SU977466A1 - Polyacrylamide gel for medical and biological uses and process for producing the ssame - Google Patents

Description

The invention relates to the chemistry of polymers, in particular to a polyacrylamide gel, intended for use in pedicine and biological purposes.

Polyacrylamide gel has been known for a relatively long time and is currently used in medicine and biology exclusively for the separation of various substances.

However, the possibilities of using polyacrylamide gel for other purposes in medicine and biology are limited by the presence of toxic starting monomers

A polyacrylamide gel is known, containing as oqHOBH a copolymer of acrylamide and N, N-methylene bis-acrylamide, which is used to grow various microorganisms.

This gel is obtained by radical copolymerization of acrylamide with N, N-methylene bis-acrylamide in the presence of an animator and radical polymerization activator in aqueous solutions with a content of 3-20 parts by weight. water for 1 h, comonomers, the mixture of comonomers contains 0.01 0, 25 h N, N-methylene-bis-acrylamide for 1 h acrylamide.

At the same time, inorganic salts can be introduced into the initial medium during the synthesis of the gel at a relatively high end of 1 stration, as well as various other additives to ensure the nutritional needs of certain types of fl microorganisms.

The disadvantage of these gels is that they contain a certain amount of toxic starting monomers. In addition, since the gels are acidic (pH 3.5-4), not any nutrient substrates can be introduced into them. This circumstance limits their use.

The closest to the technical essence and the achieved result is a polyacryl20 gel for biomedical purposes, including a net copolymer of acrylamide and N, N-petylene bis-acrylamide and water as a swelling medium. This gel is used as a dense base for preparing the nutrient medium used to grow various microorganisms.

The gel is obtained by radical copolymerisation by acrylagdization with N, N-methylenebis-bis-acrylamide and a material solution in the presence of an initiator and radical polymerization activator, followed by washing the gel with water from 1ecomeolar impurities. The known method allows to obtain a non-toxic gel, which can be used in the cultivation of most species of microorganisms 2. However, the desired product can be removed while removing toxic substances, but it does not ensure the contact of the obtained gel with animal cells, tissues and organs. and man, because it does not provide isoosmoticity, which limits the scope of its use in medicine and biology. In addition, the quality of the polyacrylamide gel, which is used as the basis for growing the microorganism, is not high enough. The purpose of the invention is to expand the functional capabilities of the gel and improve its performance. This goal is achieved by the fact that polyacrylamide gel for medicinal and biologic purposes, including a net copolymer of acrylamide with α-methylene-bis-acrylamide and a bootable medium, contains saline as the swelling medium in the following ratio of components,% by weight: Net copolymer 3, 0-28,0 Physiological solution 72,0-97,0 The proposed gel is obtained by radical copolymerization of acrylics with N, N-methylene-bis-acrylamide in the presence of an initiator and an activator of radical polymerization followed by washing the gel from low in this process, the impurities are crushed, while the gel is washed in a physiological solution at the following initial monomer concentration, in May.%: Acrylamide 0.5-40 N, N-methylene bis acrylamide 2.5-12 The resulting polyacrylamide gel does not toxic and has a high porosity, hydrophilicity, elasticity, transparency, thermal stability. In addition, it has isosmoticity with biological systems (microorganisms, cells, tissues, organs), which makes it possible to stabilize its size in biological systems, as well as to increase its saturation with solutions of various substances. All this makes it possible to make a harmless contact of the polyacrylic and gel with biological systems and thus greatly expand the possibilities of using it in medicine and biology. It is advisable to expand the use of polyacrylamide gel in medicine and biology as a physiological solution to use a 0.5% aqueous solution of sodium chloride or a 0.9% aqueous solution of sodium chloride, Ringer-Lok solution, Earl solution, HeH1 $ sa solution , Wednesday 199, Wednesday Needle, 5% aqueous solution of glucose. It is recommended that polyacrylamide gel as a physiological solution contains 0.5% aqueous solution of sodium chloride, with the following content of the component 1t, wt.%: Polyacrylamide 6.0-15.0 0.5% aqueous solution of sodium chloride 84- 94 The indicated modification of a polyacrylamide gel allows one to obtain the most optimal isoosmosis with microorganisms, which makes it possible to use it as a dense basis of nutrient media for the cultivation of microorganisms. It is possible that polyacrylamide gel as a saline solution contained a 0.9% aqueous solution of sodium chloride, with the following content of components, wt%: Polyacrylamide 5.0-13.0 0.9% aqueous sodium chloride solution 82.0-95.0 This modification of a polyacrylamide gel allows one to obtain the most optimal isoosmoticity with animal and human cells, which makes it possible to use it as a carrier of nutrient substrates when cultivating cell cultures. It is proposed that polyacrylamide gel as a physiological solution contains a 5% aqueous glucose solution with the following content of components, wt.%: Polyacryllylide 4.0-20.0 5% aqueous glucose solution 80.0-96.0 Specified modification polyacrylamide gel provides optimum isoosmosis with biological properties and is used in cases where the presence of sodium chloride is beneficial; It is advisable to use polyacrylamide gel as a nutrient medium. Nutrient media, where polyacrylamide gel containing physiological saline is used as a dense base, do not contain impurities, have microbiological reproducibility and stability. They hold well in Petri dishes, are resistant to drying, are amenable to long-term storage and have a number of significant advantages over lime media.

In order to decrease; traumatizing the eye tissues during implantation by making minimal incisions, ensuring: complete reactivity of the eye membranes, it is recommended to use the above-mentioned polacrylamide gel as an artificial crystalline lens.

In order to ensure long continuous wear when correcting the refractive error over a wide range, it is proposed to use a polyacrylamide gel as a contact lens.

The polyacrylamide gel is obtained by polymerizing acrylamide and α-methylene bis acrylaglide. The reaction monomer mixture typically contains 80-99.5 wt.% Acrylamide, 0.520 wt.% N N-methylene bis-acrylamide. The initial monomers are dissolved in physiological solution. As a physiological solution, a 0.5% aqueous solution of sodium chloride or a 0.9% aqueous solution of sodium chloride, a 5% aqueous solution of glucose, or Ringer-Locke solution, Hanks, are used. Earla, or Wednesday 199, Needle and others.

By varying the amount of the initial monomers in the reaction mixture, a Polyacrylamide gel of different density and elasticity can be obtained.

The polymerization of the starting monomers can proceed without heating or by heating the reaction mixture, adding known initiators, catalysts. The rate of the polymerization reaction is directly proportional to the temperature, the amount of the catalyst. O6tJ4Ho catalysts are added in the amount of 0.05-0.1 wt.% Of the amount of the initial monomers.

The reaction mixture can be prepared by mixing dry powdered monomers, followed by dissolving in saline. The saline is heated to speed up the dissolution. Polymerization of the reaction mixture can be carried out in a volume of a predetermined shape — in glass, metal, ceramic containers, as well as containers made of synthetic materials. The gel obtained during the polymerization process repeats the shape and dimensions of the container used.

The polymerization process can be carried out in volumes simulating forgda known contact lenses, or in the form of known monolithic artificial crystalline lenses, which makes it possible to use Polyacrylamide gel as a wet contact lens, or as an artificial crystalline lens. The conditions for the polymerization process are similar.

above.

Washing the gel from toxic products can be carried out under normal conditions and at ambient temperature. In this case, the source (toxic)

the components are dissolved and transferred from the gel to the physiological solution. By replacing the saline solution, it is possible to completely remove toxic products from the gel. Usually for this is enough to change the solution three times. Heating accelerates the oTtiHBKH gel process. The process of washing the gel is carried out as described above and when obtaining a soft

tact lenses or crunch / tick. The resulting gel is amenable to stamping, cutting.

Sterilization of the gel can be accomplished by means of tag, radiation, and chemical means. The choice of the sterilization method and its mode are determined by the conditions of the particular task. Thus, the gel becomes suitable for use as a solid basis for obtaining nutrient media, for the purpose of cultivating microorganisms, either an artificial lens or a soft contact lens, and can be stored until and use.

Saturation of the gel with substrates for feeding microorganisms and cell cultures can be carried out before or after sterilization. Method selection

saturation is determined by the specific composition of the nutrient substrate. In the presence of thermolabile components in the nutrient substrate, saturation is carried out after sterilization.

The composition of nutrient substrates is determined by the nutritional needs of specific groups or species of microorganisms and cells. Nutrient substrates can be used to saturate the polyacrylamide gel prepared in accordance with the invention to meet the nutritional needs of virtually all known types of microorganisms and cells, including

natural, semi-synthetic and synthetic compositions of substrates or their mixtures.

To determine the quality of nutrient media, where polyacrylamide gel containing saline is used as a solid base, as well as studying the biological properties of microorganisms, animal cells and humans, known r-5 research methods are used. The optical properties of soft contact lenses and artificial polyacrylamide gel crisps are also determined by known methods, containing a physiological solution. Polyacrylamide gel is used as a carrier (dense substrate) of nutrient substrates necessary for growth, reproduction and development of microorganisms. The polyacrylate plates 1 {one gel can be made round in diameter of the cups, square-Elg or rectangular and in the sizes of cover and slide, as well as blocks of various sizes and shapes and used after saturation with nutrient substrates for macro-micro-cultivation of various groups, microorganism, animal and human cells. The use of polacrylamide gel as a dense basis of nutrient media provides the latter with microbiological inertness, which increases the biomass yield of microorganisms. The preparation of polyacrylamide gel plates can be automated,. Gel plates do not require special sterilization methods. Their sterilization can be carried out; General information 1 and methods and under normal conditions. For example, in an autoclave at a pressure of 1-1.5 atm or flowing steam. It is possible to store ready-to-use plates of polyacrylamide gel for a long time. Polyacrylamide gel has a known and permanent composition that ensures the reproducibility of the dense base of nutrient media and the associated standardization of microbiological studies, which allows comparing the results of various studies. The use of a polyacrylamide gel as a soft contact lens can provide a correction of from -20 OD to + 20 OD .. In addition, soft contact lenses can be used simultaneously with the therapeutic purpose for the prolonged action of medicinal substances (antibiotics sulfanilamides, mydriatic, myotin Cove and other drugs). It is possible to use soft contact lenses as an additional link when sealing a wound with emergency and planned eye surgery or as a self-contained method in the same situations. The lens of the proposed polyacryl gel can be used as follows for cosmetic purposes and as a means of reducing the light scattering of aniridia, iridodilase, persistent mydriasis, and albinism. Polyacrylamide gel can be used as an artificial lens and used to correct refractive errors after extracapsular cataract extraction simultaneously or as a second step. The lens can have a predetermined shape, size and optical power. Example 1. To obtain a polyacrylamide gel according to the invention, three solutions (A, B and C) were prepared according to the following procedure (the quantities of the starting components are indicated per 1000 ml of the basic solution). Preparation of Solution A: 5 ml of tetramethylethylenediamine is dissolved in 995 ml of a 0.5% aqueous solution of sodium chloride and stored in a dark container at 4 ° C under refrigerated conditions until consumed (shelf life 6-8 months). Preparation of Solution B: 7.35 g of methylene bis-acrylamide is dissolved in 350 MP of a 0.5% aqueous solution of sodium chloride heated to before and then 280 g of acrylamide are added, which is stirred until complete dissolution. The resulting solution is filtered through a cotton-gauze filter, up to 1000 ml of a 0.5% aqueous solution of sodium chloride is added and stored in a dark container under refrigerator conditions until used (storage time 6-8 months). Preparation of Ct solution 1.4 g of ammonium persulfate is dissolved in 1000 ml of a 0.5% aqueous solution of sodium chloride and stored in a dark container until consumed (storage time 4-6 weeks). From the prepared solutions 1A, B, and c) the reaction mixture is prepared. To do this, 2 volumes of solution B and 4 volumes of solution C are added to 1 volume of solution A (Volumetric ratios of the main solutions can be altered depending on the required elasticity or gel density). The reaction mixture is poured into a slot formed by two plane-parallel glass plates 3 mm thick. The polymerization process takes place within 15 minutes. The glass plates disassemble and release the polyacrylamide gel plate that forms. Round plates with a diameter of 70 mm are punched from a gel plate. The resulting discs are placed in a container and poured with a 0.5% aqueous solution of chlorine: addition of sodium at the rate of 20 ml of solution per disc. - Disks Next, scrape in 0.5% with Mr. solution of sodium chloride for 12 hours solution every 4 hours. After 12 hours the solution is drained.

The resulting polyacrylamide gel discs contain May.%

Polyacrylamide 11.0 0.5% aqueous solution of sodium chloride 89.0 The resulting polyacrylamide gel is non-toxic, has a high porosity, hydrophilicity, elasticity, transparency, thermal stability.

The cells of various types of microorganisms, which were applied to the gel for a long time (3 months or more) time, remained in shape, size and viability, which indicates that this gel is isosmotic with the cells of microorganisms.

The gel was well saturated with substrates for nourishing microorganisms, for example, peptone broth, and therefore it was used as a solid basis for obtaining nutrient media for the cultivation of various groups of microorganisms (Escherichia, Salmonella, Shigella, Protey, Staphylococcus, etc.).

To saturate, washed discs are filled with Hottinger's broth with amine.

300 mg of nitrogen at the rate of 10 ml of broth per disc and sterilized with steam under pressure at 30 min. During this time, the discs were filled with broth and sterilized. Saturated broths and sterile discs, being sterile, were placed in sterile tea, poured and sown with E. coli Microorganisms are incubated at 37 ° C for 24 hours, during which time the culture of Escherichia coli in the form of colonies grew on the surface of the disks.

With the growth on the gel, the microorganisms retained their biological properties: the nature of growth and reproduction, the shape of cells and colonies, morphological, tinctorial, cultural, biochemical, serological properties, antigenic structure, phagolysis. At the same time, the biomass of the grown microorganisms with the same sowing dose exceeded the biomass of the same microorganisms grown on the m-peptone agar.

five

The survival rate of the studied microorganism species was determined on a polyacrylamide gel according to the invention and on a known gel (2) 1

0 Results are tabulated.

Staphy1OCOCCUS Example 2. A polyacrylamide gel containing, in wt.%: Polyacrylamide 8.0, physiological saline, is prepared by the method described in Example 1. A 0.9% aqueous solution of sodium chloride is used as a physiological solution. The resulting polyacrylamide gel was not toxic, had a high

passion, hydrophilicity, elasticity, transparency, thermostability.

The gel is well saturated with substrates for nourishing microorganisms, animal cells and humans. . .

A 2% suspension of sheep erythrocytes (0.2 ml) applied to the surface of the gel was not subjected to hemolysis, and the applied fibroblasts, HEI and KB cells, remained in shape, size and viability for up to 2 days at. The intracirculation and grafting of white plates of the obtained gel with dimensions of 1 cm x 1 cm, 3 cm for 5 days did not change the initial 2; sizes of plates. The plates remained transparent, did not cause reactive changes from the surrounding organs and tissues, were well tolerated by animals even with one-parameter implantation of 2-3 plates to one animal,

Example 3. Polyacrylamide gel in accordance with the invention containing, may. %: polyacrylamide 0.3 physiological solution 97.0, obtained by the method described in example 1. As a physiological solution using RingerLock solution,

The properties of the resulting polyacrylamide gel are similar to those described in 2.

In addition, the shape, size and viability of fibroblasts, cells of IEI a and CB were maintained on this gel for up to 4 days at.

Example 4. Polyacrylamide gel, in accordance with the invention, containing, in May.%: Polyacrylamide 1.0 physiological solution 89.0, is obtained by the method described in Example 1 Hanks solution is used as physiological solution,

The properties of the obtained polyacrylamine gel were similar to those described in example 3.

In addition, the gel plates were stained in pink, and the shape, size, and viability of fibroblasts, Hea and CB cells, were maintained for up to 6 days at.

The obtained gel plates are saturated with growth medium for cell cultures containing 60% of medium 199, 20% of lactalbumin hydrolyzate, 20% of bovine serum and: used for growing Hela cells. At the same time, Hela cells grew on the surface of the gel as a typical monolayer into ordinary deadlines

Example 5. Polyacrylamide gel containing wt.%: Polyacrylamide 20.0, saline 80.0, obtained by the method written off in example 1. As a saline solution is used Earl solution.

The properties of the obtained polyacrylamide gel were similar to the properties described in examples 2 and 4.

Example 6. Polyacrylamide gel containing wt.%: Polyacrylamide 5.0, saline 95, was obtained by the method described in example 1.

As a saline solution using medium 199.

The properties of the obtained polyacrylamide gel were similar to the properties described in examples 2 and 4.

In addition, the shape, size, and viability of fibroblasts, Hela cells and CBs were maintained for up to 3-10 s at 4 ° C.

The cells grown on the gel plates were well attached to the gel, which made it possible to transfer gel blocks cut out of plates with cells and examine them under a microscope, as well as enclose the blocks with cells in microchambers ..

Example 7. Polyacrylamide gel containing, in May.%: Polyacrylamide 15.0, saline 85.0, is obtained by the method described in example 1.

B as saline using medium 199.

The properties of the obtained polyacrylamide gel were similar to the properties described in examples 2 and 4. In addition, the monolayer of cells grown on the gel plates was easily washed away, which made it possible to easily accumulate the biomass of the cells.

Example 8. Polyacrylamide gel containing, in May.%: Polyacrylamide 7.0, saline solution 93.0, obtained in the manner described with example 1.

As a physiological solution using the medium Needle.

The properties of the obtained polyacrylamide gel were similar to the properties described in examples 2 and 7.

In addition, gel plates saturated with growth medium were used to grow cell strains. A good growth of diploid cells was observed.

Example 9. Polyacrylamide gel containing Mac.% T polyacrylamide 10.0, saline 90.0, Was prepared by the method described in Example 1. A 5% glucose solution was used as saline.

The properties of the obtained polyacrylamide gel were similar to the properties described in example 2.

Claims (2)

In addition, on this gel, the shape, size, and viability of the fibroblast, But la and KP cells persisted for up to 3 days at 4 ° C. Along with this, but this gel, an acceleration of growth and an increase in the biomass of microorganisms containing saccharolytic enzymes were noted. The absence of sodium chloride in the composition of the gel significantly simplified the determination of the amount of sodium chloride in the cells of microorganisms. Example 10 (comparative). Prepare a polyacrylamide gel containing, in May.%: Polyacrylamide 2.0, saline 98.0. A 0.5% aqueous solution of sodium chloride is used as a physiological solution. At the same time, the obtained polyacrylamide gel had a semi-liquid consistency, which did not allow to obtain gel plates, to carry out their washing and saturation with substrates for nourishing microorganisms, human cells and human cells, microorganisms and cell culture. After the addition of the liquid nutrient substrates, the gel was dissolved in them and the tertiary gel structure. Example 11 (comparative). Prepared polyacrylamide gel containing, in May.%: Polyacrylamide 29.0, saline 71.0. About 9% aqueous solution of sodium chloride was used as a physiological solution. At the same time, the resulting polyacrylamide gel had a very dense consistency and was brittle at bends. Washing the gel plates from the initial components was ineffective, it took a long time — 15 or more days. The gel was poorly saturated with substrates for nourishing microorganisms and cell cultures, dried quickly with the appearance of cracks and was poorly fixed in Petri dishes. Thus, the use of isotope will make it possible to obtain polyacrylate gels with improved performance and dissolve their field of application in medicine and biology. Claim 1. Polyacrylamide gel for meico-biological purposes, comprising an acrylamide etched copolymer with an N, N-methylene bis-acryl-imide and a swelling environment, characterized in that, in order to enhance the functionality of the gel and improve its performance, medium swelling it contains saline, in the following ratio of components, wt.%: Mesh copolymer 3.0-28.0 Physiological solution 72.0-97.0 2. Method for preparing polyacrylamide gel for biomedical purposes by radic flax copolymerization of acrylamide with N, N-methyles-bis-acrylamidop in the presence of an initiator and radical polymerization activator followed by removal of the gel from low molecular weight impurities, characterized in that the copolymerization and washing of the gel is carried out in physiological solution at the next initial concentration of monomers, wt. %: AcryLamide 0.5-40 NN-methylene bis2, 5-12-acrylamide Sources of information taken into account during the examination 1. Patent RSL No. 3046201, cl. 195-100, 19G2. 2. USSR author's certificate number 659619, cl. From 12 to 1/06, 1972 (prototype).