RU2652126C1 - Method of obtaining glucose-sensitive polymer hydrogels - Google Patents

Abstract

FIELD: biochemistry.

SUBSTANCE: invention relates to the field of biochemistry and medicine, to a method for the preparation of glucose-sensitive polymeric hydrogels that can be used as carriers for controlled release of insulin upon the appearance of glucose. A process for the preparation of glucose-sensitive polymeric hydrogels is carried out by polymerization under the action of an oxidation-reduction initiator of an aqueous solution containing 0.15–0.5 % of N-(2-D-glucose)acrylamide, 3.0–5.0 % of acrylamide, 0.01–0.06 % of N, N-methylenebisacrylamide, 0.05–0.15 % of concanavalin A and 0.03–0.10 % of mercaptoacetic acid.

EFFECT: obtained hydrogel provides the release of an increased amount of insulin at the initial stage of the appearance of glucose in the subsequent regulated release in the subsequent time of action.

1 cl, 3 tbl, 7 ex

Description

The invention relates to the field of polymer chemistry, biochemistry and medicine, and in particular to a method for producing glucose-sensitive polymer hydrogels, which can be used as carriers for the controlled release of insulin when glucose appears in the environment.

As you know, the vital substances of a polypeptide nature (enzymes, inhibitors, hormones, etc.) are synthesized inside the body, and do not enter it with food. The latter is impossible, since proteolytic enzymes involved in the digestion process hydrolyze proteins to amino acids. Therefore, to eliminate the deficiency of such a protein, it is injected into the body, bypassing the digestive tract.

One of these drugs is the pancreatic hormone insulin, a polypeptide produced by the β-cells of the pancreatic islets of Langerhans. Insulin is a universal hormone that affects a large number of processes occurring in the body, such as: glucose transport through the membrane; glucose utilization; inhibition of gluconeogenesis (the formation of glucose from non-carbohydrate precursors); glucose metabolism; lipid metabolism; protein metabolism; cell reproduction, etc. Partial or complete insulin deficiency leads to diabetes mellitus. Already, according to some estimates, about 300 million people suffer from diabetes in the world, and there is a clear tendency to increase the growth in the number of patients.

Under physiological conditions, insulin is produced by the pancreas in response to an increase in glucose concentration. The threshold for insulin secretion is a glucose concentration of 80-100 mg / 100 ml of blood, and the maximum secretion rate is achieved at a glucose concentration of 300-500 mg / 100 ml. The secretion of insulin is biphasic. An immediate response or the first phase of the reaction begins within one minute after increasing the concentration of glucose, lasts the first 8-10 minutes. Then, the rate of insulin release sharply slows down and the second slower and longer phase of almost uniform release of insulin sets in, abruptly breaking off after glucose removal [Murray R., Grenner D., Meyes P. Rodwell V. Human biochemistry. - M.: Mir 1993. S. 247].

Usually (normal), insulin enters the liver through blood vessels connected to the portal hepatic vein. The liver, in turn, controls the amount of insulin reaching other organs and tissues. When injecting insulin (and this is the most common way to treat diabetes), this control is absent, the physiological relationship between the concentrations of insulin and glucose is violated, which is the reason for such complications of diabetes as cardiovascular diseases, cerebrovascular disease, etc. .d. [M. Saffran, in: Targeting of Drags: The Challenge of Peptides and Proteins, G. Gregoriadis (ed.), Plenum Press, New York (1992), pp. 89-95].

Since an increase in the concentration of glucose in the blood is the main stimulus for the secretion of insulin by the pancreas in a living organism, it seems extremely promising to create systems that, when implanted into the patient's body, would control the release of insulin in response to an increase in the concentration of glucose by a natural, two-phase mechanism.

A known method for producing glucose-sensitive polymer hydrogels by the interaction of carbohydrate derivatives of insulin with concanavalin A [Sato S., Yeong S.Y., McRea Y.C., Kim S.W. Self-regulating insulin-delivery systems. II. In vitro studies. // J. Control. Release 1984. V. 1. P. 67-77]. As carbohydrate derivatives of insulin, N-succinyl glucosamine-insulin, N-glutaryl glucosamine-insulin, n- (glutaryloamido) phenyl-α- (D-glucopyranoside) -insulin, n- (glutaryloamido) phenyl-α- (B-mannopyranoside) - insulin, n- (succinylamido) phenyl-α- (D-glucopyranoside) -insulin, n- (succinylamido) phenyl-α- (D-mannopyranoside) -insulin. Concanavalin A is a protein with a molecular weight of 102,000, having four carbohydrate binding sites. When reacting with a carbohydrate derivative of insulin, concanavalin A acts as a crosslinking agent, forming complex compounds with carbohydrate residues. When glucose appears in the environment, it displaces the carbohydrate derivative of insulin from its complex with concanavalin A, as a result of which the hydrogel decomposes and the carbohydrate derivative of insulin is released into the environment.

The disadvantage of this method is the release into the environment of all the components of the polymer hydrogel, as well as the impossibility of using this system for the controlled release of insulin, and not its carbohydrate derivative.

A known method for producing glucose-sensitive polymer hydrogels by the interaction of dextrans with concanavalin A [Tang M., Zhang R., Bowyer A., Eisenthal R., Hubble J. A reversible hydrogel membrane for controlling the delivery of macromolecules. // Biotechnol. Bioeng. 2003. V. 82. No. 1. P. 47-53].

The disadvantage of this method is the release into the environment during interaction with glucose of all components of the polymer hydrogel.

A known method for producing glucose-sensitive polymer hydrogels by the interaction of a derivative of glucose with concanavalin A [I. L. Valuev, V.V. Chupov, G.A. Sytov, L.I. Valuev, N.A. Plate. Phase reversible hydrogels based on acrylamide and N- (2-D-glucose) acrylamide. // High Molecule. soed., 1997, T. 39B, No. 4, S. 751-754].

As a derivative of glucose, a copolymer of 3.9-14.5 mol% is used. N- (2-D-glucose) acrylamide and 85.5-96.1 mol%. acrylamide, and the interaction is carried out by mixing an aqueous solution containing 10-25% wt. a copolymer of N- (2-D-glucose) acrylamide and acrylamide, with an aqueous solution of concanavalin A.

When reacting with a glucose-containing polymer, concanavalin A acts as a cross-linking agent, forming complex compounds with glucose residues belonging to different polymer chains. As a result of the reaction, a hydrogel is formed, the degree of swelling in water is determined by the content of N- (2-D-glucose) acrylamide units in the copolymer and the ratio N- (2-D-glucose) acrylamide / concanavalin A. When a certain amount of glucose is added, it displaces concanavalin And from the complex, resulting in the destruction of the hydrogel with the formation of a soluble copolymer of N- (2-D-glucose) acrylamide and acrylamide and a soluble complex of concanavalin A - glucose. If insulin was previously introduced into the hydrogel, then upon the destruction of the hydrogel, insulin is released into the solution. Thus, the synthesized system is a pancreas model capable of secreting a certain amount of insulin in response to the appearance of a certain amount of glucose in a solution.

The disadvantage of this method is the one-step isolation of insulin, as well as the formation of a soluble synthetic copolymer of N- (2-D-glucose) acrylamide with acrylamide during the release of insulin. When used in living organisms, this copolymer accumulates in the body, adsorbing on cell membranes and leading to a toxic effect.

The closest in technical essence and the achieved results is a method for producing glucose-sensitive polymer hydrogels by polymerization under the action of a redox initiator of an aqueous solution containing 0.15-0.5% wt. N- (2-D-glucose) acrylamide, 3.0-5.0% wt. acrylamide, 0.01-0.06% wt. N, N-methylenebisacrylamide and 0.05-0.15% wt. Konkanavalin A [Valuev I.L., Vanchugova L.V., Valuev L.I., Glucose-sensitive hydrogel systems // Vysokomol. conn. 2011.V. 53A. No. 5. S. 691-695].

The polymer chains in the obtained hydrogels are crosslinked by covalent bonds of N, N-methylene bisacrylamide and bonds between concanavalin A (Con A) and N- (2-D-glucose) acrylamide (GAA) units. In the glucose solution, the Con A-link GAA bond is replaced by the Con A-glucose bond and the degree of swelling of the hydrogels increases, which leads to the release of insulin previously introduced into the hydrogel. The release of insulin occurs evenly in one stage and after 60 minutes it amounts to 80% of the insulin originally introduced into the hydrogel.

The disadvantage of this method is the uniform release of insulin.

The objective of the invention is to increase the rate of release of insulin at the initial stage.

The technical result achieved by using the invention is the implementation of the physiological way of changing the concentration of insulin with increasing glucose levels in solution.

The technical result is achieved by the fact that in the method for producing glucose-sensitive polymer hydrogels by polymerization under the action of a redox initiator of an aqueous solution containing 0.15-0.5% wt. N- (2-D-glucose) acrylamide, 3.0-5.0% wt. acrylamide, 0.01-0.06% wt. N, N-methylenebisacrylamide and 0.05-0.15% wt. concanavalin A, the aqueous solution additionally contains 0.03-0.10% wt. mercaptoacetic acid.

Example 1

At room temperature and with constant stirring in 94.6 ml of distilled water, 5.0 g of acrylamide, 0.3 g of N- (2-D-glucose) acrylamide, 0.01 g of N, N-methylene bisacrylamide, 0.05 g of concanavalin are dissolved A and 0.03 g of mercaptoacetic acid. After complete dissolution, 0.04 g of ammonium persulfate and 40 μl of N, N, N ′, N′-tetramethylethylenediamine are added to the solution. The resulting solution is degassed in a round bottom vessel on a water-jet pump (pressure 50 mmHg) and left at room temperature until the reaction is completed (1.0-1.5 h). The completion of the reaction is indicated by the formation of a colorless gel. The resulting gel is removed from the vessel, crushed and washed with a 10-fold excess of distilled water. The degree of swelling of the obtained hydrogel in water is 18.1 g of water / g of dry polymer.

Examples 2-4. The process is carried out as in example 1, using various amounts of N- (2-D-glucose) acrylamide, acrylamide, N, N-methylenebisacrylamide, concanavalin A and mercaptoacetic acid (table 1).

Example 5k (control) is carried out according to the prototype method without the use of mercaptoacetic acid.

Example 6. The permeability of hydrogels for protein molecules of different molecular weights is assessed by holding the hydrogels with a protein solution at 4 ° C for 24 hours and measuring the concentration of protein in the solution spectrophotometrically at 280 them. Given the ratio of the volumes of hydrogels and solutions, calculate the number of pores available for each protein, taking as 100% the number of pores available for water. The protein used is insulin (M = 6.5 × 10 ³ ), ovomucoid (M = 31 × 10 ³ ), serum albumin (M = 67 × 10 ³ ), hexokinase (M = 96 × 10 ³ ) and alcohol dehydrogenase (M = 14 × 10 ⁴ ).

The results are shown in table 2.

It can be seen that the hydrogels obtained by the claimed method differ from the known glucose-sensitive hydrogels in their increased (on average 25%) pore content of small sizes, inaccessible to proteins with a molecular weight above 140,000.

Example 7. The saturation of hydrogels with insulin is carried out by incubating freeze-dried hydrogels with a solution of insulin (25 PIECES / ml) until equilibrium swelling. Swollen hydrogels containing 130 IU of insulin are placed in a 15 ml flow cell, through which a physiological solution (0.9% sodium chloride solution) containing 250 mg of glucose per 100 ml is pumped at a speed of 3.0 ml / min, and the amount of insulin released from the hydrogel is measured. The insulin concentration in the solution was determined spectrophotometrically at 280 nm. The rate of insulin release (U / min) is shown in table 3.

It is seen that the release of insulin into the glucose solution from the hydrogels obtained by the claimed method, occurs in two stages. The maximum speed is reached by the eighth minute and is 5.6-6.0 U / min, and then insulin accumulates in solution with a uniformly decreasing speed of 1.9-0.7 U / min. The release of insulin from hydrogels obtained by the prototype method, during the entire observation time, is uniformly reduced from 3.0-2.5 U / min to 1.5-0.6 U / min.

Thus, the present invention allows to obtain polymer hydrogels, capable of, in contrast to the known, similar to the pancreas, to secrete an increased amount of insulin at the initial stage of the appearance of glucose.

The limiting amounts of substances used in the preparation of the polymer hydrogel are determined as follows. At a concentration of N- (2-D-glucose) acrylamide below 0.15% wt. and concentrations of concanavalin A below 0.05% wt. polymer hydrogels practically do not change the degree of swelling with increasing glucose concentration, and at a concentration of N- (2-D-glucose) acrylamide above 5.0% wt. and concentrations of concanavalin A above 0.15% wt. an increase in the degree of swelling of hydrogels occurs at an extremely high glucose concentration of about 600 mg / 100 ml. At a concentration of N, N-methylenebisacrylamide below 0.01% wt. hydrogels are formed with extremely low mechanical strength, and at a concentration of N, N-methylenebisacrylamide above 0.06% wt. hard hydrogels are formed, the diffusion of insulin of which is extremely difficult. Mercaptoacetic acid in an amount below 0.03 wt. % is not able to change the nature of the distribution of pores in the hydrogel, in at a concentration above 0.10 wt. % it inhibits polymerization.

Claims (1)

A method of producing glucose-sensitive polymer hydrogels by polymerization under the action of a redox initiator of an aqueous solution containing 0.15-0.5% wt. N- (2-D-glucose) acrylamide, 3.0-5.0% wt. acrylamide, 0.01-0.06% wt. N, N-methylenebisacrylamide and 0.05-0.15% wt. concanavalin A, characterized in that the aqueous solution further comprises 0.03-0.10% wt. mercaptoacetic acid.