The inventive polyfunctional biocompatible hydrogel comprises 1.3 15 mass % of acrylamide, 0.004 0.975 mass % of cross-linking agents--N,N'-methylen-bis-acrylamide, 0.004 5.1 mass % of N,N'-ethylen-bis-acrylamide, 0.002 0.45 mass % of poviargol and water up to 100 mass %. Said hydrogel is produced by sopolymerising acrylamide with the cross-linking agents in an aqueous medium in the presence of peroxide polymerisation initiator, the incubation of reaction mixture being carried out in two stages. The first stage is carried out at a temperature ranging from 20 to 90.degree. C. during a time of 2 to 24 hours, the second stage being carried out at a temperature ranging from 107 to 130.degree. C. during a time equal to or less than 2 hours.

SUMMARY OF THE INVENTION

The present invention is aimed at the reduction of the resorption and shrinkage rate of biocompatible hydrogels based on cross-linked polyacrylamides after implantation into a patient's body.

A second aim is to reduce the possibility of colonization and proliferation of microorganisms within the hydrogel, including microorganisms already present in the patient.

Another aim is to diminish the concentration of free radicals and monomers in the hydrogel thereby reducing the possibility of adverse tissue responses to the implant.

These goals are achieved by a polyfunctional biocompatible hydrogel which contains copolymers of cross-linked acrylamide with a cross-linking agent and water. According to the invention, one suitable hydrogel comprises a copolymer containing a mixture of N,N'-methylene-bis-acrylamide, N,N-ethylene-bis-acrylamide and poviargolum with the following ratio of the components in percent by mass percent: Acrylamide--65.0 99.5, N,N'-methylene-bis-acrylamide--0.2 6.5, N,N'-ethylene-bis-acrylamide--0.2 34.0, and Poviargolum--0.1 3.0.

As a cross-linking agent, the above-mentioned copolymer can also contain vinylpyrrolidone or ethylene-bis-(oxyethylenenitrilo)-tetraacetic acid or a mixture thereof with the following ratio of the components in percent by mass percent: Acrylamide--65.0 99.4, N,N'-methylene-bis-acrylamide--0.2 6.5, N,N'-ethylene-bis-acrylamide--0.2 34.0, Poviargolum--0.1 3.0, and Vinylpyrrolidone or ethylene-bis-(oxyethylenenitrilo)-tetraacetic acid, or a mixture thereof--0.1 2.5.

Wherein the above-mentioned biocompatible hydrogel: contains twice-distilled pyrogen-free water, has a pH of 3.5 7.5, and the cross-linked copolymer constitutes from 2.0 to 15.0% of the total mass of the hydrogel.

Another suitable biocompatible hydrogel according to this invention contains the following ratio of the components by mass percent: Acrylamide--1.3 15, N,N'-methylene-bis-acrylamide 0.004 0.975, N,N'-ethylene-bis-acrylamide 0.004 5.1, Poviargolum--0.002 0.45, and Water--up to 98.69.

The biocompatible hydrogel can alternatively contain the following ratio of the components in mass percent: Acrylamide--1.3 15, N,N'-methylene-bis-acrylamide 0.004 0.975, N,N'-ethylene-bis-acrylamide 0.004 5.1, Poviargolum--0.002 0.45, Vinylpyrrolidone or ethylene-bis-(oxyethylenenitrilo)-tetraacetic acid, or a mixture thereof--0.002 0.375, Water--up to 98.688.

The challenges identified above are also solved by the proposed method of production of the polyfunctional biocompatible hydrogel. The copolymerization of acrylamide with the cross-linking agent(s) is performed in an aqueous medium in the presence of a peroxide polymerization initiator and incubation of the reaction mixture is done in two stages. The first stage is conducted at the temperature of 20 90.degree. C. for 2 24 hours. In accordance with the invention, the cross-linking agent may be a mixture of N,N'-methylene-bis-acrylamide, N,N'-ethylene-bis-acrylamide and poviargolum with the following ratio of the components in mass percent. Acrylamide--1.3 15, N,N'-methylene-bis-acrylamide 0.004 0.975, N,N'-ethylene-bis-acrylamide 0.004 5.1, Poviargolum--0.002 0.45, Water--up to 98.69,

The second stage of the reaction mixture's incubation is conducted at a temperature of 107 130.degree. C. for not more than 2 hours.

The method can be used even when the cross-linking agent is a mixture of N,N'-methylene-bis-acrylamide, N,N'-ethylene-bis-acrylamide, poviargolum, vinylpyrrolidone and/or ethylene-bis-(oxyethylenenitrilo)-tetraacetic acid with the following ratio of the components in mass percent: Acrylamide--1.3 15, N,N'-methylene-bis-acrylamide 0.004 0.975, N,N'-ethylene-bis-acrylamide 0.004 5.1, Poviargolum--0.002 0.45, Vinylpyrrolidone or ethylene-bis-(oxyethylenenitrilo)-tetraacetic acid, or a mixture thereof--0.002 0.375, Water--up to 98.688.

After the first stage of the incubation, the hydrogel is washed in hot water at 70 110.degree. C. for at least 3 hours. The mass ratio of the hydrogel and water may be from about 1:8 to about 1:10.

The initiator of polymerization may be hydrogen peroxide and/or ammonium persulfate in the amount not more than 0.33 mass percent of the total of the original components.

Twice-distilled pyrogen-free water may form the aqueous medium.

It is known that hydrogels formed from a copolymer of acrylamide and cross-linking agents create a three-dimensional network of cross-linked copolymer of acrylamide with cross-linking agents. The voids present in such a three-dimensional network are filled with the aqueous medium in which polymerization occurred. Inevitably, this aqueous component contains a certain amount of initiator. Additionally, some amount of the initiator is incorporated directly into the copolymer's structure (see Savitskaya M. N., Kholodova Y. D. "Polyacrylamide", Publishing House "Tekhnika", 1969, p.103) or because it was rinsed out of the hydrogel during its washing.

The biological activity characteristics of such hydrogels depend largely on the structure of the cellular polymer, which in turn depends on the conditions of its synthesis, i.e. the quantitative and qualitative ratio of original reagents, including cross-linking agents and initiators of polymerization, which by means of covalent and hydrogen bonds get built into the copolymer's structure (by groups NH, CH, COOH, NH.sub.2, CH.sub.2), as well as the temperature of polymerization.

The main advantages of the present invention lie in the inclusion of N,N'-ethylene-bis-acrylamide and poviargolum as cross-linking agents into a hydrogel, based on an acrylamide and N,N'-methylene-bis-acrylamide copolymer, and in selection of conditions for copolymerization that mimimize the concentration of unreacted amides, free NH.sub.2 radicals, and residual double bonds. The invention also makes possible an increase in the extent of cross-linking. Effectively increasing the formation of the cross-linking functional groups including:

(HC--NH--CH), (--CO--NH--CR--O--R), (--CO--NH--NH--CO--), (H--COR--NH--CR--O--R), (--CONH--R--NH--CO), where R=CH.sub.3, CH.sub.2, NH.sub.2, C.sub.2H.sub.5, and increasing the number of cross-linking (N--N) connections.

These features reduce the tissue response of the patient upon implantation of the hydrogel, provide for high stability during implantation by reducing the rate of resorption and shrinkage, and reduce the possibility of colonization and proliferation of microorganisms within the hydrogel, including those from the recipient's body.

DETAILED DESCRIPTION OF THE INVENTION

In order to produce the biocompatible hydrogel of the present invention the following reagents were used: Acrylamide: C.sub.3H.sub.5NO, molecular weight--71.08, white crystalline powder without any smell; melting temperature 84.5.degree. C.; made by Sigma (Catalogue <<Reagents for Biochemistry and Research in the Area of Natural Sciences>> SIGMA, 1999, p. 47, catalogue No.A8887); N,N'-methylene-bis-acrylamide: C.sub.7H.sub.10N.sub.2O.sub.2, molecular weight--154.16, white crystalline powder without any smell; melting temperature 85.degree. C., made by Sigma (Catalogue <<Reagents for Biochemistry and Research in the Area of Natural Sciences>> SIGMA, 1999, p. 696, catalogue No.M7256);

N,N'-ethylene-bis-acrylamide: C.sub.8H.sub.12N.sub.2O.sub.2, molecular weight--168.2, made by Sigma (Catalogue <<Reagents for Biochemistry and Research in the Area of Natural Sciences>> SIGMA, 1999, p. 428, catalogue No.E2763); Poviargolum--argentiferous detergent, powder, No.97/167/7 from Institute of High-Molecular Compounds of Russian Academy of Sciences (Russia), F. G.: 11.2 (Register of Drugs, Used in Russia--Aptekar, 2001, p.1067), which is a superfine metal silver, stabilized by poly-N-vinylpyrrolidone-2-ethylenebis(oxyethylenenitrilo)tetraacetic acid (see WWW:http;//home.comset.net/poviarg/; RU 2088234, published on Aug. 27, 1997); Ethylene-bis(oxyethylenenitrilo)tetraacetic acid, molecular weight 380.35, melting temperature 249.degree. C., made by Aldrich (Catalog handbook of Fine Chemicals Aldrich, 1994 1995, p.664, catalogue No.23,453-2); 1-vinyl-2-pyrrolidone: C6H9NO, molecular weight 111.4; melting temperature 93.degree. C.; white powder; made by Fluka (Catalogue Fluka Chemika-Biochemika, Switzerland, sFr, 1993/94, p.1384, catalogue No.95060); Ammonium persulfate: (NH4)2 S2O8--molecular weight 228.19; colourless flat crystals; decomposition temperature 120.degree. C.; made by Sigma (Catalogue <<Reagents for Biochemistry and Reasearch in the Area of Natural Sciences>> SIGMA, 1999, p.117); Hydrogen peroxide: H.sub.2O.sub.2--molecular weight 34.0; colourless fluid, density at 0.degree. C. 1,465; melting temperature -0.89.degree. C.; made by Sigma (Catalogue <<Reagents for Biochemistry and Research in the Area of Natural Sciences>> SIGMA, 1999, p. 556, catalogue No.H6520);

All above-mentioned monomers shall be suitable for biological purposes and shall not require additional cleaning.

Water shall be twice-distilled and pyrogen-free (pH=5.6).

The method must be conducted the following way:

In order to prepare the compound we take twice-distilled pyrogen-free water with pH of 5.6.

We prepare an aqueous solution of acrylamide and cross-linking agents, for example, the mixture of N,N'-methylene-bis-acrylamide, N,N'-ethylene-bis-acrylamide and poviargolum (taken in the given mass ratio within the following limits: 65.0 99.5:0.2 6.5:0.2 34.0:0.1 3.0 respectively), or a mixture of N,N'-methylene-bis-acrylamide, N,N'-ethylene-bis-acrylamide, poviargolum, vinylpyrrolidone or ethylenebis(oxyethylenenitrilo) tetraacetic acid, or combinations of the latter two (taken in the given mass ratio within the following limits: 65.0 99.4:0.2 6.5:0.2 34.0:0.1 3.0:0.1 2.5 respectively), such that the total mass of monomers in the solution makes up 2.0 15.0%. (By modifying the amount of the monomers in the solution, we get the hydrogel of different density and flexibility).

Into this solution we introduce the polymerization initiators hydrogen peroxide in the amount of 0.1 0.3 mass % or ammonium persulfate in the amount of 0.0006 0.03 mass %, or a mixture of these two in any correlation and amount, not exceeding the sum of their individual maximum vales. By modifying the amount of hydrogen peroxide and ammonium sulfate, we receive a substance with a pH within the desired range of 3.5 7.5.

The above mixture is filtered through a bactericidal polymer filter, (for example Sigma (USA) part number F8273 with a pore size of 0.45 .mu.m) and then incubated at a temperature of 20 90.degree. C. for 2 24 hours. After incubation, the product, which looks like a gel, is washed with hot water. For that purpose the gel is placed into a reservoir with water of 90 100.degree. C. with the ratio of the gel and water of from 1:8 to 1:10 for 4 6 hours. Then the second stage of the incubation is carried out at the temperature of 107 130.degree. C. for 1.0 1.5 hour.

The gel thus produced is packed in the necessary amount into bottles or syringes and sterilized by autoclaving (at the temperature of 120.degree. C. and a pressure of 1.2 atm.) for 20 minutes.

We carried out physical and chemical, medical and toxicological examinations of the gel samples, including those received in the below-mentioned Examples 1 5 (see Original Patent), in accordance with ISO 10993 "Evaluation of Biological Effect of Medical Devices", "Methodical Directions on Sanitary and Hygienic Evaluation of Rubber and Latex Devices, Meant for Medical Purposes" (Ministry of Public Health of USSR, Moscow, 1988) and methodological recommendations in "Admissible amount of migration of chemical agents, that are exuded from polymer and other materials in contact with food hydrogels and methods of their determination" Sanitary Rules and Standards 42-122-42-40-86.

The determination of the concentrations of the monomers for acrylamide, N,N'-methylene-bis-acrylamide, N,N'-ethylene-bis-acrylamide and ethylenebis(oxyethylenenitrilo) tetraacetic acid were carried out in accordance with the methods, described in the work of V. V. Kuznetsov et al. <<Determination of Acrylamide in Polyacrylamide gels"// The 52-nd Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy.--New Orleans, La., 2001, Abstract Book, N.1648.

These researches revealed that the hydrogel of the present invention has the following physical and chemical characteristics: Appearance--gel Colour--from colourless to semitransparent dark brown, opalescent; Refractive index--1.328 1.360; Density--1.0 1.2 g/cm3; pH--3.5 7.5; Monomer content--up to 0.4 ppm; Bromation rate--not more than 1.0 (mg of bromine for 1 L of gel).

Sanitary and chemical examination revealed the following: Migration of metals--Cu, Fe, Ni, Zn, Al, Ti, Ag from the hydrogel into the aqueous extract, determined by an atomic absorptive method, was found to be below the limits of detection for the instrument (0.02; 0.05; 0.05; 0.02; 0.005; and 0.04 mg/l respectively), which is a figure considerably lower than those allowable for drinking water; Migration of sodium constituted not more than 0.12 mg/l while the admissible level is 200 mg/l for drinkable water;

Toxicological examination showed that aqueous extracts from the hydrogel did not reveal a hemolytic effect in "in vitro" experiments with isolated erythrocytes of rabbits. The hemolysis rate was found to be 0.04% while the maximum admissible rate is 2%.

Zone of growth inhibition of Staphylococcus aureus by the agar disk diffusion method, determined as described in the guide "Method for Determination of Microorganisms' Antibiotic Perceptibility by Disk Diffusion Technique", Moscow, Ministry of Public Health, 1984, was 1.5 3 mm, depending on the density of the sample (polyacrylamide content).

In an acute toxicity study in white mice with parenteral introduction of hydrogel samples in the amount of 50.0 ml per 1 kg of animal weight, there were neither loss of animals nor clinical signs of intoxication: general condition of the mice under the experiment, their behaviour, eating, state of hair had no difference from those in an untreated control group.

Autopsy of the mice from this experiment showed that the tissues at the site of hydrogel injection, regional lymph nodes, internals (liver, kidneys, spleen) were within the boundaries of physiologic norms and control.

We have not found any statistically relevant differences in dynamics of body weight, clinical and biochemical blood indices, or coefficients of internals of the animals under experiment in comparison with the control group with the subcutaneous implantation of the gel for 2.5 months.

We have not found any sensitizing effect of the hydrogel in an immunologic diagnostic reaction of mast cells' degranulation reaction.

Microkernel test on bone marrow showed no mutagenic effect of the hydrogel. Histological examination of area where the hydrogel was implanted, and internals (liver, kidneys, spleen, testicles) showed a slight tissue response to the hydrogel only in the first days after the implantation. There were no dystrophic and necrotic changes in the organs.
 

Claim 1 of 18 Claims

1. A polyfunctional biocompatible hydrogel comprising: water; and a cross-linked copolymer comprising: acrylamide; N,N'-methylene-bis-acrylamide; N,N'-ethylene-bis-acrylamide; and poviargolum.

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