Multifunctional biocompatible hydrophilic gel contains, in % (w/w), 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 and water--q.s. to 100. The hydrophilic gel is produced by co-polymerization of the above mentioned monomers in water medium in the presence of a peroxide initiator of polymerization in three stages which include (1) incubation of the reaction mixture at 20-30.degree. C. for 12-24 hours, (2) exposure to .gamma.-radiation at a dose of 0.4-1.0.times.10.sup.6 rads, and (3) incubation at a temperature of 100-130.degree. C. and pressure of 0-1.2 atm. for 20-40 minutes.

DISCLOSURE OF THE INVENTION

The applied invention is primarily aimed at increasing the time of physiological activity of heterogeneous cells placed in a polyacrylamide gel when in a recipient's body.

The second goal is to reduce resorption of a hydrophilic gel and make it less permeable for macrophages after implantation into a recipient's body.

The third goal is to reduce tissue response to gel implantation by minimizing the contents of free radicals and monomers in a hydrophilic gel.

The problems are solved by proposing a multifunctional biocompatible hydrophilic gel consisting of polyacrylamide and water, which, according to the invention, it contains as the polyacrylamide a cross-linked copolymer of acrylamide, methacrylamide and cross-linking agents--2-hydroxyethyl methacrylate and N,N'-methylene-bis-acrylamide.

The above-mentioned polyacrylamide contains components in the following proportions, in % (w/w): 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.

The above mentioned polyacrylamide makes up from 3.0 to 10.0% (w/w) of the total weight of the biocompatible hydrophilic gel.

The above-mentioned biocompatible gel contains components in the following proportions, in % (w/w): 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--sufficient quantity (hereafter q.s.) to 100.

The hydrophilic gel contains bidistilled apyrogenic water as the water.

The multifunctional biocompatible hydrophilic gel has pH 3.5-4.5.

The above-mentioned hydrophilic gel is suitable for injection and packed in syringes.

The above mentioned hydrophilic gel is easy for capsules to form around it in a human or animal body, for which purpose it is implanted into the human or animal body, with subsequent placement of a selected cell culture into it.

The above mentioned goals are also accomplished by proposing a method for manufacturing a multifunctional biocompatible hydrophilic gel by the way of multistage copolymerization of monomers with cross-linking agents in an aqueous medium in the presence of a peroxide initiator of polymerization, in which, according to the invention, acrylamide and methacrylamide as monomers and 2-hydroxyethyl methacrylate and N,N'-methylene-bis-acrylamide as cross-linking agents are taken in the following proportions, in % (w/w): 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--q.s. to 100, and copolymerization is run in three stages which include (1) incubation of the reaction mixture at 20-30.degree. C. for 12-24 hours, (2) exposure to .gamma.-radiation at a dose of 0.4-1.0.times.10.sup.6 rads, and (3) incubation at a temperature of 100-130.degree. C. and pressure of 0-1.2 atm. for 20-40 minutes.

After the first stage of copolymerization, the half-finished product is washed with hot water with a temperature of 70-110.degree. C. for at least 3 hours at a pressure of 0-1.2 atm., the gel and water being taken in the ratio by weight 1:8-10.

Hydrogen peroxide and/or ammonium persulfate used as initiators of copolymerization are added at the rate of no more than 0.33% (w/w) of the total weight of starting materials.

Bidistilled apyrogenic water is used as aqueous medium.

The gel obtained by this method is placed or packed in syringes and used for implantation into a human or animal body where it becomes a basis for formation of a capsule and serves as a carrier for subsequently placed selected cell cultures.

The applied hydrophilic gel can be produced by different methods, and the applied method in no way excludes other approaches to its making, either direct or indirect, and presents one of possible ways of manufacturing a hydrophilic gel of the given composition and properties.

Obviously biological active properties of a hydrogel made on the basis of a three-dimensional net of monomers cross-linked by cross-linking agents will largely depend on the structure of the cross-linked polymer.

The structure of the cross-linked polyacrylamide, in turn, will depend on the conditions its synthesis, namely, (1) the quality and proportions of the starting materials, including cross-linking agents and initiators of polymerization which fit into the copolymer structure (at NH--, CH--, COOH--, NH.sub.2-- and CH.sub.2-groups) by means of chemical and hydrogen bonds, and (2) the mode of polymerization. This is because the cross-linking process is known to produce substantial changes in physical and chemical properties of polyacrylamide derivatives, caused by branching of the main chains, formation of cross-linked structures, and partial destruction (see Savitskaya, M. N., and Kholodova, Yu. D. Polyacrylamide, Tekhnika Publishing House, 1969, p. 113).

The substance of the invention lies in that the formulation of a polyacrylamide gel and the operating conditions of co-polymerization are chosen, which serve to increase the time of physiological activity of heterogeneous cells when in a recipient's body.

The use of the applied method for manufacturing the applied polyacrylamide gel allowed a final product to be obtained with fewer unbound amino groups, free NH.sub.2-radicals and unsaturated double bonds. Also, a higher degree of cross-linking is achieved as a result of both formation of structural groups such as (--H.sub.2C--NH--CH.sub.2--), (--CO--NH--CR.sub.2--O--R), (--CO--NH--NH--CO--), (H--COR--NH--CR--O--R), (--CONH--R--NH--CO), where R is CH.sub.3, CH.sub.2, NH.sub.2, C.sub.2H.sub.5, or C.sub.3H.sub.7, and an increase in number of cross links, i.e. N--N bonds.

All this taken together made it possible to obtain a hydrophilic gel with higher resistance to resorption and shrinking while in a recipient's body, and to create conditions for survival of the cells placed in the gel.

EXAMPLES OF EXERCISE OF THE INVENTION

Reagents used for manufacture of the applied biocompatible hydrophilic gel:

Acrylamide: C.sub.3H.sub.5NO, molecular weight 71.08, white crystalline odorless powder; melting point 84.5.sup.0N; supplied by Sigma (Catalogue <<Biochemicals and reagents for molecular biology and life sciences research>> SIGMA, 1999, p. 47, cat. No. 8887);

Methacrylamide: C.sub.3H.sub.7NO; molecular weight 73.08, white powder; melting point 111.degree. C.; supplied by Fluka (Fluka Katalogue "Chemica-Biochemica", Fluka AG, Switzerland, 1986/87, p. 1151);

2-hydroxyethyl methacrylate: C.sub.6H.sub.10O.sub.3, molecular weight 130.1, liquid; boiling point 205-208.degree. C.; density 1.07 g/ml; supplied by Sigma (Catalogue <<Biochemicals and reagents for molecular biology and life sciences research>> SIGMA, 1999, p. 567, cat. No. H8633);

N,N'-methylene-bis-acrylamide: C.sub.7H.sub.10N.sub.2O.sub.2, molecular weight 154.16, white crystalline odorless powder; melting point 185.degree. C., supplied by Sigma (Catalogue <<Biochemicals and reagents for molecular biology and life sciences research>> SIGMA, 1999, p. 696, cat. No. M7256);

Ammonium persulfate: (NH.sub.4).sub.2 S.sub.2O.sub.8--molecular weight 228.19; colorless plane crystals; breaking temperature 120.degree. C.; supplied by Sigma (Catalogue <<Biochemicals and reagents for molecular biology and life sciences research>> SIGMA, 1999, p. 117);

Hydrogen peroxide: H.sub.2O.sub.2-- molecular weight 34.0; colorless liquid, density at 0.sup.0N -1.465; melting point -0.89.degree. C.; supplied by Sigma (Catalogue <<Biochemicals and reagents for molecular biology and life sciences research>> SIGMA, 1999, p. 556, cat. No. H6520).

All the above mentioned monomers must be of biological grade and suitable for use without additional purification.

Water must be bidistilled and apyrogenic (pH=5.6).

The method is exercised as follows:

To prepare a reaction mixture, bidistilled apyrogenic water with pH 5.6 is taken.

An aqueous solution is prepared which contains acrylamide, methacrylamide, 2-hydroxyethyl methacrylate and N,N'-methylene-bis-acrylamide taken in the ratio 65.0-80.0:18.0-30.0:0.1-4.0:0.2-6.0.

The starting monomers make up from 3.0 to 10.0% of the total weight of solution. (By varying proportions between monomers in the starting mixture, hydrophilic gels of different densities and elasticities can be obtained).

To the obtained solution is added an initiator of polymerization which is either hydrogen peroxide 0.1-0.3% (w/w) or ammonium persulfate 0.0006-0.03% (w/w) or a mixture of both of them taken in any proportions but in the amounts not to exceed the sum of their maximal values. By varying proportions between hydrogen peroxide and ammonium persulfate, hydrophilic gels with pH-values in the range from 3.5 to 4.5 are obtained.

The prepared reaction mixture is caused to pass through antiseptic polymer filters, such as F8273, with filtration rating of 0.45 mm CA/CN (Sigma, USA), and then incubated at 20-30.degree. C. for 12-24 hours for co-polymerization of the starting monomers. After that, the obtained half-finished product, which looks like a gel by that time, is washed with hot water. For this purpose, it is placed in a vessel with water with a temperature of 70-110.degree. C. at a pressure of 0-1.2 atm. for 4-6 hours, the gel and water being taken in the ratio 1:8-10.

This is followed by the second stage of co-polymerization for which purpose the half-finished product is exposed to y-radiation at a dose of 0.4-1.0.times.10.sup.6 rads. After that, the half-finished product is packed into bottles or syringes, and the third stage of co-polymerization is carried out by incubating the gel at 120.degree. C. and 1.2 atm. for 20-40 minutes.

Studies of physicochemical and chemical characteristics and toxicity of the applied hydrophilic gel were conducted in accordance with ISO Standards 10993 "Biological Evaluation of Medical Devices", "Guidelines on Sanitary and Hygienic Assessment of Medical Rubber and Latex Devices" (USSR Ministry of Health, M., 1988) and Guidelines "Allowable Migration of Chemical Substances Contacting Food Hydrophilic Gels and Assay Methods", SanPiN 42-122-42-40-86 (Sanitary and Hygienic Regulations).

Assays of acrylamide monomers and N,N'-methylene-bis-acrylamide in the obtained hydrophilic gel were made following a procedure described in V. V. Kuznetsov et al. <<Determination of Acrylamide in Polyacrylamidic gels"// The 52-nd Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy.--New Orleany, La., 2001, Abstract Book, .sup.1 1648.

In these studies, the applied hydrophilic gel was found to have the following characteristics: Appearance--gel; Color--from colorless to semitransparent opal; Refraction index--1.328-1.360; Density--1.0-1.2 g/cm.sup.3; pH--3.5-4.5; Monomer contents--up to 0.4 ppm; Bromine value--max. 1.0 (mg of bromine per 1). Chemical studies showed that: no migration of metals (Cu, Fe, Ni, Zn, Al, Ti, Ag) from the hydrophilic gel to an aqueous extraction determined by the atomic absorption method is found within response limits of the analytical method (0.02; 0.05; 0.05; 0.02; 0.005; 0.04 mg/l, respectively), which values are much lower that the corresponding maximal permissible concentrations set for potable water; migration of sodium does not exceed 0.12 mg/l, which is lower than the maximal permissible concentration of 200 mg/l set for potable water.

In vitro studies of toxicity on rabbit isolated red blood cells did not reveal any hemolytic effect of aqueous extractions from the hydrophilic gel. The hemolytic index was found to be 0.04% vs. 2% set as permissible limit.

In a study of acute toxicity on white mice, no case of death or appearance of clinical signs of intoxication in the animals was reported after parenteral administration of the hydrophilic gel at a dose of 50.0 ml/kg body weight: there were no differences in general condition, behavior, food uptake and hair condition as compared to controls.

In a study after dissection of the mice, the status of tissues at the site of injection, regional lymphatic nodes, the internal organs condition (liver, kidneys, spleen) were found normal.

No statistically significant differences in body weight gain, clinical and biochemical blood test results, internal organs' condition were found between the animals with subcutaneously injected gel and controls 2.5 months post-injection.

No sensitizing effect of the hydrophilic gel was found in mast cell degranulation immunological test (MCDIT).

Micronuclear tests of the bone marrow preparations did not reveal any mutagenic effect of the hydrophilic gel. A histological assessment of the tissue samples taken at the site of injection and from the internals (liver, kidneys, spleen, testes) showed a mild tissue response only during the first days after the transplantation and no dystrophic or necrotic changes in the internals.
 

Claim 1 of 8 Claims

1. Multifunctional biocompatible hydrophilic gel containing polyacrylamide and water, distinguished by that it contains as the polyacrylamide a cross-linked copolymer of acrylamide, methacrylamide and cross-linking agents which are 2-hydroxyethyl methacrylate and N,N'-methylene-bis-acrylamide, wherein it contains components in the following proportions (w/w %): 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--sufficient quantity to 100.

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