The method of formation of heparinized surface

 

(57) Abstract:

The invention relates to the field of biology, in particular biochemistry. The method of formation of heparinized surface, consisting of sequential application of water rasters dye methylene blue (toluidine blue) and heparin on the surface, is able to adsorb or chemically bind the dye molecules. Technical result: method offers speed and ease of formation of the surface with anticoagulant properties. table 1.

The present invention relates to the field of biology, in particular biochemistry.

Progress angiosurgery has made significant progress in the development and implementation of reconstructive operations on the vessels. However, thrombosis of the reconstructed vessels that occur both in the near future mainly in the late postoperative periods, significantly reduce the effectiveness of surgical interventions. In this regard, despite the numerous studies conducted in this direction, the problem of forming atrombogennoe, in particular, heparinized surface of the vascular prosthesis, vessels with acetaminophenol the intima, ALLO - and xenotransplantation m is s used in the practice (a), following similar methods of forming atrombogennoe (heparinised) surface:

a) local treatment of blood vessels and anastomoses with a solution of heparin during surgery (Operative surgery /Ed. by I. Littmann. - Budapest: Publishing house of the Academy of Sciences of Hungary, 1985. -1175 C.). Heparin is not capable of strong interaction with elements of the connective tissue, therefore this measure is short-term: most of the anticoagulant is not delayed when restoring blood flow;

b) physico-chemical and chemical methods of surface treatment of blood vessels or ALLO - and xenografts. For example, in order to prevent thrombosis wall endarterectomies arteries exposed to ultrasound in the presence of heparin for 30 sec. Heparin retained in the vessel wall is not more than 12 hours (Antijew A. F. Prevention of infection of vascular prostheses and thrombosis endarterectomies arteries /Diss. for obtaining the academic degree of Cand. Sciences.-1985). The umbilical vein or the carotid artery of dogs, pigs, proposed for use as vascular grafts, chemically modified heparin using carbodiimide for at least 5 hours, reaching a concentration of heparin of 31.5 ág-p 735-741);

C) surface heparinization of different synthetic (e.g., polyurethane) vascular prostheses by chemical modification of functional groups (PCT WO 90 00, 343; Heyman P. W., C. S. Cho, J. C. McRea et al. Heparinized polyurethanes: in vitro and in vivo stadies //J. of Biomedical Materials Research.-1985.-V. 19.- P. 419-436), impregnation of synthetic grafts heparin-collagen copolymer (Shankar N., Senatore F., Wu D. R. et al. Co-immobilisation and interaction of heparin and plasmin on collageno-elastic tubes //Biomaterials, Artificial Cells and Artificial Organs.-1990.-V. 18. -# 1. -P. 59-73), etc., These methods heparinisation or are in the process of scientific development, or have not received practical application because of some disadvantages.

Also there is a method of binding heparin, which is a polyanionic electrolyte, with cationizing surface. For example, heparin or heparansulfate (endothelium bovine aorta), immobilized on a partially cationizing using 3-chloro-2-hydroxypropyltrimethylammonium the surface of the pulp, possess anticoagulant activity and can be used as biomaterials, membranes with atrombogennoe surface similar to the surface of blood vessels (Baumann N., Keller R., Ruzicka E. Partially cationized cellulose for non-thrombogenic membrane in the presence of heparin and endothelial-cell-surface-heparansulfate //J. Membr. Sci.-the military chromatography. Also describes the interaction of heparin with poly surface formed immobilized on polietileniminov alcohol poly-L-lisinopril (Xinghang M, Fazal M. S. , Wan K. S. Heparin binding on poly(L-lysine)-immobilized surface //J. Colloid Interface Sci. -1991.- V. 147.-#l.-P. 251-261). On the smooth surface of the polymer for 30 minutes from blood or plasma at a speed of 100 ml/min heparin concentration reaches a value of 0.52 ág/cm2on porous was 1.69 mg/cm2. For the manifestation of the surface of the anticoagulant properties of heparin concentration should not be less than 5 ág/cm2(E. S. Venkataramani, Fred Senatore, Mario Feola et al. Nonthrombogenic small-caliber human umbilical vein vascular prosthesis //Surgery.-1986.-V. 6.-P. 735-741).

Methods describing the formation of heparinized surface biological or polymeric materials using any dyes in the literature were not found. The authors proposed a method of forming a heparinized surface by applying a solution with heparin pretreatment with an aqueous solution of the dye methylene blue (MTB) any surface can either adsorb or chemically bind the dye molecules. Thus, with the help of molecules MS, which is the Quaternary ammonium salts initially formed cationization paolicelli MS, attaching the surface of the anticoagulant properties. The described mechanism of the interaction of the dye and heparin underlies the phenomenon of metachromasia (Konorski A. I. the Histochemistry. - Kiev: high school, 1976.- 280s.) (schema, see end of text).

The advantages of this method is speed and ease of formation of the surface with anticoagulant properties, and the ability in a wide range to change the surface concentration of heparin, depending on the concentration of dye solution (see table).

Methylene blue (blue) refers to the so-called vital dyes. For nearly 70 years it was used as an antiseptic for bowel and urinary tract. Currently, the MS does not find such widespread use, but has not lost value in the treatment of cyanosis caused by lack of oxygen or blood poisoning poisons (intravenously) (Mashkovsky M. D. Medicines. - Kharkov: Torsing, 1997.-T. 2.-S). MS is also used in histology as metachromatic dye for the detection of acid mucopolysaccharides and other components of the connective tissue.

To make the surface of the anticoagulant properties can handle posledovateli, for example, endarterectomies blood vessels, ALLO - and xenografts.

The invention is illustrated by the following example. Aqueous solutions of MS with different concentrations (from 0.1 to 1.0%) was applied on a surface area equal in fragments artery or aorta with acetaminophenol the intima (cadaver material) within 2-5 minutes, the excess dye was removed with filter paper and treated with a solution of heparin (5000 units/ml) for 2-5 minutes. Unreacted with the surface of the media (media) dye and heparin was washed with running water for 10-15 minutes. For the quantitative determination of heparin dye on the surface of the fragments of the vessel was restored Zn in hydrochloric acid environment to leucosolenia. After bleaching MS heparin in solution was determined carbazolyl method by the reaction of the DISHA on uronic acid Carbohydrate analysis /Ed. by M. F. Chaplin and Kennedy J. F.-Oxford, Washington, 1986.-228p) and counted on 1 cm2the surface of the media (see table).

As can be seen from the table results, the concentration of heparin immobilized on the surface of the tissue with methylene blue, more than 5 g/cm2and is sufficient for the manifestation of surface anticoagulant heparin, characterized in that the surface is pre-treated with an aqueous solution of the dye methylene blue at a concentration of 0.1 - 1.0%.

 

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