M.m. 10 kDa, at a concentration of 0.0002% in the composition of the ointment. Combined ointment treatment increases the specific wound healing rate by 10.5% compared to the group treated with ointment with copper nanoparticles of sample No. 2, increases the specific wound healing rate by 81.7% compared to the group treated with ointment with chitosan nanoparticles, and by 40.9% compared to the control group (untreated animals).

When treating with a combined ointment containing chitosan NPs and copper NPs of sample No. 2, the wound healing effect is enhanced due to the synergistic action of the components of the organic (chitosan nanoparticles) and inorganic (copper nanoparticles) nature, contributing to an increase in the rate of wound healing.

III. Antimicrobial activity of ointments with copper nanoparticles

Medium suitable for growing test microbes is poured into sterile Petri dishes of the same diameter with an even flat bottom: Staphylococcus albus (St. albus) and Escherichia coli AB 1157 (E. coli AB1157). The composition of the growth medium of microorganisms (in g): peptone - 10, yeast extract - 5, sodium chloride - 5, agar-agar - 20 in 1 liter of distilled water. After solidification of the agar-agar in a Petri dish, a test culture of microorganisms is seeded. Sowing is carried out evenly and equally in all cups from a previously prepared suspension. Sowing density should be sufficient for continuous lawn growth. The cups are ground with a sterile spatula. Samples of copper nanoparticles with different physicochemical characteristics were placed on paper glycerin disks in an amount of 5 mg / disk, a reference standard for samples of streptomycin nanopowders in an amount of 100 μg / disk. Ointments developed by us with copper nanoparticles and ointments-standards: Solcoseryl and Levomekol, were placed on paper disks in the amount of 50 mg / disk. Disks were laid on the surface of the agar in pre-prepared Petri dishes, seeded with test cultures. Next, Petri dishes for a day were placed in a thermostat at a temperature of 37 ° C. After a day, the cups were removed from the thermostat and photographed. If the test sample affects the growth of the test microbe, a cell growth inhibition zone is formed. The intensity of the influence of the test sample on test microbes was estimated by the area of the growth inhibition zone (mm ² ) [9].

Example 9. Evaluation of the antimicrobial activity of copper nanoparticles by the disk diffusion method

Samples of copper nanoparticles with different physicochemical characteristics at a concentration of 5 mg / disk were placed on a disk, the reference standard was streptomycin was placed on a disk at a concentration of 100 μg. The discs were laid on agar petri dishes, seeded with test cultures: St. albus and E. coli AB 1157. The antimicrobial activity of copper nanoparticles and reference standards - streptomycin was evaluated by the size of the zones of growth inhibition of bacteria.

The results of the study are shown in table 8. We found that samples of copper nanoparticles have antibacterial activity against test culture cells of both gram-positive and gram-negative microorganisms. However, there are differences in the effectiveness of nanoparticles. It is seen that the areas of the zones of growth inhibition of bacteria are more pronounced under the action of copper nanoparticles in relation to the cells of the test culture St. albus no in comparison with E. coli AB1157 cells. Moreover, the nanoparticles of copper samples have different effects on bacteria: the greatest antimicrobial effect on the cells of the test culture St. albus have copper nanoparticles of samples No. 5 and No. 6, and on the cells of the test culture of E. coli AB1157 - copper nanoparticles of sample No. 4. The least pronounced antibacterial effect on the cells of the test culture St. albus possess copper nanoparticles of samples No. 1 and No. 2, and in relation to cells of the test culture of E. coli AB1157 - copper nanoparticles of sample No. 1. Nanoparticles of samples No. 3 and No. 7 have the same antimicrobial effect in relation to cells of test cultures. In these studies, the classic antibiotic streptomycin at a concentration of 100 μg / disk was used as a reference standard. The antimicrobial activity of the antibiotic at this concentration is comparable to the antimicrobial effect of copper nanoparticles.

Therefore, copper nanoparticles (7 samples), regardless of their size and structure, have an antimicrobial effect, slightly differing from each other in efficiency.

Example 10. Antimicrobial effect of ointments with copper nanoparticles of sample No. 2, ointments with copper nanoparticles of sample No. 1, ointments with O-sulfochitosan, ointments with chitosan nanoparticles, combined ointments with copper nanoparticles of sample No. 2 and O-sulfochitosan, combined ointment with copper nanoparticles sample No. 2 and nanoparticles of chitosan, factory ointments-standards of comparison "Solcoseryl" and "Levomekol" in terms of areas of growth inhibition zones of test cultures St. albus and E. coli AB1157

Ointments developed by us with copper nanoparticles of sample No. 2 in concentrations,%: 2 × 10 ^-2 , 2 × 10 ^-3 , 2 × 10 ^-4 , 2 × 10 ^-5 , ointment with copper nanoparticles of sample No. 1 at a concentration of 2 × 10 ^-3 %, ointment with O-sulfochitosan at a concentration of 0.05%, ointment with chitosan nanoparticles at a concentration of 0.0002%, combined ointment with copper nanoparticles of sample No. 2 at a concentration of 2 × 10 ^-3 % and with O-sulfochitosan at a concentration of 0.05%, a combined ointment with copper nanoparticles of sample No. 2 at a concentration of 2 × 10 ^-3 % and with chitosan nanoparticles at a concentration of 0.0002% was applied to disks in an amount of 0.2 g / disk. The discs were placed on agar petri dishes seeded with test cultures: St. albus and E. coli AB 1157. The antimicrobial activity of the developed ointments and factory ointments-standards “Solcoseryl”, “Levomekol” was evaluated by the size of the zone of growth inhibition of bacteria.

The results are shown in table 9 and FIG. 3. Our studies showed that the ointments developed by us with copper nanoparticles of sample No. 2, in concentrations,%: 2 × 10 ^-2 , 2 × 10 ^-3 , 2 × 10 ^-4 , 2 × 10 ^-5 , ointment with copper nanoparticles of sample No. 1 at a concentration of 2 × 10 ^-3 %, ointment with O-sulfochitosan at a concentration of 0.05%, ointment with chitosan nanoparticles at a concentration of 0.0002%, combined ointment with copper nanoparticles of sample No. 2 at a concentration of 2 × 10 ^-3 % and O-sulfochitosan at a concentration of 0.05%, a combined ointment with copper nanoparticles of sample No. 2 at a concentration of 2 × 10 ^-3 % and chitosan nanoparticles at a concentration of 0.0002% possess antimicrobial action, as evidenced by the formation of zones of growth inhibition of cells of the test culture St. albus ranging from 34.2 mm ² (ointment with O-sulfochitosan at a concentration of 0.05%) to 88.6 mm ² (combined ointment with copper nanoparticles of sample No. 2 at a concentration of 2 × 10 ^-3 % and O-sulfochitosan at a concentration of 0.05%) and delay zones growth of E. coli AB1157 test culture cells ranging in size from 22.3 mm ² (ointment with O-sulfochitosan at a concentration of 0.05%) to 29.1 mm ² (ointment with copper nanoparticles at a concentration of 2 × 10 ^-2 %). Comparison of the antimicrobial action of the ointments we developed with the action of factory ointments-standards showed that the growth retardation zones of St. test cells Albus and E. coli AB1157 when applied to discs of factory ointments-standards of comparison of Solcoseryl and Levomekol above.

Therefore, ointments of various compositions that we developed, containing copper nanoparticles, derivatives of low molecular weight chitosans and nanoparticles thereof, as well as combined ointments, have antimicrobial activity against test culture cells of gram-positive and gram-negative microorganisms.

Table 8 Area (mm ² ) of zones of growth inhibition of cells of test cultures St. albus and E. coli AB1157 when exposed to copper nanoparticles at a concentration of 5 mg / disk, streptomycin 100 μg / disk Copper LF Sample Growth Retardation Area albus, mm ² The zone of growth retardation E. coli AB1157, mm ² Sample No. 1 1004 ± 42 805 ± 57 Sample No. 2 1014 ± 72 857 ± 15 Sample No. 3 1001 ± 88 868 ± 33 Sample No. 4 1054 ± 79 940 ± 44 Sample No. 5 1210 ± 32 839 ± 10 Sample No. 6 1207 ± 40 835 ± 60 Sample No. 7 1103 ± 59 868 ± 25 Reference standard - streptomycin 794 ± 16 361 ± 14

Table 9 Area (mm ² ) of zones of growth inhibition of cells of test cultures St. albus and E. coli AB 1157 under the action of ointments with copper nanoparticles of sample No. 2 in concentrations,%: 2 × 10 ^-2 , 2 × 10 ^-3 , 2 × 10 ^-4 , 2 × 10 ^-5 , with copper nanoparticles of the sample No. 1 at a concentration of 2 × 10 ^-3 %, with O-sulfochitosan at a concentration of 0.05%, with NP of chitosan at a concentration of 0.0002%, a combined ointment with copper NPs of sample No. 2 at a concentration of 2 × 10 ^-3 %, and with O-sulfochitosan at concentration of 0.05%, a combined ointment with copper NPs of sample No. 2 at a concentration of 2 × 10 ^-3 %, and with chitosan NPs at a concentration of 0.0002%, and factory ointments-reference standards (Solcoseryl, Levomekol) No. Samples Area of growth inhibition zone (mm ² ) Test culture St. albus E. coli AB 1157 one Ointment with copper NPs of sample No. 2 at a concentration of 2 × 10 ^-2 % 82.4 ± 3.4 29.1 ± 2.9 2 Ointment with copper NPs of sample No. 2 at a concentration of 2 × 10 ^-3 % 72.8 ± 19.8 28.5 ± 0.9 3 Ointment with copper NPs of sample No. 2 at a concentration of 2 × 10 ^-4 % 71.4 ± 6.7 26.7 ± 1.2 four Ointment with copper NPs of sample No. 2 at a concentration of 2 × 10 ^-5 % 74.7 ± 9.4 27.2 ± 4.0 5 Ointment with copper NPs of sample No. 1 at a concentration of 2 × 10 ^-3 % 82.3 ± 16.1 23.4 ± 1.6 6 Ointment with O-sulfochitosan at a concentration of 0.05% 34.2 ± 10.1 22.3 ± 10.1 7 Ointment with NP chitosan at a concentration of 0.0002% 37.6 ± 4.8 25.9 ± 1.6 8 Combined ointment with copper NPs of sample No. 2 at a concentration of 2 × 0 ^-3 % and with O-sulfochitosan at a concentration of 0.05% 88.6 ± 14.5 24.3 ± 4.1 9 Combined ointment with copper NPs of sample No. 2 at a concentration of 2 × 10 ^-3 % and with NP chitosan at a concentration of 0.0002% 81.4 ± 25.5 26.6 ± 4.5 10 Comparison standard - Solcoseryl ointment 120.5 ± 5.2 180.0 ± 44.6 eleven The standard of comparison - ointment "Levomekol" 648.5 ± 42.3 747.3 ± 65.3

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. (A): (SEM) * image of copper nanoparticles of sample No. 2,

(B): (TEM) ** image of copper nanoparticles of sample No. 2.

* (SEM) - scanning electron microscopy,

** (TEM) - transmission electron microscopy.

Figure 1 presents photographs of copper nanoparticles of sample No. 2, obtained by scanning electron microscopy (A) and transmission electron microscopy (B). It is seen that the copper nanoparticles of sample No. 2 are single crystal structures of circular regular shape (A), coated with a translucent film of copper oxide (B).

FIG. 2. Kinetic curves of changes in the area of experimental full-layer wounds under the action of an ointment base without nanoparticles (curve 1), under the action of an ointment with copper nanoparticles of sample No. 7 (curve 2).

Figure 2 presents the kinetic curves of changes in the area of experimental full-layer wounds under the action of an ointment base without nanoparticles (curve 1) and under the action of an ointment with copper nanoparticles of sample No. 7 (curve 2). It is seen that the kinetics of changes in the area of wounds during treatment with ointments with copper nanoparticles indicates the effective effect of ointments on the course of the wound process.

FIG. 3. Photos of Petri dishes in the study of the antimicrobial activity of copper nanoparticles (sample No. 2), reference standards - ointments "Solcoseryl" and "Levomekol" disk diffusion method.

A-B Ointment with copper nanoparticles of sample No. 2 in concentrations,%: 2 × 10 ^-3 (disk No. 1), 0 (disk No. 2), 2 × 10 ^-4 (disk No. 3), 2 × 10 ^-5 (disk number 4);

С-D Reference standard - factory ointment "Solcoseryl";

E-F Reference standard - factory ointment "Levomekol".

FIG. 3 shows photographs of Petri dishes seeded with a Staph.albus test culture (Photos A, C, E) and E. coli 1157 test culture (Photos B, D, F). Near the Petri dishes are lids with markers on which the disc numbers are marked. Photographs A and B show Petri dishes, on the surface of which there are disks, with an ointment dosed in an amount of 0.2 g with copper nanoparticles of sample No. 2 in concentrations, in%: 2 × 10 ^-3 (disk No. 1), 0 (disk No. 2), 2 × 10 ^-4 (disk No. 3), 2 × 10 ^-5 (disk No. 4). Photographs C and D show Petri dishes on the surface of which there are disks dosed with Solcoseryl ointment in the amount of 0.2 g. Photographs E and F show Petri dishes with disks on their surface dosed with Levomekol ointment applied amount of 0.2 g. It is seen that the ointments developed by us, containing copper nanoparticles in different concentrations, have antimicrobial activity against test culture cells of gram-positive and gram-negative microorganisms.

FIG. 4. Changes in the specific rate of wound healing during treatment with a combination ointment containing a derivative of low molecular weight chitosans - O-sulfochitosan and copper nanoparticles of sample No. 1 compared with an ointment containing a copper nanoparticle of sample No. 1, an ointment containing a derivative of low molecular weight chitosans - O-sulfochitosan, and control (untreated animals).

Figure 4 presents data on the change in the specific rate of wound healing of the control group (untreated animals), the group treated with ointment with copper nanoparticles of sample No. 1 at a concentration of 0.002%, the group treated with gel with O-sulfochitosan at a concentration of 0.05%, and the group treated combined ointment containing copper nanoparticles of sample No. 1 at a concentration of 0.002% and a derivative of low molecular weight chitosans - O-sulfochitosan at a concentration of 0.05%. It is seen that treatment with a combined ointment leads to an increase in the specific rate of wound healing by a factor of 1.5 compared with the group treated with an ointment with O-sulfochitosan, by 1.4 times compared to the group treated with an ointment with copper nanoparticles of sample No. 1, and by 1.4 times compared with a control group (untreated animals).

FIG. 5. Changes in the specific rate of wound healing during treatment with a combination ointment containing a derivative of low molecular weight chitosans-N-sulfosuccinoyl-N-carboxymethylchitosan and copper nanoparticles of sample No. 2, compared with an ointment containing a copper nanoparticle of sample No. 2, an ointment containing a derivative of low molecular weight chitosans - N-sulfosuccinoyl -N-carboxymethylchitosan, and control (untreated animals).

Figure 5 presents data on the change in the specific rate of wound healing of the control group (untreated animals), the group treated with ointment with copper nanoparticles of sample No. 2 at a concentration of 0.002%, the group treated with gel with N-sulfosuccinoyl-N-carboxymethylchitosan at a concentration of 0.05%, and the group treated with a combined ointment containing copper nanoparticles of sample No. 2 at a concentration of 0.002% and a derivative of low molecular weight chitosans - N-sulfosuccinoyl-N-carboxymethylchitosan at a concentration of 0.05%. It is seen that treatment with a combined ointment leads to an increase in the specific rate of wound healing by 26% compared with an ointment containing N-sulfosuccinoyl-N-carboxymethylchitosan at a concentration of 0.05%, by 16.5% compared with an ointment containing copper nanoparticles of sample No. 2, and by 48.5% compared with the control group (untreated animals).

FIG.6. Change in the specific rate of wound healing during treatment with a combination ointment containing copper nanoparticles of sample No. 2 and chitosan nanoparticles synthesized from chitosan SD 89% and M.m. 10 kDa, at a concentration of 0.0002% in the composition of the ointment compared to an ointment containing copper nanoparticles of sample No. 2, an ointment containing chitosan nanoparticles synthesized from chitosan SD 89% and M.m. 10 kDa, and control (untreated animals).

Figure 6 presents data on the change in the specific wound healing rate of the control group (untreated animals), the group treated with ointment with copper nanoparticles of sample No. 2 at a concentration of 0.002%, the group treated with a gel with chitosan nanoparticles synthesized from chitosan SD 89% and M. m 10 kDa, at a concentration of 0.0002%, and the group treated with a combined ointment containing copper nanoparticles of sample No. 2 at a concentration of 0.002% and chitosan nanoparticles synthesized from chitosan SD 89% and M.m. 10 kDa, at a concentration of 0.0002%. It can be seen that treatment with a combined ointment increases the specific healing rate by 10.5% compared with the group treated with ointment with copper nanoparticles of sample No. 2, it increases the specific healing rate by 81.7% compared with the group treated with ointment with chitosan nanoparticles, and by 40.9% by compared with the control group (untreated animals).

LITERATURE

1. Alekseeva T.P., Rakhmetova A.A., Bogoslovskaya O.D., Olkhovskaya I.P., Levov A.N., Ilyina A.V., Varlamov V.P., Baitukalov T.A., Glushchenko N.N. Wound healing properties of chitosan and its N-sulfosuccinoyl derivatives. // Proceedings of the RAS. Biological Series. - 2010. - No. 3. - p. 1-7.

2. Baitukalov T.A., Bogoslovskaya O.A., Olkhovskaya I.P., Glushchenko N.N., Ovsyannikova M.N., Lopatin S.A., Varlamov V.P. Regenerating activity and antibacterial effect of low molecular weight chitosan. // Proceedings of the Academy of Sciences. Biological Series. - 2005. - No. 6. - p. 659-663.

3. Glushchenko NN, Bogoslovskaya OA, Olkhovskaya I.P. Physico-chemical laws of the biological action of finely divided metal powders. // Chemical physics. - 2002. - T. 21. - No. 4. - p. 79-85.

4. Zavadsky S.P., Cherkasova O.G., Kharitonov Yu.Ya. Quality control criteria and properties of magnetic rectal suppositories. // Chem. - farm. Journal. - 2000. - No. 10.

5. Patent for invention No. 2296571. Wound healing composition and method for its preparation. Baitukalov T.A., Glushchenko N.N., Bogoslovskaya O.A., Olkhovskaya I.P., Folmanis G.E., Arsentieva I.P. Application No. 2006120516. Priority of the invention June 14, 2006. Registered in the State register of inventions of the Russian Federation on April 10, 2007.

6. Patent for the invention No. 2306141. A drug that accelerates wound healing. Baitukalov T.A., Glushchenko N.N., Bogoslovskaya O.A., Olkhovskaya I.P., Leipunsky I.O., Zhigach A.N., Shafranovsky E.A. Application No. 2005141039. Priority of invention December 28, 2005. Registered in the State Register of Inventions of the Russian Federation September 20, 2007.

7. For 1977-2011 publications, see the website: http // www.nanobiology.narod.ru

8. Rakhmetova A.A., Alekseeva T.P., Bogoslovskaya O.A., Leipunsky I.O., Olkhovskaya I.P., Zhigach A.N., Glushchenko N.N. Wound healing properties of copper nanoparticles depending on their physicochemical characteristics. // Russian nanotechnology. - 2010. - Volume 5. - No. 3-4. - p. 102-107.

9. Handbook of microbiological and virological research. / Ed. Birgera M.O. - M .: Medicine, 1973.- 455 p.

Claims (16)

1. A pharmaceutical composition that accelerates wound healing and has antimicrobial activity and includes an active substance and a base that includes copper nanoparticles as an active substance, while copper nanoparticles have the following characteristics: particle size 33.8 ÷ 103 nm; the thickness of the oxide film is 6 ÷ 10 nm and the phase composition: crystalline copper is 67 ÷ 96%, and copper oxide CuO is 4 ÷ 33% or the particle size is 77 ÷ 119 nm, phase composition: crystalline copper is 0.5 ÷ 3.3%, CuO - 27.1 ÷ 90% and Cu ₂ O - 9.05 ÷ 69.5, and are contained in a concentration of from 0.00002 to 0.002%. 2. The pharmaceutical composition according to claim 1, which further comprises derivatives of low molecular weight chitosans. 3. The pharmaceutical composition according to claim 1, which further comprises chitosan nanoparticles. 4. The pharmaceutical composition according to claim 1, which contains as a base methylcellulose MTs-100 and water. 5. The pharmaceutical composition according to claim 4, which further comprises liquid paraffin, tween-80 and a preservative. 6. The pharmaceutical composition according to claim 5, which contains nipagin as a preservative in the following ratio of components, g per 100 g of composition:
Copper nanoparticles 0.0002-0.002 Vaseline oil 10.0-15.0 MTs-100 3.0-4.0 Twin 80 2.0-3.0 Nipagin 0.1-0.3 Purified water Up to 100.0 7. The pharmaceutical composition according to claim 2, which contains, as derivatives of low molecular weight chitosans, O-sulfochitosan or N-sulfosuccinoyl-N-carboxymethylchitosan and, as a base, methylcellulose MTs-100 and water. 8. The pharmaceutical composition according to claim 7, which further comprises liquid paraffin, tween-80 and a preservative. 9. The pharmaceutical composition of claim 8, which contains nipagin as a preservative in the following ratio of components, g per 100 g of composition:
Copper nanoparticles 0.00002-0.002 Derivatives of low molecular weight chitosans 0.005-0.05 Vaseline oil 10.0-15.0 MTs-100 3.0-4.0 Twin 80 2.0-3.0 Nipagin 0.1-0.3 Purified water Up to 100.0 10. The pharmaceutical composition according to claim 3, which contains as particles of chitosan nanoparticles synthesized from chitosan SD 89% and M.m. 10 kDa and as a base methylcellulose MTs-100 and water. 11. The pharmaceutical composition of claim 10, which further comprises liquid paraffin, tween-80 and a preservative. 12. The pharmaceutical composition according to claim 11, which contains nipagin as a preservative in the following ratio of components, g per 100 g of composition:
Copper nanoparticles 0.00002-0.002 Chitosan Nanoparticles 0.01-0.0002 Vaseline oil 10.0-15.0 MTs-100 3.0-4.0 Twin 80 2.0-3.0 Nipagin 0.1-0.3 Purified water Up to 100.0 13. The pharmaceutical composition according to claim 7, which contains O-sulfochitosan or N-sulfosuccinoyl-N-carboxymethylchitosan as a derivative of chitosan and MT-100 methyl cellulose and water as a basis. 14. The pharmaceutical composition according to item 13, which contains nipagin as a preservative in the following ratio of components, g per 100 g of composition:
Derivatives of low molecular weight chitosans 0.005-0.05 MTs-100 3.0-40.0 Nipagin 0.1-0.3 Purified water Up to 100.0 15. The pharmaceutical composition of claim 10, which contains as particles of chitosan nanoparticles synthesized from chitosan diabetes 89% and M.m. 10 kDa and as a base methylcellulose MTs-100, water and a preservative. 16. The pharmaceutical composition according to claim 19, which contains nipagin as a preservative in the following ratio of components, g per 100 g of composition:
Chitosan Nanoparticles 0.01-0.0002 MTs-100 3.0-4.0 Nipagin 0.1-0.3 Purified water Up to 100.0

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