儿茶酚-(胺)表面化学介导的一氧化氮催化释放涂层研究

Chemical-mediated Nitric Oxide-generating Coatings on Catechol-(Amine)Surface

目的在316L不锈钢(SS)表面构建原位一氧化氮(NO)催化释放涂层,使其能特异选择性地抗凝血、抑制平滑肌细胞(SMCs)增生,从而促进内皮细胞(ECs)生长。方法在弱碱性水溶液中,选用多巴胺(DA)和己二胺(HD)为前驱体,利用多巴胺邻二苯酚结构自聚合沉膜的能力、多巴胺与己二胺的酚-(胺)表面化学,通过简单的一锅法在316L SS表面构建富氨基粘附涂层DA/HD。再通过碳二亚胺化学交联反应,共价接枝螯合Cu^(2+)的1,4,7,10-四氮杂环十二烷-1,4,7,10-四羧酸(DOTA),最后获得均匀且稳定的NO催化释放涂层(命名为Cu-DOTA@DA/HD)。结果DA/HD涂层表面的氨基密度高达22 nmol/cm^(2),实现了Cu-DOTA的有效固定,其NO催化释放速率可达5.2×10^(-10)mol/(cm^(2)·min)。Cu-DOTA@DA/HD涂层显著地抑制了血小板的粘附和激活,也能有效抑制血栓的形成,其表面血栓总质量由316L SS的(40.3±10.3)mg降低至(3.0±0.4)mg。Cu-DOTA@DA/HD涂层催化释放的NO赋予了316L SS选择性地抑制SMCs粘附和增殖,从而促进了ECs生长的功能。结论基于儿茶酚-(胺)表面化学构建的NO催化涂层,成功地实现了316L SS表面持续、稳定的NO催化释放,赋予了其抗凝血、选择性抑制SMCs增殖和促进EC生长等多重生物学功能,在血液接触类器械(如体外循环导管和血管支架表面)应用方面具有潜在价值。

It is an advanced surface technology that can improve the biocompatibility and hemocompatibility of blood contact-like material surfaces by constructing an in situ nitric oxide(NO)catalytic release coating on the surface of 316L stainless steel(SS)without changing the physical properties of the substrate material.The biocompatibility and hemocompatibility of unmodified 316L stainless steel(SS)are too poor to cause complications such as acute thrombosis and smooth muscle cell proliferation after implantation,which can inhibit the regenerative repair of diseased tissue.Thus,in this work,a NO-catalyzed coating based on the chemical construction of catechol-(amine)surface was investigated,and a sustained and stable NO-catalyzed release from the surface of 316L SS was successfully achieved,endowing it with multiple biological functions such as anti-coagulation,selective inhibition of SMCs proliferation and promotion of EC growth.In this work,316L SS disc with 1 cm diameter was used as the study object.First,the samples were cleaned by sonication with acetone and anhydrous ethanol,dried sufficiently and then prepared for use.Subsequently,the samples were immersed in the reaction solution of DA(1 mg/mL)and HD(2.44 mg/mL)dissolved by Tris-base,then the solution reacted at a constant temperature of 37℃ for 24 hours.After the reaction,the DA/HD-coated modified samples were removed and washed thoroughly with distilled water.Subsequently,DOTA(0.01 mg/mL),EDC(0.0066 mg/mL),NHS(0.0033 mg/mL)and MES(0.65 mol/L)were fully dissolved in deionized water and the pH of the solution was adjusted to 5.4.After the reaction at 37℃for 30 min,the appropriate amount of Cu Cl2-2H2O was fully dissolved in the above solution to prepare Cu-DOTA solution with a concentration of 0.003 mg/mL.Finally,1 mL of Cu-DOTA solution was added to the surface of each sample,and after grafting reaction at 25℃ for 24 hours,the samples were removed,then washed with distilled water.Finally,Cu-DOTA@DA/HD coating modified samples were obtained,and a series of characterization analysis was subsequently performed on the resulting coatings.The composition and structure of the coating were analyzed by X-ray photoelectron spectroscopy(K-Alpha),water contact angle tester(DSA100)and enzyme standard(HM-SY96S);The adhesion and activation of platelets on top of the functional coating were observed by scanning electron microscope(JSM 7800F)and the proliferation of smooth muscle cells and endothelial cells were observed by fluorescence microscope(Y-E68).Finally,in order to simulate the most realistic circulatory state of the functional coating in animals,a New Zealand rabbit arteriovenous short-circuit parallel shunt model was used to perform semi-internal circulation(ECC)experiments to realize the circulation of closed blood circulation,and therefore to verify the hemocompatibility of the functional coating.The results showed that the HD introduced by this modification strategy provided a large amount of amino groups to the coating surface,and the amount of surface amino groups increased from 2 nmol/cm^(2) to 22 nmol/cm^(2) compared to the bare 316L SS substrate.The large amount of exposed amino groups provided the active site for the grafted functional molecule Cu-DOTA,resulting in a catalytic release rate of 5.2×10^(-10)mol/(cm^(2)·min)from the coating,which was close to the NO release rate from normal endothelial cells.In vitro results showed that Cu-DOTA@DA/HD coating significantly inhibited platelet adhesion,with the number of adhesions decreasing from 23 cells/1000 cm^(2) to 6 cells/1000 cm^(2) compared to 316L SS,and inhibited platelet activation;cellular experiments showed that the number of adhesions of HUVSMCs on the NO-catalyzed release surface was only 526 cells/mm^(2),compared to 1182/mm^(2) on the surface of 316L SS,and the number of HUVECs adhering was 4251/mm^(2),which was 2.5 times higher than that on the surface of 316L SS(1700/mm^(2));the semi-in vivo results showed that the Cu-DOTA@DA/HD coating significantly inhibited thrombus formation,compared to 316L SS,the blockage rate of the coated modified samples was reduced from 80% to 5%,and the total surface thrombus mass was reduced from(40.3±10.3)mg to(3.0±0.4)mg.The NO-catalyzed coating based on catechol-(amine)surface chemistry successfully achieves sustained and stable NO-catalyzed release from the surface of 316L SS,endowing it with multiple biological functions such as anti-coagulation,selective inhibition of SMCs proliferation and promotion of EC growth,which are potentially valuable for the application on the surface of blood-contacting devices such as extracorporeal circulation catheters and vascular stents.