AT10166U1 - HYDROGEL BASED ON A POLYACRYLAMIDE AND A METHOD FOR THE PRODUCTION THEREOF - Google Patents

Description

2 AT 010 166 U1

The invention relates to a formulation and a method for obtaining biocompatible polyacrylamide hydrogel, as a material for medical purposes, in particular as a carrier of human and animal cells, which are implanted in the mammalian organism, as a depot for medical preparations during a longer , drug treatment, such as tumors or abscesses can be used.

It is known that the polyacrylamide hydrogels (PAAG) are sufficiently inexpensive and simple to produce and have chemical and biological inertness. They are readily synthesized at the desired density and in a form suitable for subcutaneous insertion and / or soft tissue insertion with minimal trauma to the patient's body.

In medicine, RU 2165263 discloses a method of treating insulin-dependent diabetes in which heterogeneous pancreatic beta cells are transplanted into the polyacrylamide gel previously subcutaneously introduced into the patient around which a capsule has been formed.

The RU 2152800 also discloses a method of culturing mammalian heterogeneous cells, in particular leididal cells and melanoma cells, by transplanting them into the polyacrylamide gel previously introduced into the mammals.

This opens up the possibility of treating a number of diseases by transplanting the heterogeneous cells into the patient's organism, which produces the necessary enzymes and / or hormones for him; this also allows a vaccine treatment of oncological diseases.

It has been established experimentally that the duration of production of the cells required by the heterogeneous cells of the cells necessary for the patient's organism depends on the properties of the polyacrylamide gel (PAAG) under the same conditions.

As is known, the implantation of PAAG in the mammalian organism around the gel forms a junction capsule (AB Shekhter et al., &Quot; Injectable hydrophilic polyacrylamide gel formaciyl and tissue response to its implantation ", in the journal " Annals of Plastic, Reconstructive and Aesthetic Surgery ", 1997, No. 2, page 19), which for a time prevents T lymphocytes from entering the cells implanted in the PAAG and destroying the implanted cells.

However, not only the presence of the tissue-binding capsule influences the duration of production of the substances required by the heterogeneous cells, necessary for the patient's organism.

EP 742022 discloses a biocompatible polyacrylamide hydrogel which contains from 3.5 to 9.0% by mass of the copolymer acrylamide with the crosslinking agent methylene-bis-acrylamide and 96.5-99.0% by mass of water.

This hydrogel is obtained by a process described in the same document (EP 742022) and which consists in reacting the copolymerization of acrylamide with methylene-bis-acrylamide in the water in the presence of polymerization peroxide initiators with delay of the reaction mixture at room temperature within 20 minutes for the crosslinking of the polymer. The process of copolymerization is carried out in one stage. As peroxide polymerization initiators, a mixture of peroxodisulfate ammonium and tetramethylethylenediamine is used. As the aqueous medium, apyrogenic water or a sodium chloride solution is taken.

The hydrogel obtained by this method has insufficient 3 AT 010 166 U1

Degree of crosslinking, which is due to the low temperature behavior in the implementation of the copolymerization process and the uniformity of the process. This leads to a rapid penetration of the connective tissue into the implanted gel and to its rapid shrinkage and rapid absorption (ABShekhter et all "Injectable hydrophilic polyacrylamide de gel Formacryl and tissue response to its implantation", in the journal "Annalen der plastischen , Reconstructive and Aesthetic Surgery ", 1997, No. 2, page 19).

In addition, the hydrogel obtained by this method contains unbound molecules of tetramethylethylenediamine, free NH 2 radicals, and monomers of acrylamide in an amount of 1.0-1.2 pg per 1 gram of polymer (1.0-1.2 ppm), resulting in a active, aseptic and inflammatory reaction in the early stage of introduction of the hydrogel into the organism can be caused (see AB Shekhter et al "Injectable hydrophilic polyacrylamide gel Formacryl and tissue response to its implantation", in the journal "Annalen der plastischen, reconstructive and Aesthetic Surgery ", 1997, No. 2, page 19).

Patent RU 2127129 discloses a biocompatible hydrogel containing from 1.0 to 8.0 mass% cross-linked copolymer of acrylamide with the crosslinking agent methylene-bis-acrylamide and 92.0-99.0 mass% water. A process for recovering this material is also described in patent RU 2127129 and involves the copolymerization of acrylamide with methylene-bis-acrylamide in the aqueous dispersion medium in the presence of a peroxide polymerization initiator. This is taken as the aqueous medium of the electrolysis submerged water with a pH of 9.0-9.5. Crosslinking of the copolymer is carried out by incubating the reaction mixture in two stages; and at a temperature of 20-90 ° C within 2-24 hours and then at a temperature of 100-105 ° C over 2-4 hours.

The hydrogel obtained by this method does not contain tetramethylethylenediamine but contains 1% of free NH 2 radicals and monomers of acrylamide in an amount of 0.6-0.8 pg per 1 gram of the polymer (0.6-0.8 ppm). However, this hydrogel is primarily designed and suitable for soft tissue plasticity and does not ensure a sufficient duration of active function of the heterogeneous cells when used as a support for the implanted cells.

US Pat. No. 5,338,492 discloses a polyacrylamide hydrogel for irradiation color filters. The hydrogel comprises a crosslinked copolymer of the acrylamide (50-70 mass%) with hydroxyethyl methacrylate and crosslinking agents, namely methylene-bis-acrylamide or methylene-bis-methacrylamide, wherein a weight ratio of the crosslinking agent to the acrylamide is about 1: 5 to about 1:12 is used. Weight ratios of about 1: 8 to about 1:12 are preferred. Particularly preferred is a weight ratio of about 1: 9. The solids content is preferably in the range of about 9 mass%. This hydrogel is prepared using a one-step reaction wherein water solutions of the corresponding monomers are mixed with the cross-linking agents, the polymerization initiators benzoin methyl ether, benzoin ethyl ether, succinic acid peroxide, 2-hydroxy-2-methyl-1-phenylpropan-1-one, 4- ( 2-hydroxyethoxy) -phenyl (2-propyl) ketone, 2,2'-azobis (2,4-dimethyl-4-methoxy-valero) -nitrile or azo-bis-isobutyonitrile, and organic solvents ( Isopropyl alcohol or ethylene glycol), which aim to improve the dispersion of the initiator in the aqueous solution. This hydrogel is toxic to cells and tissues and its utility is limited to technical use. Furthermore, because of the one-step polymerization, it has only a low degree of cross-linking.

WO 02/16453 discloses a polyacrylamide hydrogel which is used as an injectable or implantable endoprosthetic adjuvant and has crosslinked polyacrylamide. This hydrogel is produced by combining acrylamide and N, N'-methylene-bis-acrylamide and using it for cross-linking in a particular weight ratio. This hydrogel is produced by reacting an aqueous monomer solution of the acrylamide (AM) and the Ν, Ν'-methylene-bis-acrylamide (BISAM) as crosslinking agent with Ν, Ν, Ν ', Ν'-tetramethyleneethylenediamine (ATO10166U1). TMED) as Kostarter and ammonium persulfate (APS) as a radicalless starter (redox system) are mixed. By degassing a voluminous solution with nitrogen (maintained at a temperature of 45 ° C and degassed with nitrogen for a period of 20 s), the polymerization (which takes place for a period of 0.5-1.5 hours) starts. After a final polymerization, the gel is filled into a wash tank with mesh cups into which the gel is taken up. During washing of the gel with water, the gel expands and monomer residues are removed by equilibration with water for a period of 92 hours, with the water exchanged several times. The hydrogel produced has the same disadvantages as the hydrogel described in EP Application 742022. The hydrogel has an insufficient degree of cross-linking due to the low temperature conditions of the copolymerization process and its one-step performance, so that rapid penetration of the connective tissue into an implanted hydrogel and rapid shrinkage and rapid resorption of the hydrogel can occur. This hydrogel may comprise unbound molecules of tetramethylethylenediamine, free NH 2 radicals and acrylamide monomers, so that an active, aseptic inflammatory reaction can take place immediately after implantation of the hydrogel in the organism (see AB Shekhter et al., "Injectable Hydrophilic Polyacid"). rylamide gel Form Acrylic and Tissue Response to its Implantation " in " Annals of Plastic, reconstructive and esthetic surgery ", 1997, No. 2, page 19).

Further, by the article by Olga Garcia et al. " 5-fluorouracil trapping in poly (2-hydroxyethyl methacrylates-coacrylamides) hydrogels: in vitro drug delivery studies ", European Polymer Journal, Vol. 36, 2000, No. 1, pp. 111-122, polyacrylamide hydrogels known in the art Copolymer acrylamide, 2-hydroxyethyl methacrylate (HEMA) and ethylene glycol dimethacrylate (EGDMA). The process for producing this gel comprises recovering a water mixture of the components described above, adding ammonium peroxodisulfate and degassing the mixture with nitrogen for 5 minutes. The mixture is then held at a temperature of 50 ° C (323 ° F) for three hours. The use of ethylene glycol dimethacrylate (EGDMA) as a crosslinking agent allows the recovery of a highly expanding hydrogel that can be easily absorbed by macrophages when the hydrogel is implanted in the recipient organism. Furthermore, this hydrogel has an insufficient degree of cross-linking due to the one-step performance of the polymerization process, which can result in rapid penetration of the connective tissue into the implanted gel and rapid shrinkage and resorption of the hydrogel.

Finally, WO 00/45792 discloses a corrosive polyacrylamide hydrogel which is crosslinked and is formed by copolymerizing acrylamide with Ν, Ν'-methylene-bis-acrylamide. The usual hydrogel monomers have 1-hydroxyethyl methacrylate (HEMA). The common cross-linking agents include tetraethylene glycol dimethacrylate (TEGDMA) and Ν, Ν'-methylene bis-acrylamide. Also, this hydrogel can not guarantee long viability of the heterogeneous cells in the recipient organism due to rapid decomposition in the living organism, as expressly confirmed in this document by the presence of erosion-prone hydrogels in which hydrolytic instability occurs in the crosslinks, a cross-linked product, which is prepared by copolymerizing N-vinylpyrrolidone or acrylamide with N, N'-methylene-bis-acrylamide. The use of tetraethylene glycol dimethacrylate (TEGDMA) as a cross-linking agent gives an increasing likelihood of pronounced tissue reaction caused by the unavoidable presence of residual TEGDMA monomers.

The main object to be solved by the proposed invention is to increase the active viability of heterogeneous cells housed in the polyacrylamide in the organism of the recipient.

Another object is to lower the resorption level of the hydrogel and the possibility of macrophage penetration upon implantation of the hydrogel into the recipient's organism.

Another object is to reduce the tissue response of the organism to the implanted hydrogel by reducing free radicals and monomers.

The stated objects are achieved by proposing a polyfunctional, biocompatible hydrogel comprising polyacrylamide and water, polyacrylamide according to the invention comprising the copolymer of acrylamide and methacrylamide and the crosslinking agent 2-hydroxyethyl methacrylate and N, N'-methylene bis-acrylamide contains.

The polyacrylamide contains the following proportions of the components in mass.%:

Acrylamide 65.0-80.0,

Methacrylamide 18.0-30.0, 2-hydroxyethyl methacrylate 0.1-4.0 Ν, Ν'-methylene-bis-acrylamide 0.2-6.0.

The proportion of polyacrylamide in the total mass of the biocompatible hydrogel is 3.0 to 10.0 mass.%.

The biocompatible hydrogel contains the following proportions of the components in mass.%:

Acrylamide 1.95-8.0,

Methacrylamide 0.54-3.0, 2-hydroxyethyl methacrylate 0.003-0.4, N, N'-methylene-bis-acrylamide 0.006-0.6,

Water rest on 100.

As water, the hydrogel contains bi-distilled apyrogenic water. The multi-functional, biocompatible hydrogel has a pH of 3.5-4.5.

Said hydrogel is suitable for injection, packaged in syringes and well suited for the formation of a capsule in the organism of animals and humans. For this purpose, it is implanted in the organism, and then it is housed in the desired cell culture.

The tasks are solved as follows. A process is proposed for obtaining a polyfunctional, biocompatible hydrogel by copolymerizing monomers and a crosslinking agent in an aqueous medium in the presence of peroxide initiators. This procedure takes place in several stages. Acrylamide and methacrylamide are used according to the invention as monomers in this process, and hydroxyethyl methacrylate and Ν, Ν'-methylene-bis-acrylamide are used as crosslinking agent in the following proportions of the components in% by weight:

Acrylamide 1.95-8.0,

Methacrylamide 0.54-3.0, 2-hydroxyethyl methacrylate 0.003-0.4, N, N'-methylene-bis-acrylamide 0.006-0.6,

Water rest on 100.

The copolymerization is carried out in three stages: the first stage is carried out at a temperature of 20-30 ° C within 12-24 hours, the second stage is carried out by irradiation with γ-rays at a dose of 0.004 -0.01 MGy, and the third stage takes place at a temperature of 100-130 ° C and a pressure of 0-1.2 bar within 20-40 minutes.

Said hydrogel is after the first stage of the copolymerization with hot water having a temperature of 70-110 ° C for at least 3 hours at a mass ratio of the hydrogel to the water of 1: 8-10 and a pressure of 0-1, 2 bar rinsed.

As a copolymerization initiator, hydrogen peroxide and / or peroxydisulfate ammonium is taken in an amount of not more than 0.33% by mass of the total weight of the starting components. As an aqueous medium, bi-distilled apyrogenic water is taken.

The recovered hydrogel is filled in syringes and used for implantation into the organism of animals and humans to form a capsule. Thereafter, the desired cell cultures are introduced into the capsule.

The proposed hydrogel can be obtained by different methods, and the method of the invention for obtaining polyacrylamide hydrogel does not limit the other possible variants of recovery, both direct and indirect, of the hydrogel. This process represents one of the possible ways to recover the hydrogel of this composition with predetermined properties.

As is known, the hydrogel containing polyacrylamide constitutes a triaxial network of monomers crosslinked with the crosslinking agent in whose cells the aqueous medium is contained. This aqueous medium contains an undetermined amount of non-combined polymerization initiators since some, even unconfined, amount of polymerization initiators are incorporated directly into the structure of the copolymer (see MN Sa-witzkaya, Ju.D. Holodova " Polyacrylamide " , Verlag " Technik " 1969, p. 103) or washed out of the hydrogel during its rinsing.

The biologically active properties of such a gel depend substantially on the structure of the network polymer, which in turn depends on the conditions for the synthesis of the network polymer, the qualitative and quantitative ratio of the starting reagents, including the crosslinking agent and the polymerization initiators, with their chemical and hydrogen compounds are incorporated into the structure of the polymer (by groups NH, CH, COOH, NH 2, CH 2). These properties also depend on the type of polymerization.

The essence of the invention is that a qualitative and quantitative composition of the polyacrylamide gel as well as the conditions of carrying out the copolymerization were found, which allow to increase the duration of the active life function of heterogeneous cells implanted in the organism.

The conditions for obtaining the proposed polyacrylamide hydrogel have allowed to reduce the number of unlinked amino groups, the free NH 2 radicals and the unsaturated double bonds. It has also been possible, the degree of crosslinking by the formation of structural groups (H2C-NH-CH2-), (-CO-NH-CR2-0-R), (-CO-NH-NH-CO-), (H-COR -NH-CR-OR), (-CONH-R-NH-CO), where R is CH3, CH2, NH2, C2H5 or C3H7, and by increasing the number of cross-links of NN bonds.

This can ensure a high resistance of the proposed hydrogel in terms of absorption and shrinkage in the patient's body as well as conditions for the survival of the cells implanted in the gel. 7 AT010 166U1

Abstract of the drawings

The invention will now be explained in more detail with reference to exemplary embodiments. Show it:

FIG. 1a shows an infrared (IR) absorption spectrum of the proposed gel; FIG.

Figure 1b is an infrared (IR) absorption spectrum of a prototype of the gel marketed in Russia under patent RU 2127129 under the Trade Mark "Form Acrylic". where both IR spectra are in the range of 4000-500 cm'1, on the X axis is the length of the light wave (cm'1) and on the Y axis is the degree of light absorption T (in%) ;

Fig. 2a is a photograph of the proposed hydrogel taken with an electronic scanning microscope; and

Fig. 2b is a picture of the hydrogel " formacrylic " which was also taken with an electronic scanning microscope.

To obtain the proposed biocompatible gel, the following is taken:

Acrylamide: C3H5N03, molecular weight 71.08, white crystal powder without odor, melting temperature 84.5 ° C, production by Sigma (catalog "Reagents for Biochemistry and Studies in the Natural Sciences", SIGMA, 1999, p. 47) , Catalog number No. A8887),

Methacrylamide: C3H7NO, molecular weight 73.08, white powder, melting temperature 111 ° C, production by Fluka (Fluka Katalogue " Chemica-Biochemica ", Fluka AG, Switzerland, 1986/87, P-1151), 2-hydroxyethyl -Methacrylate: C6H10O3, Mol mass 130.1, liquid, boiling temperature 205-208 ° C, density 1.07g / ml, production by Sigma (Catalog "Reagents for Biochemistry and Science Examinations", SIGMA, 1999, p. 567, catalog number No. H8633), N, N'-methylene-bis-acrylamide: C7H10N2O2, mol mass 154.16, white crystal powder odorless, melting temperature 185 ° C, production by Sigma (catalog " Reagents for biochemistry and investigations in the field of natural sciences ", SIGMA, 1999, p. 696, catalog no. 7256),

Peroxodisulfate ammonium: (NH4) 2-S208, molecular weight 228.19, colorless, flat crystals, destruction temperature 120 ° C, production by Sigma (Catalog " Reagents for Biochemistry & Science Investigations ", SIGMA) 1999, p. 117), hydrogen peroxide: H 2 O 2, molecular weight 34.0, colorless liquid, density at 0 ° C: 1.465, melting temperature 0.89 ° C, production by Sigma (catalog " Reagents for Biochemistry and Examinations in the field of science ", SIGMA, 1999, p. 556, catalog number H6520).

All of the above monomers are considered to be suitable for the biological targets, they do not require biological purification. The water used is bidistilled apyrogenic water (pH 5.6).

The process is carried out as follows: For the preparation of the reaction mixture, bidistilled apyrogenic water with a pH of 5.6 is taken. A water solution is prepared containing acrylamide, with methacrylamide, 2-hydroxyethyl methacrylate and Ν, Ν'-methylene-bis-acrylamide in the ratio 65.0-80.0: 18.0-30.0: 0.1- 4.0: 0.2-6.0 to each other. The total mass of the starting monomers in the solution is 3.0 to 10.0% (by varying the starting monomers in the solution, a hydrogel of different density and elasticity is obtained). In the solution obtained, the polymerization initiators are hydrogen peroxide in the amount of O, 1-0.3 Mass.% Or peroxodisulfate ammonium in the amount of 0.0006-0.03 Mass.% Or a mixture thereof in any ratio and in a Quantity introduced that does not exceed the sum of their maximum values. By varying the amount of hydrogen peroxide and peroxodisulfate ammonium, material is obtained which has the desired pH in the range of 3.5-4.5. The final reaction mixture is filtered through bactericidal polymer filters, for example type F8273 with a pore size of 0.45 mm, CA / CN, Sigma (USA). The mixture is brought for the copolymerization of the monomers for incubation at 5 a temperature of 20-30 ° C within 12-24 hours. After this incubation, the hydrogel, which already looks like a gel, is rinsed with hot water. For this purpose, the gel is placed in a container with water at a temperature of 70-110 ° C, at a pressure of 0-1.2 bar and a ratio of gel content to water of 1: 8-10 for 4-6 Hours. Then, the second stage of the copolymerization is carried out, whereby the semi-product is processed with irradiation by γ-rays in a dose of 0.004-0.01 MGy. Thereafter, the product is filled to the required extent in bottles or syringes. Thereafter, the third stage of the copolymerization is carried out, wherein the gel is exposed to a temperature of 120 ° C and a pressure of 1.2 bar within 20-40 minutes. 15 Physical-chemical, hygienic-chemical and toxicological investigations of samples of the proposed hydrogel were carried out in accordance with the standard ISO 10993 "Evaluation of the biological effect of medicinal products", "Methodological notes on the hygienic evaluation of rubber and latex products for medical purposes " (Ministry of Health USSR, M., 1988) and in accordance with the methodological recommendations "Permitted amounts of migration of chemical substances, which are made of polymer and other materials and which contact with food hydrogels, and the methods for their determination "; SanPiN 42-122-42-40-86.

The determination of the content of the monomers acrylamide and Ν, Ν'-methylene-bis-acrylamide in the samples of the recovered hydrogel was carried out in accordance with the methodology used in the work of V.V. Kuznetsov et al., "Determination of acrylamides in polyacrylamide dicels", The 52nd is Pittsburgh Conference on Analytical Chemistry and Applied Spectrosco-py, New Orleans, LA, 2001f Abstract Book, No. 1648. 30 In this investigation, it was found that the proposed hydrogel has the following physicochemical properties:

Appearance: gel

Color: from colorless to semi-translucent dark brown, opalescent, 35 refraction ratio: 1.328-1.360,

Density: 1.0-1.2 g / cm3, pH: 3.5-4.5,

Content of monomers: up to 0.4 ppm,

Bromination level: not more than 1.0 (mg of bromine per 1 liter). 40

Hygienic tests have shown that the migration of the metals Cu, Fe, Ni, Zn, Al, Ti, Ag in an aqueous extract of hydrogel, which is determined by the atomic absorption method, was not found within the limits of the sensitivity of the method (each 0 , 02, 0.05, 0.05, 0.02, 0.005 and 0.04 mg / l). These values are much lower than the permitted values for drinking water. The migration of sodium was not more than 0.12 mg / l with a permissible level in the drinking water of 200 mg / l.

The technological experiments have shown that the aqueous extracts of hydrogel have no hemolytic effect in the experiments "in vitro". exhibited by means of rabbit-isolated E-50 Rythrozyten. Haemolysis of 0.04% at a permissible value of 2% was noted.

In an experiment with white mice in the parenteral introduction of the hydrogel pattern in a dose of 50.0 ml per 1 kg of body mass neither the death of the animals nor the clinical signs of intoxication were found: the general condition of the experimental mice, their 9 AT 010 166 U1

Behavior, the feed intake and the condition of the fur did not differ from the control conditions. In the autopsy of the experimental mice, it was found that the tissues at the site of introduction of the hydrogel were the regional lymph nodes and internal organs (liver, kidneys, spleen) within the limits of the physiological norm and control. There were no statistically significant differences in body mass dynamics, blood biochemical data, internal organ function in experimental animals compared to the control values for subcutaneous implantation of the gel for 2.5 months.

No sensitizing effect of the hydrogel was detected in carrying out the immunological diagnostic reaction in the degranulation of the mast cells. In a microkernel trial on the bone marrow preparation, no mutagenic effect of hydrogel activity was noted. The histological examination of the area of hydrogel implantation and the internal organs (liver, kidneys, spleen, seminal vesicle) showed the presence of a weak tissue response to hydrogel only in the first days after implantation and the absence of dystrophic and necrotic changes in the organs.

The following are specific examples for obtaining the proposed biocompatible hydrogel and its application for the cultivation of heterogeneous cells in the recipient's organism.

example 1

To obtain hydrogel, 384 ml of bidistilled apyrogenic water with a pH of 5.6 were taken. 11.2 g of acrylamide, 3.6 g of methacrylamide, 0.48 g of 2-hydroxyethyl methacrylate and 0.72 g of Ν, Ν'-methylene-bis-acrylamide, which are suitable for biological purposes, were then taken into the water. Thereafter, 0.04 g of peroxodisulfate ammonium and 2 ml of 30% hydrogen peroxide were added to the starting solution. The recovered mixture was filtered through a bactericidal polymer filter, type F8273 with a pore size of 0.45 mm, CA / CN, manufacturer Fa. Sigma (USA). The mixture was placed in a container which was brought to incubation in a water bath at a temperature of 30 ° C within 22 hours.

Thereafter, the hydrogel in the form of the gel was rinsed with hot water in the ratio of water to gel of 10: 1 at a temperature of 90 ° C within 4 hours. Then, the second stage of the copolymerization was carried out, whereby the half-product was processed with irradiation by γ-rays in a dose of 0.008 MGy. Thereafter, the product was filled in syringes of 0.5-3.0 ml. Then, the third stage of the copolymerization was carried out, wherein the gel was exposed to a temperature of 120 ° C and a pressure of 1.2 bar for 30 minutes.

The recovered gel contains 96% by mass of the water phase and 4% by mass of copolymer, with 70.0% by mass of acrylamide 22.5% by mass of methacrylamide, 0.3% by mass of 2-hydroxyethyl methacrylate, 0.4% by mass .% N, N'-methylene-bis-acrylamide omitted. The gel has a pH of 4.3.

The obtained pattern of the hydrogel has the following physicochemical properties:

Appearance: colorless, semi-transparent, opalescent gel, refraction ratio: 1.348, pH value: 4.3, density: 1.0 g / cm3, content of monomers: 0.1 ppm, bromination level: 0.1 (mg bromine to 1 l ).

The IR spectrum was obtained and electron microscopic examinations of the dried sample of this hydrogel were carried out, which are respectively shown in FIGS. 1a and 2a. For comparison, Figures 1b and 2b show the infrared spectrum and a scanning micrograph of the extract of the famous hydrogel prototype made in Russian patent RU 2127129 under the trademark " Formacryl " is produced, shown. This gel contains 96% by mass of the water phase and 4% by mass of copolymer, with 96% by mass of the water phase containing 4.0% by mass of N, N'-methylene-bis-acrylamide with a pH of 5.4 and a bromination level of 0.27 (mg of bromine to 1 l) accounted for. The gel was incubated in the incubation of the starting mixture in the presence of hydrogen peroxide and peroxodisulfate ammonium in the total amount of 0.3% by mass at a temperature of 60 ° C within 12 hours and then at a temperature of 100 ° C within 2 hours received.

As can be seen from the spectrum in Fig. 1a, the peak in the region of the strip is missing at 1620 cm'1. This indicates that the structure of the polymer lacks the free NH 2 radicals that could form the coordination compounds with the structural network of the hydrogel.

As can be seen from the spectrum in Fig. 1b, there is a stripe at 1620 cm'1. This indicates the presence of NH2 radicals in an amount of just over 1%.

In order to conduct the electron microscopic examination of the registered hydrogel and the hydrogel " Farmokrir " the patterns of the gel obtained according to the example described were dried to a constant weight. The patterns are in the form of a film. The structure of the hydrogels was investigated by the method of electronic scanning microscopy (SEM) using an S 405A electron microscope. For this purpose, the preparation was carried out by the process of chipping the dried-up hydrogel patterns frozen in liquid nitrogen. Before the electronic scanning microscopy method was used, gold was sputtered onto the surface of the samples.

The electron micrographs of the proposed hydrogel and hydrogel " Formakril " are each shown in Figures 2a and 2b. As can be seen from the comparison of the images shown in Fig. 2, the proposed hydrogel has a finer cell structure compared to the hydrogel " Formakril ".

Example 2

The proposed hydrogel is used for the cultivation of xenogenic tumor cells.

The hydrogel obtained according to Example 1 described was implanted subcutaneously in an amount of 1 ml of mice of the C57-Black line. After the lapse of 1.5 months, human melanoma cells of the SKMEL-1 line, 1 ml each, were placed on each hydrogel capsule in the implanted gel around which a connective tissue capsule was formed. After 3, 6 and 9 months, the capsules were removed from the animals and the viability and physiological activity of the human melanoma cells contained in these capsules were tested.

It has been found that the human melanoma cell of the SKMEL-1 line has maintained its viability even 9 months after culturing in the capsule of the proposed hydrogel. Upon transfer of the capsule-secreted melanoma cells into the culture, the conventional method of polymerase chain reaction was to establish the full identity of the human conserved human moglobin in the melanoma cells cultured in the culture medium and the identity of the cells within 9 Months were cultivated in the capsule of the hydrogel, which were implanted in the organism of a mouse.

This example demonstrates the ability of the proposed hydrogel to permanently cultivate the cells implanted in the recipient's organism, with the cells maintaining their viability.

Example 3

The proposed hydrogel is used to cultivate pig liver cells in the human body for the treatment of infertility.

The hydrogel obtained according to Example 1 described was implanted in sick male patients. After the formation of the connective tissue capsule around the gel, the cells were introduced into the gel taken from mature piglets.

The testosterone level in the patient's blood was determined by means of a diagnostic kit from ChemaMedika (Russia) according to the manufacturer's instructions. The testosterone level in the patient after implantation of the pig liver cells is given in the following table.

Testosterone level Duration of implantation of cells in the proposed hydrogel 0 months 3 months 6 months 10 months 12 months 16 months 22 months 1 patient 52 years old 4.8 nmol / L 8.6 nmol / L 10, 8 nmol / l 16.0 nmol / l 12.0 nmol / l 16.2 nmol / l 18.0 nmol / l 2nd patient 38 years old 6.8 nmol / l 12.6 nmol / l 16.4 nmol / l 18.6 nmol / l 22.4 nmol / l

Duration of implantation of the cells in the hydrogel " Formakril " (Prior art) 0 Mon. 3 Mon. 6 Mon. 10 Mon. 12 Mon. 16 Mon. 22 Mon. 3. Patient 50 years old 3.8 nmol / l 5.8 nmol / l 10.8 nmol / l 10.6 nmol / l 8.0 nmol / l 6.2 nmol / l 0 4th patient 36 years old 5.6 nmol / l 8.6 nmol / l 13.4 nmol / l 12.6 nmol / l 9 , 5 nmol / l 8.4 nmol / l 5.2 nmol / l

As can be seen from this table, the proposed hydrogel assures the possibility of cultivating leidogenic pig cells in the human organism while preserving the ability of these cells to synthesize the testosterone over 22 months, while implanting the cells into the gel According to the closest prior art (Formakril) the ability to actively synthesize the hormone begins to decline after only 10 months.

Example 4

The proposed hydrogel is used to cultivate pancreatic heterogeneous cells in the human body for the treatment of insulin-dependent diabetes. 1. In the patient F., 37 years old, was an insulin-dependent diabetes 11 years ago, a 12 AT 010 166 U1

Year after delivery, diagnosed. The patients' pregnancy was severe with toxicosis in the second half of gestation, nephropathology and a significant weight gain of 26 kg. The disease had a non-stable character all these years, resulting in much effort in choosing the appropriate insulin therapy. Consumption of exogenous insulin varied from 58 units / day to 30 units / day. In the last two years pathological changes of the kidneys have been diagnosed and these changes could be defined as diabetic nephropathy. The urine analyzes showed an increase in the upper limit of proteinuria by 10-12 fold. The arterial blood pressure increased up to 170/110 mm Hg.

The hydrogel obtained as described in the example was implanted in this patient. After the formation of the connective tissue capsule around the gel, a cell culture of the pancreas of newborn rabbits was introduced into the gel.

As early as 7 days after the pancreatic cells were introduced, the patient noted an improvement in the general condition, a reduction in the feeling of thirst and dryness of the oral mucosa and a lowering of the arterial blood pressure up to 140/90 mm Hg. In 15 days, the patient's condition allowed to reduce the requirement for exogenous insulin from 30 units to as much as 18 units (control of blood and urine). In 30 days, the need for exogenous insulin decreased to 12 units / day and up to 4 units / day by the end of the second month.

After introduction of cells of a pancreas the patient was observed within 12 months. Clinical phenomena of nephropathy could not be detected, arterial pressure was within limits of age norms. The patient was placed on peroral anti-diabetic drugs with the obligatory requirement of diabetes diet and glucose control in the blood, urine and glycated homoglobin. 2. Sick K., 52 years old, has been diagnosed with insulin-dependent diabetes since the age of 18 with severe background stress. The nature of the disease was initially very unstable. Doses of exogenous insulin reached 70 units / day.

Over time, the course of the disease stabilized, the worsening of the condition emerged after stressful situations and failures in the diet. The last three years have seen a worsening of lower limb vessels, a decrease in libido and a deterioration in the erection and quality of the sex act. Diabetic angiopathy of the lower limbs and penis has been diagnosed. The demand for exogenous insulin was 20 units / day to 40 units / day in the last year. The hydrogel obtained according to Example 1 described was implanted in this patient. After the formation of the connective tissue capsule around the gel, the cell culture of the pancreas of 14-day piglets was introduced into the gel.

In two weeks after the pancreatic cells were introduced, the patient noted an improvement in the general condition. In one month, the need for exogenous insulin decreased to as much as 12 units / day and to 6 units / day in two months. In 4 months after transplantation, the patient was placed on peroral anti-diabetic drugs. The sexual life of the patient has normalized, the condition of the lower limbs has improved considerably.

The subjective and objective symptoms of the patients studied and the indications of additional research methods (blood, urine) allow to talk about the high effectiveness of treatment of diabetes by introducing heterogeneous cells of the pancreas into the hydrogel of the proposed composition.

Claims (9)

13 AT 010 166 U1 The therapeutic effect usually lasts for 10 to 20 months, depending on the severity of the disease. The number of cells to be transplanted is also determined by the severity of the course of diabetes, especially by the amount of insulin taken by the patient. Industrial Applicability The mentioned embodiments confirm that the proposed biocompatible hydrogel can be obtained by the proposed method. The proposed hydrogel produces virtually no tissue reaction, no organism sensitization, no dystrophic and no necrotic changes, and can be used for implantation into the animal and human organism to form the capsule and to further culture the desired cell cultures in the capsule , Compared to the known hydrogel prototype (hydrogel " Formakril "), the structure of the proposed hydrogel lacks the free NH 2 radicals which could form the coordinate bond in the structural network of the hydrogel. The proposed hydrogel, in contrast to the prototype, has a better fine cell structure and ensures the possibility of prolonged cultivation of the implanted cells in the recipient's organism while preserving the viability of the cells and the ability to produce the products required for humans, particularly testosterone and insulin. Claims 1. A polyfunctional, biocompatible hydrogel containing polyacrylamide and water, characterized in that the polyacrylamide is the copolymer of acrylamide and methacrylamide and the crosslinking agent is 2-hydroxyethyl methacrylate and N, N'-methylene-bis-acrylamide in the following ratio of the components in% by mass contains: acrylamide: 65.0-80.0, methacrylamide: 18.0-30.0, 2-hydroxyethyl methacrylate: 0.1-4.0 -N, N'- Methylene-bis-acrylamide: 0.2-6.0. 2. Multi-functional, biocompatible hydrogel according to claim 1, characterized in that the proportion of the polyacrylamide to the total mass of the biocompatible hydrogel 3.0 to 10.0 mass.% Is. 3. Multi-functional, biocompatible hydrogel according to one of claims 1 to 2, characterized in that it contains the following ratio of the components in mass.%: - acrylamide: 1.95-8.0, - methacrylamide: 0.54-3.0 , - 2-hydroxyethyl methacrylate: 0.003-0.4, - N, N'-methylene-bis-acrylamide: 0.006-0.6, - water: remainder to 100. 4. Multi-functional, biocompatible hydrogel according to one of claims 1 to 3, characterized in that it contains bidistilled water as apyrogenes water. 5. Multi-functional, biocompatible hydrogel according to one of claims 1 to 4, characterized in that it has a pH of 3.5-4.5. 6. A method for obtaining polyfunctional, biocompatible hydrogel by copolymerization in several stages of monomers and crosslinking agents in aqueous medium in the presence of a peroxide initiator of the polymerization, characterized in that acrylamide and methacrylamide as monomers and hydroxyethyl methacrylate and as crosslinking agents AT010 166U1 N, N'-methylene-bis-acrylamide are taken at the following ratio of the components in% by weight: - acrylamide: 1.95-8.0, - methacrylamide: 0.54-3.0, - 2-hydroxyethyl methacrylate : 0.003-0.4, - Ν, Ν'-methylene-bis-acrylamide: 0.006-0.6, - water: balance to 100, wherein the copolymerization is carried out in three stages, namely the first stage at a temperature of 20 -30 ° C within 12-24 hours, the second stage by irradiation with γ-rays in a dose of 0.004-0.01 MGy and the third stage at a temperature of 100-130 ° C and a pressure of 0-1 2 bar within 20-40 minutes. 7. The method according to claim 6, characterized in that the hydrogel after the first stage of the copolymerization with hot water having a temperature of 70-110 ° C for at least 3 hours at a pressure of 0-1.2 bar and at a mass ratio of the hydrogel to the water of 1: 8-10 is rinsed. 8. The method according to claim 6, characterized in that is taken as a copolymerization initiator hydrogen peroxide and / or peroxodisulfate ammonium in an amount of not more than 0.33 mass.% Of the total weight of the starting components or be. 9. The method according to claim 6, characterized in that is taken as an aqueous medium bidestilated lately apyrogenic water. No drawing