CN101934080B - Method for improving material blood compatibility by using electrostatic self-assembled coating - Google Patents

Abstract

Translated fromChinese

本发明公开了一种利用静电自组装涂层改善材料血液相容性的方法，其方法是将聚阴离子二元共聚物通过静电相互作用吸附在聚阳离子材料表面，在聚阳离子材料表面形成仿细胞外层膜结构。本发明将含有两性离子、阴离子的丙烯酸类单体的二元共聚物作为聚阴离子，与聚阳离子通过静电自组装在聚阳离子材料表面构建仿细胞外层膜结构，聚阴离子二元共聚物通过静电相互作用吸附在聚阳离子表面，条件简单，适应性强，可实现对具有复杂体型结构的聚阳离子材料表面修饰。与聚阳离子材料相比，改性后的表面对蛋白吸附、血小板黏附明显降低，血液相容性显著提高。The invention discloses a method for improving the blood compatibility of materials by using an electrostatic self-assembly coating. The method is to adsorb a polyanionic binary copolymer on the surface of a polycation material through electrostatic interaction, and form a cell-like structure on the surface of the polycation material. outer membrane structure. In the present invention, the binary copolymer of acrylic monomers containing zwitterions and anions is used as polyanions, and self-assembled with polycations on the surface of polycation materials through electrostatic self-assembly to construct a structure imitating the outer layer of cells. The interaction is adsorbed on the surface of polycations, the conditions are simple, and the adaptability is strong, which can realize the surface modification of polycation materials with complex body structures. Compared with polycation materials, the modified surface has significantly reduced protein adsorption and platelet adhesion, and significantly improved blood compatibility.

Description

A kind of method of utilizing static self assembly coating to improve the material blood compatibility

Technical field

The present invention relates to a kind of method of utilizing static self assembly coating to improve the material blood compatibility, belong to Surface Science and biological medical polymer material technical field.

Background technology

Gene therapy is considered to the effective way of range gene disease (comprising infectious disease, cancer etc.) treatment.In the gene therapy process, must nucleic acid drug be transported to lesions position with genophore (comprising viral vector and non-virus carrier).The former is the high transfection efficiency carrier, but immunoreation, gene mutation have limited its application.Therefore, People more and more is paid close attention to safety, hypotoxic non-virus carrier, and wherein polycation gene carrier is one of research focus in the last few years.

In the application of reality, the polycation non-viral gene vector also exists certain problem.Specifically, the composite surface that polycation and DNA form has the positive charge of part, and easy adhesion protein in the circulation causes platelet, cell adhesion then in vivo, causes forming thrombosis, immunoreation.Therefore, will have the Polyethylene Glycol (PEG) that suppresses non-specific adsorption ability, good biocompatibility and be applied to cation radical, become the emphasis of research in the last few years because of the carrier surface modification.Yet, compare the transfection efficiency that PEG modifies the back polycation decrease (macromolecule journal, 2009,6,499-505 with this polycation; Biomaterials, 2009,30 (34): 6655-6664).

(Phosphorycholine is the Main Ingredients and Appearance of biological cell outer membrane PC) to Phosphorylcholine, is the outer functional group in the tunic of extracellular, is the terminal hydrophyllic group of forming cell membrane elementary cell lecithin.From structure, Phosphorylcholine has the amphion end group, has positive and negative two kinds of electric charges simultaneously, thereby has the ability of very strong bound water.This character makes the material surface that is rich in the Phosphorylcholine group be difficult for absorption and reaches biological components such as heavy egg collection white matter, liposome, shows excellent biological compatibility.Research group (Biomaterials, 2009,30 (28): 4930-4938 of Japan and Britain; Biomaterials, 2004,25 (19): 4785-96) protein adsorption, cellular adhesion studies are being carried out in the surface of containing cation, neutrality, anionic Phosphorylcholine polymer.The result shows: have the blood compatibility that electrostatic charge Phosphorylcholine base polymer all can be used to improve material.Therefore, utilize and to contain Phosphorylcholine and derivant thereof and make up imitating cell outer-layer membrane structure at the polycation material surface and have important significance for theories and great application prospect.

Chinese scholars adopts grafting to contain the micromolecular method modification of chitosan of Phosphorylcholine group, makes its biocompatibility, particularly blood compatibility be significantly improved (Carbohydrate Polymers, 2007,70 (1): 82-88; ZL 200410054022.2; Journal of Applied Polymer Science, 2003,88 (2): 489-493; Polymer International, 2003,52 (1): 81-85; Journal ofbiomaterials science, Polymer edition, 2002,13 (5): 501-510; Colloids andSurfaces B:Biointerfaces, 2009,71 (2): 268-274; Applied Surface Science, 2008,255 (2): 538-540).Yet; Phosphorylcholine group surface is rich in the difficult acquisition of grafting method, also limited further raising biocompatibility simultaneously, and graft modification receives equipment; Condition effect such as base material composition, shape; Application (Langmuir, 2009,25 (6): 3610-3617 have been limited in biomaterial surface modification field; Science, 1997,277 (5330): 1232-1237; Biomacromolecules, 2009,10 (8): 2275-2283; Biomacromolecules, 2007,8 (10): 3169-3176).Through the static self-assembling method of electrostatic interaction, the polyelectrolyte of band xenogenesis electric charge in powered surfaces absorption, condition is simple, and adaptability is strong, has become the effective means of bio-medical material surface modification.

In order to reduce absorption of polyelectrolyte multilayer film nonspecific proteins and cell adhesion; People (Langmuir such as Andreas Reisch; 2009; 25 (6): the polymer that 3610-3617) will contain Phosphorylcholine group acrylamide monomers is adsorbed onto PSS/PAH (PSS, poly (styrenesulfonate) through electrostatic interaction; PAH, poly (allylamine hydrochloride)) biocompatibility of polyelectrolyte multilayer film has significantly been improved on surface.Molecule graft modification chitosan (235th ACS National Meeting, United States 2008, the 6-10 of aldehyde radical Phosphorylcholine will be contained in Canadian scientific research group; Biomacromolecules, 2007,8 (10), 3169-3176; Biomacromolecules, 2006,7 (11): 3151-3156; Journal ofColloid and Interface Science, 2009,336 (1): 125-133), then chitosan after the modification and hyaluronic acid are passed through the self assembly layer by layer of multi-layer polyelectrolyte deposition technique, improved the blood compatibility of coating.The Phosphorylcholine chitosan modified can be compounded to form nano-particle with DNA and be used for gene therapy.Equally; The negative charge Polyethylene Glycol will be had and the transfection efficiency (Biomacromolecules of gene can be improved at PEI/DNA nano-particle gene carrier surface through the electrostatic interaction self assembly; Doi:10.1021/bm1002569), opened up the new direction of gene controlled release.(one Chinese patent application CN 200710047123.0 for people such as Liu Jianping; One Chinese patent application CN 200710047124.5) utilize static self assembly layer by layer to be adsorbed on the intravascular stent surface silk polypeplide and chitosan.Compare with unmodified intravascular stent, the material surface after the modification has good blood compatibility, can reduce late restenosis rate of post-operative heart.People (one Chinese patent application CN 03116748.9) such as meter sword adopt surface modifying method to obtain the surface with positive charge or negative charge at biomaterial surface; Through the polyelectrolyte layer by layer self assembly of electrostatic interaction, improve the anticoagulant property of material with reactance blood-clotting agent (albumin, heparin, sulfonation glucosan, urokinase, kayexalate) and band xenogenesis electric charge.People such as Gao Changyou (one Chinese patent application CN200510061354.8) improve the biocompatibility of microcapsule through introducing the polyelectrolyte that is grafted with Polyethylene Glycol at surface of microcapsule, avoid the untoward reaction of intravital tissue.

Summary of the invention

The purpose of this invention is to provide a kind of through making up the method that imitating cell outer-layer membrane structure improves polycation material surface blood compatibility.The polyanion bipolymer that will contain the amphion hydrophilic radical is adsorbed on positively charged polycation material surface through electrostatic interaction, forms imitative cell membrane layer structure, reaches the purpose to material surface modifying.

Implementation procedure of the present invention:

A kind of method of utilizing static self assembly coating to improve the material blood compatibility, specific as follows: as the polyanion bipolymer to be adsorbed on the polycation material surface through electrostatic interaction, to form imitating cell outer-layer membrane structure at the polycation material surface.

Described polyanion bipolymer is randomcopolymer or the block copolymer that contains the amphion hydrophilic radical, and the amphion hydrophilic radical is the acrylic monomer that has Phosphorylcholine group, sulphur ammonium class or carboxylic ammonium class.

Anionic group is carboxylic acid group, sulfonic group, sulfate or phosphate in the polyanion bipolymer, and to account for the polymer monomer molar content be 50～90% to anionic monomer in the polyanion bipolymer.

Described polycation is chitosan, PEI, polylysine or branch polyurethane.

Preferred scheme is; The polycation material is a chitosan; The polyanion bipolymer is methacrylic acid-methylacryoyloxyethyl Phosphorylcholine bipolymer, and the amino on the chitosan chain makes up imitating cell outer-layer membrane structure through the carboxyl on the electronegative binary polymer that contains the Phosphorylcholine group of electrostatic interaction absorption on its surface automatically; Form compound polyelectrolyte, compound polyelectrolyte is nano-particle, hydrogel, microcapsule and film form.

(MA-co-MPC PMA) makes up imitating cell outer-layer membrane structure through the principle absorption methacrylic acid-methylacryoyloxyethyl Phosphorylcholine bipolymer of static self assembly on the chitosan surface.PMA is a kind of anionic polyelectrolyte, has the Phosphorylcholine zwitterionic pendant groups, is applicable to chitosan and other polycation material surface are carried out the modification of imitating cell outer-layer membrane structure.Anionic carboxylic acid root among the PMA can adsorb amino or positively charged polycation through electrostatic interaction, contains amino and other polycation material surface structure imitating cell outer-layer membrane structure so be used in.

The preparation list of references of chitosan film (Colloidsand Surfaces B:Biointerfaces, 2009,71 (2): 268-274; One Chinese patent application CN 200910020872.3): with the acetate dissolution certain amount of chitosan of 1% (V/V), obtain the chitosan solution of 0.1～0.8wt%.After above-mentioned chitosan solution filtration, the standing and defoaming, solid substrate such as microscope slide are vertically immersed chitosan solution, slowly propose then.After the chitosan film that applies on the base material at room temperature dries, be soaked in sodium hydroxide solution 1～1.5h of 0.5wt%, fully soak with redistilled water afterwards, till the washing electrical conductivity of water is suitable with distilled water, last 30 ℃ of vacuum dryings.

The static self assembling process is following: chitosan film is vertically immersed in the NaCl aqueous solution of PMA absorption 10-30min.Afterwards, print is taken out, NaCl aqueous solution, the careful drip washing of distilled water successively, lyophilization can obtain the static self-assembly modified chitosan film of PMA.

The present invention will contain the bipolymer of amphion, anionic acrylic monomer as polyanion, make up imitating cell outer-layer membrane structure through the static self assembly at the polycation material surface with polycation.The polyanion bipolymer is adsorbed on the polycation surface through electrostatic interaction, and condition is simple, and adaptability is strong, can realize the polycation material surface with complex shape structure is modified.Compare with the polycation material, the surface after the modification obviously reduces protein adsorption, platelet adhesion reaction, and blood compatibility significantly improves.

Description of drawings

Fig. 1. (CS is a) with through chitosan film (CS-PMA, b) surperficial platelet adhesion reaction shape appearance figure after the modification in order to apply chitosan film;

Fig. 2. be the influence of salt pair modified coating dynamic contact angle;

Fig. 3. for chitosan film (CS) and through chitosan film (CS-PMA) coating after the modification in PBS (pH=7.4) stability.

The specific embodiment

The MPC monomer can be by method (Polymer Journal, 1990,22 (5): 355-360 of bibliographical information; Makromolekulare Chemie, Rapid Communications, 1982,3 (7): 457-459) synthetic.Methacrylic acids (MA) etc. all can be bought or the purchase of Sigma company from domestic production company.Methacrylic acid-methylacryoyloxyethyl Phosphorylcholine bipolymer (MA-co-MPC, but PMA) list of references reported method (Biomacromolecules, 2002,3 (1): 100-105; Biomaterials, 2010,31 (14): 4009-4016) synthetic.

Utilize dynamic contact angle tester (DCA), x-ray photoelectron spectroscopy (XPS), AFM (AFM) respectively the chitosan film dynamic contact angle before and after the modification, surface-element composition, pattern to be characterized.Advancing angle 84.0 ± 2.5 is reduced to 48.7 ± 0.6 before the modification, and receding angle is reduced to 6.5 ± 0.3 by 11.4 ± 0.6; Surperficial phosphorus molar content is 3.23% after the modification; Pattern is different before and after the modification, all explanation: the chitosan surface obtains imitating cell outer-layer membrane structure through the static self assembly.

Protein adsorption experiment reference literature (Biomacromolecules, 2009,10 (2): 267-274.) carry out by method.Use buffer (pH=7.4) to dissolve a certain amount of bovine serum albumin (BSA) and Fibrinogen (Fg) respectively, obtain certain density protein solution.With sample balance 1h in PBS buffer solution, careful buffer wiped clean with sample surfaces, afterwards with sample transfer in contactor, add a certain amount of BSA and Fg protein solution, hatch 2h for 37 ℃.After hatching end, desorption adopts the protein content of micro-BCA method calculation sample surface adsorption at the albumen of material surface absorption.

Platelet adhesion reaction experiment reference literature (Biomaterials, 2007,28 (10): 1752-1760.) carry out by method.Sample is balance 2h in PBS buffer solution, and the careful buffer wiped clean with sample surfaces is got afterwards and is rich in hematoblastic blood plasma (PRP, Platelet-rich plasma) 20 μ L and is added drop-wise on the sample strip, cultivates 2h in 37 ℃ of CO2 gas incubators.After cultivate finishing, sample strip is soaked in 2.5% the glutaraldehyde solution fixedly 1h.The sample strip that fixes is used PBS, distilled water flushing, lyophilization successively.After the sample strip lyophilization, observe the material surface pattern behind the platelet adhesion reaction with scanning electron microscope (SEM).

Embodiment 1:

The 0.5g chitosan is dissolved in 100mL, and in 1% aqueous acetic acid, room temperature condition stirred 24 hours down.After the chitosan dissolving, filter, leave standstill 30min and eliminate bubble with the sand core funnel.After treating that bubble is eliminated, adopt the dip-coating mode on microscope slide, to apply chitosan film, it was hung 24 hours at ambient temperature.After chitosan film dries, be placed on the solution soaking 1 hour of 0.1% sodium hydroxide, afterwards, use distilled water immersion, whenever change first water at a distance from 4-5h, accumulative total was soaked 48 hours.Last 30 ℃ of vacuum dryings 8 hours can obtain chitosan film.

Embodiment 2:

Take the chitosan film of method for preparing, be soaked in 20mL, in 1mg/mL PMA30, PMA50, the PMA70 aqueous solution (30; 50; 70 respectively representation polymer synthetic in monomer M PC feed intake account for the monomeric percentage molar content of total throwing), absorption 15min uses distilled water drip washing then; Lyophilization can obtain the chitosan modified film.Compare with chitosan film, the advancing angle that the film after the modification contacts with water reduces about 20 degree.

Embodiment 3:

Take the chitosan film of method for preparing, be soaked in 20mL, 1mg/mL PMA30, in the 0.14MNaCl aqueous solution, absorption 15min uses 0.14M NaCl aqueous solution, distilled water drip washing successively, and lyophilization can obtain the chitosan modified film.Compare with chitosan film, the film after the modification to the protein adsorption amount obviously reduce, platelet is not assembled that a small amount of adhesion is only arranged, blood compatibility significantly improves (Fig. 1).

Embodiment 4:

Take the chitosan film of method for preparing, be soaked in 20mL, 1mg/mL PMA50, in the 0.14MNaCl aqueous solution, absorption 15min uses 0.14M NaCl aqueous solution, distilled water drip washing successively, and lyophilization can obtain the chitosan modified film.

Embodiment 5:

Take the chitosan film of method for preparing, be soaked in 20mL, 1mg/mL PMA70, in the 0.14MNaCl aqueous solution, absorption 15min uses 0.14M NaCl aqueous solution, distilled water drip washing successively, and lyophilization can obtain the chitosan modified film.

Compare with embodiment 2; PMA30 under the salt condition, PMA50, PMA70 (30 are being arranged; 50,70 respectively representation polymer synthetic in monomer M PC feed intake account for the monomeric percentage molar content of total throwing) can obtain having the advancing angle lower (Fig. 2) that imitative membrane structure surface contacts with water.

Sample strip was soaked in PBS (pH=7.4) solution after a period of time, uses distilled water drip washing, lyophilization.The test dynamic contact angle according to the hydrophilic, hydrophobic relation on dynamic contact angle and surface composition and surface, is weighed stability (Applied Surface Science, 2008,255 (2): 538-540) of Modified Membrane front and back.Compare with PMA50, PMA70, the advancing angle in 40 days of the coating after the PMA30 modification is constant basically, coating stability higher (Fig. 3).

Claims (5)

Translated fromChinese

1.一种利用静电自组装涂层改善材料血液相容性的方法，其特征在于：将聚阴离子二元共聚物通过静电相互作用吸附在聚阳离子材料表面，在聚阳离子材料表面形成仿细胞外层膜结构；1. A method for improving the hemocompatibility of materials by using an electrostatic self-assembled coating, characterized in that: the polyanionic binary copolymer is adsorbed on the surface of the polycation material by electrostatic interaction, and an imitation cell is formed on the surface of the polycation material. Layer film structure;所述的聚阴离子二元共聚物为含有两性离子亲水性基团的随机共聚物或嵌段共聚物，所述的两性离子亲水性基团带有磷酰胆碱基团，聚阴离子二元共聚物中阴离子基团为羧酸基、磺酸基、硫酸基或磷酸基。The polyanionic binary copolymer is a random copolymer or a block copolymer containing zwitterionic hydrophilic groups, and the zwitterionic hydrophilic groups have phosphorylcholine groups, and the polyanionic binary copolymers The anionic group in the copolymer is carboxylic acid group, sulfonic acid group, sulfuric acid group or phosphoric acid group.2.根据权利要求1所述的利用静电自组装涂层改善材料血液相容性的方法，其特征在于：聚阴离子二元共聚物中阴离子单体占聚合物单体摩尔百分含量为50～90%。2. The method for improving the hemocompatibility of materials by using an electrostatic self-assembled coating according to claim 1, characterized in that: in the polyanionic binary copolymer, the anionic monomer accounts for 50 to 50 mole percent of the polymer monomer. 90%.3.根据权利要求1所述的利用静电自组装涂层改善材料血液相容性的方法，其特征在于：所述的聚阳离子为壳聚糖、聚乙烯亚胺、聚赖氨酸或树枝状聚氨酯。3. The method for improving the hemocompatibility of materials by using an electrostatic self-assembled coating according to claim 1, characterized in that: said polycation is chitosan, polyethyleneimine, polylysine or dendritic Polyurethane.4.根据权利要求1至3任意之一所述的利用静电自组装涂层改善材料血液相容性的方法，其特征在于：聚阳离子材料为壳聚糖，聚阴离子二元共聚物为甲基丙烯酸-甲基丙烯酰氧乙基磷酰胆碱二元共聚物，壳聚糖链上的氨基通过静电相互作用吸附带负电荷含有磷酰胆碱基团的二元聚合物上的羧基，在其表面自动构建仿细胞外层膜结构，形成聚电解质复合物。4. The method according to any one of claims 1 to 3, wherein the electrostatic self-assembled coating is used to improve the blood compatibility of materials, wherein the polycationic material is chitosan, and the polyanionic binary copolymer is methyl Acrylic acid-methacryloyloxyethyl phosphorylcholine binary copolymer, the amino group on the chitosan chain adsorbs the carboxyl group on the negatively charged binary polymer containing phosphorylcholine group through electrostatic interaction, in Its surface automatically builds a structure imitating the outer layer of the cell and forms a polyelectrolyte complex.5.根据权利要求4所述的利用静电自组装涂层改善材料血液相容性的方法，其特征在于：聚电解质复合物是纳米颗粒、水凝胶、微胶囊及膜形态。5. The method for improving blood compatibility of materials by using electrostatic self-assembled coatings according to claim 4, characterized in that: the polyelectrolyte complex is in the form of nanoparticles, hydrogels, microcapsules and membranes.

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