CS270919B1 - Method for tromboresistive treatment of products being used in medicine in contact with blood - Google Patents

Abstract

This innovation concerns the method of tromboresistant surface treatment of products used in medicine in contact with blood, their treatment by albumin, heparin and glutaric aldehyde solutions, the essence of which rests in the fact the product surface gets alternately submitted 1 to 50 times to the impact of the albumin solution of 0.1 to 0.5 mass % concentration for a duration between 5 and 30 minutes, and of a heparin solution of 50 to 100 mass units/ml concentration for a duration between 5 and 30 minutes while the solutions of pH reach between 3.0 and 4.8 and their ionic potential is less than 0.5, whereupon a final fixation is made by the glutaric aldehyde solution of 0.5 to 1.0 mass 5 concentration.

Description

The present invention relates to a method of thromboresistant treatment of the surface of medical products in contact with blood by treating them with albumin, heparin and glutaraldehyde solutions, characterized in that the surface of the product is treated 1 to 50 times alternately with an albumin solution of 0.1 to 0 5% by weight, for 5 to 30 minutes and a heparin solution of 50 to 100 IU / ml for 5 to 30 minutes, the solutions having a pH of 3.0 to 4.8 and an ionic strength of less than 0.5, followed by final fixation is carried out in a solution of glutaraldehyde at a concentration of 0.5 to 1.0% by volume.

CS 270919 Вб

The invention relates to a method of thromboresistant treatment of surfaces of products used in medicine in contact with blood, by treating them with solutions of albumin, heparin and glutaraldehyde.

Products made of foreign materials activate the processes leading to thrombus formation in contact with blood. Therefore, products with reduced thrombogenicity are used for contact with blood. Such products can be obtained both by selecting the materials and technological conditions of production (Biomer, Avcothane, Pellethane) and by subsequently finishing the surfaces of already finished products. An effective way of treating products is to immobilize anticoagulants on their surfaces. The most promising is the natural anticoagulant heparin. Methods for direct ionic or covalent binding of heparin to the surface are known. However, in ionic binding, heparin is rapidly eluted from the surface in physiological solutions; in covalent binding, heparin often loses its activity.

The most technologically simple and effective surface treatment is surfactant-bound heparin. For example, surface treatment with cetylammonium chloride, heparin and glutaraldehyde (Lagergren H., Eriksson JC, Trans. Amer. Intern. Organs, 1971, 17, 10-19; Larsson, N., Stenius P., Eriksson JC, Maripuu R. , Lindberg B., Colloid Interface Sci, 1982, 90, 127). The disadvantages of this method are the use of a toxic organic surfactant amino compound, insufficient bond strength of the surfactant to the surface, the use of a high temperature (60-90 ° C) and in many cases the necessity of pretreating the surface in an oxidizing environment.

There is known a method of immobilization by physical adsorption of a covalently bound heparin complex with a natural surfactant (albumin), wherein the bound complex is preformed in solution (Hennink WE, Kim SW, Feijen J., J. Biomed. Mater. Res. 1984 18,911-926 ). The drawbacks of this method are: 1. The use of carboimide to bind heparin to albumin induces the breakdown of the carboxyl groups of heparin, which reduces the anticoagulant activity of heparin. 2. formation of a covalently bonded complex reduces the ability of the albumin to hydrophobically bind to the surface - in aqueous solutions, the complex elutes from the surface.

A method for obtaining thromboresistant polymeric products by treating their surfaces with albumin followed by heparin adsorption and glutaraldehyde fixation in water is known (Novikova S.P., Dobrova)

N. B., Popov S.A., Selezneva M.N., USSR Authentication 1 097 336 A. This method allows the bonding between the surface and the heparin-albumin complex without heparin inactivation. The disadvantages of this method are: 1. the unevenness of the complex layer formed by adsorption of heparin at neutral pH to the adsorbed albumin and the associated variation of the resulting amount of bound complex, which may have a negative effect on thromboresistant properties. 2. Low thickness of anticoagulant coating consisting of a single layer of albumin and heparin molecules, which may affect the duration of the anticoagulant effect, especially for products that have long been in contact with the organism. The above-mentioned drawbacks are eliminated by the method of thromboresistant treatment of the surfaces of medical products in contact with blood according to the invention, which consists in that the surface of the product is alternately treated with an albumin solution with a concentration of 1 to 50 times.

0.1 to 0.5 wt.%, For 5 to 30 minutes and with a heparin solution t

CS 270919 B1 2 at a concentration of 50 to 100 IU / ml for 5 to 30 minutes, the solutions having a pH of 3.0 to 4.8 and an ionic strength of less than 0.5, followed by a final fixation in a glutaraldehyde solution of 0 5 to 1.0 vol.

Albumin solutions, in addition to the first adsorption solution, heparin solutions, wash solutions, and glutaraldehyde solution have a pH of 3 to 4.8 to ensure the stability of the albumin-heparin complex. At higher pHs, the adsorbed heparin is rapidly released into solution, glutaraldehyde crosslinking is less effective as the complex decomposition process competes with the crosslinking process. The instability of the complex makes it impossible to perform multiple adsorption at a pH greater than 4.8.

An albumin concentration of greater than 0.01% ensures complete adsorbed albumin layers.

A concentration of heparin of 50-100 IU / ml ensures the formation of complete adsorbed layers of heparin. For economical reasons, it makes no sense to use higher concentrations.

The temperatures of adsorption of albumin up to 35 ° C ensure the formation of a uniform adsorbed layer. At higher temperatures, aggregates rapidly form in acidic pH solutions.

The ionic strength of the aqueous solutions is less than 0.5, because at a higher ionic strength the adsorbed complex is unstable.

Times of 5 to 30 minutes (depending on the concentration and flow of the solution at the surface) ensure the formation of individual adsorbed layers.

The proposed method can be used for thromboresistant treatment of materials of different types that adsorb albumin, the further process being determined by the first adsorbed layer of albumin. Known materials include, for example, polyolefins, polyesters, polymethacrylates, polyvinyl chloride, fluorinated polymers, silicone rubber, and inorganic materials such as metals, glass, ceramics, or materials whose surfaces have been heparinized by any of the methods used hitherto. In the proposed manner, the surfaces of blood-contacting products of any geometric shape and size, such as catheters, vascular prostheses, artificial heart valves, oxygenators, blood-contacting device components, blood storage containers, can be treated. Depending on how the product is used, the number of adsorbed albumin and heparin layers can be selected to be optimal for the application. For example, polyethylene pipes coated with a double or quadruple layer of complex (albumin-heparin) did not find a difference at 20 min. ex-vivo experiments, while the quadruple layer showed a better effect on two-month in-vivo implantation.

The advantages of the proposed process, which produce a new qualitative effect compared to the method used so far using albumin, heparin and glutaraldehyde in water without pH adjustment, are:

1) Increased thromboresistant properties due to

(a) uniformity and planarity of coverage;

b) multilayered adsorption of albumin and heparin.

2) Possibility to control the amount of immobilized albumin and heparin according to the product use.

3) Simplification of the process technology by performing adsorption of albumin and heparin at temperatures of 0 to 35 ° C.

4) Reduce the consumption of biological preparations (albumin up to 10 times, heparin 30 to 50 times), reduce the concentration of the solutions used, which was made possible by carrying out the whole process at pH 3.0 to 4.8.

Example 1

Double-layer (albumin-heparin) - (albumin-heparin) coating of tubes of polyethylene approved for medical use. The first layer of adsorbed albumin was obtained by contacting the tubes with 0.5 wt. % solution of albumin in 0.1 M citrate buffer pH 3.9 for 30 min at 20 ° C.

After adsorption, the tubes were washed three times with 0.1 M citrate buffer pH 3.9.

The first layer of heparin was obtained by contacting the surface of the albumin-treated tubes with a heparin solution of 0.1 M citrate buffer pH 3.9 with a heparin concentration of 100 IU / ml for 20 min at 20 ° C.

The tubes were washed three times with 0.1 M citrate buffer pH 3.9.

The first layer of adsorbed complex contained 0.15 μg / cm 3 albumin and 0.07 цд / ст ² heparin.

A second layer of adsorbed albumin was obtained by contacting the tubes with a 0.5 solution of albumin in 0.1 citrate buffer pH 3.9 for 20 min at 20 ° C.

The tubes were washed three times with 0.1 M citrate buffer pH 3.9.

A second layer of heparin was obtained by contacting the tubes with a heparin solution of 0.1 M citrate buffer pH 3.9 with a heparin concentration of 100 IU / ml for 20 min at 20 ° C.

The tubes were washed three times with 0.1 M citrate buffer pH 3.9.

The second layer contained 0.35 ид complex / спг albumin and 0.15 ц.д / ² ст heparin.

The adsorbed double layer (albumin-heparin) - (albumin-heparin) was fixed in a 1% v / v solution of glutaraldehyde in 0.1 M citrate buffer pH 3.9 for 30 min at 60 ° C.

The tubes were washed first with a stream of water and then placed in larger amounts of distilled water for 20 min. with five water changes at 20 ° C.

The samples were dried at 40 G.

plexus containing 0.5 цд / ст ² albumin and 0.2 μg / cm ² heparin. Thromboresistant properties of treated tubes in contact with native blood were evaluated by ex-vivo dog experiments. Blood was circulated through the test tubes mounted in a special holder attached to the animal by the arterio-venous shunt method. The holder allowed parallel monitoring of the treated and control untreated tubes in one experiment. In the course of time, the amount of thrombosic material deposited on the tube walls was determined. The maximum blood contact time was 20 min.

The thrombosic mass on samples coated with a double layer of albumin and heparin was less than 5% compared to the amount of thrombosic mass on the untreated control tubes; on tubes coated with a single layer of albumin and heparin, there was about 20% thrombosis mass relative to the control tubes.

Example 2

Covering the surface of polyethylene pipes with four layers of complex (albumin-heparin).

* The tube surface was contacted with 0.1wt% albumin in 0.1M citrate buffer pH 3.9 for 20 min at 18 ° C. The tubes were washed three times with 0.1 M citrate buffer pH 3.9 over 1 min. The tubes were contacted with a solution of 50 IU / ml heparin in 0.1 M citrate buffer pH 3.9 for 10 min at 18 ° C. The tubes were washed three times with 0.1 M citrate buffer pH 3.9 over one minute. The described procedure was repeated three times, with the contact time with the albumin solution being reduced to 10 min. The resulting quadruple layer of adsorbed albumin and heparin was fixed with 1 vol% glutaraldehyde in 0.1 M citrate buffer pH 3.9 for 30 min at 55 ° C. The tubes were washed thoroughly with distilled water for 20 min and dried. The quadruple layer of the albumin-heparin complex contained 1.2 Cd / cm ² albumirium and 0.47 Cd / ст ² heparin.

The amount of thrombosis mass deposited on the tubes in the ex-vivo tests was less than 5% of the thrombosis mass deposited in the control or conditioned tubes.

Example 3

Covering the surface of the polyvinyl chloride sheet with a triple layer of albumin-heparin complex.

The film surface was contacted with 0.1 wt. % albumin solution in 0.1 M citrate buffer pH 4 for 20 min at 23 ° C. The films were washed three times with a stream of 0.1 M citrate buffer pH 4 for 1 min. The films were contacted with a solution of 50 IU / ml heparin in 0.1 M citrate buffer pH 4 for 10 min at 23 ° C. The films were washed three times with 0.1 M citrate buffer pH 4 over one minute. The described procedure was repeated twice more, with the contact time with the albumin solution being reduced to 10 min. The resulting quadruple layer of adsorbed albumin and heparin was fixed with 1% v / v glutaraldehyde in 0.1 M citrate buffer pH 4 for 30 min at 55 ° C.

The films were washed with distilled water and dried. The triple layer of the albumin-heparin complex contained 0.8 cc / cm ² albumin and 0.35 cc / ст ² heparin.

Example 4

Covering the surface of the silicone rubber foil with a triple layer of albumin-heparin complex.

The film surface was contacted with 0.1 wt. % albumin solution in 0.1 M citrate buffer pH 4 for 20 min at 23 ° C. The films were washed three times with a stream of 0.1 M citrate buffer pH 4 for 1 min. The films were contacted with a solution of 50 IU / ml heparin in 0.1 M citrate buffer pH 4 for 10 min at 23 ° C. The films were washed three times with 0.1 M citrate buffer f

pH 4 within one min. The described procedure was repeated twice more, with the contact time with the albumin solution being reduced to 10 min. The resulting quadruple layer of adsorbed albumin and heparin was fixed with 1 vol% glutaraldehyde in 0.1 M citrate buffer pH 4 for 30 min at 55 ° C. The films were washed with distilled water and dried. The triple layer of the albumin-heparin complex contained 1 цд / ст ² albumin and 0.4 цд / ст ² heparin.

Example 5

Covering the surface of inorganic material - germanium - with a five-fold layer of the complex (albumin-heparin).

The crystal surface was contacted with 0.1 wt. % solution of albumin in 0.1 M citrate buffer pH 3.9 for 20 min at 30 ° C. The crystals were washed three times with a stream of 0.1 M citrate buffer pH 3.9 over 1 min. The crystals were contacted with a solution of 50 IU / ml heparin in 0.1 M citrate buffer pH 3.9 for 10 min at 30 ° C. The crystals were washed three times with 0.1 M citrate buffer pH 3.9 over one minute. The described procedure was repeated four times, with the contact time with the albumin solution being reduced to 10 min. The resulting quadruple layer of adsorbed albumin and heparin was fixed in 1% v / v glutaldehyde in 0.1 M citrate buffer pH 3.9 for 30 min at 55 ° C.

The amount of adsorbed complex in this case in the first layer of albumin 0.15 цд / ст ² , heparin 0.08 in the second layer of albumin 0.35 цд / ст ² , heparin 0.14 in the third layer of albumin 0.21 цд / ст ² , heparin 0,10 in the 4th layer of albumin 0,30 цд / ст ² , heparin 0,12 in the 5th layer of albumin 0,35 цд / ст ² , heparin 0,14 consisted of цд / ст ² μ-g / cmид / сп / цд / ст * цд / ст ^

Example б

Covering a woven vascular prosthesis of ftorlon-lavsan (polyethylene terephthalate fibers and tetrafluoroethylene-1,1-difluoroethylene copolymers) of the USSR with a four-layer complex (albumin-heparin).

The surface of the prostheses was contacted with 0.1 wt. % albumin solution in 0.1 M citrate buffer pH 3.9 for 20 min at 18 ° C. The prostheses were washed three times with 0.1 M citrate buffer pH 3.9 over 1 min. The prostheses were contacted with a solution of 50 IU / ml heparin in 0.1 M citrate buffer pH 3.9 for 10 min at 18 ° C. The prostheses were washed three times with 0.1 M citrate buffer pH 3.9 over one minute. The described procedure was repeated three times, with the contact time with the albumin solution being reduced to 10 min. The resulting quadruple layer of adsorbed albumin and heparin was fixed in 1% v / v glutaraldehyde in 0.1 M citrate buffer pH 3.9 for 30 min at 55 ° C. The prostheses were washed with a stream of water for 30 min and dried.

The amount of bound heparin in the first layer was 48.4 цд / д, in the second layer 58.9 цд / д, in the third layer 59.2 ид / д, in the fourth layer 59.8 цд / д. The amounts of heparin are based on 1 g of dry prosthesis.

Sterilization of albumin-heparin treated products was performed by t-irradiation (2.5 Mrad) or ethylene oxide. Both sterilization methods do not affect the coating properties and are selected

CS 270919 Bl

so as not to damage the material from which the product is made.

The amount of albumin and heparin gradually increasing as the adsorption was repeated was determined by infrared reflection spectroscopy in Examples 1 to 5. In Example 6, where the material was a large surface, the amount of immobilized heparin was determined by specific toluidine blue adsorption using visible spectroscopy. areas.

Claims (1)

Method of thromboresistant treatment of surfaces of products used in medicine in contact with blood, their treatment with albumin, heparin and glutaraldehyde solutions, characterized in that the surface of the product is treated 1 to 50 times alternately with an albumin solution with a concentration of 0.1 to 0.5% by weight. for 5 to 30 minutes and a heparin solution of 50 to 100 IU / ml for 5 to 30 minutes, the solutions having a pH of 3.0 to 4.8 and an ionic strength of less than 0.5, followed by final fixation in the solution of glutaraldehyde at a concentration of 0.5 to 1.0% vol.