基于聚乙二醇和不同多肽构建的生物涂层及其内皮细胞选择性研究

利用自由基聚合反应将甲基丙烯酸聚乙二醇酯(PEGMA)和甲基丙烯酸缩水甘油酯(GMA)的二元共聚物接枝在基材表面,并通过开环反应分别固定精氨酸-甘氨酸-天冬氨酸(RGD)、精氨酸-谷氨酸-天冬氨酸-缬氨酸(REDV)和酪氨酸-异亮氨酸-甘氨酸-丝氨酸-精氨酸(YIGSR)3种可特异性黏附内皮细胞的多肽.通过核磁共振检测合成的聚合物分子结构,并进一步通过X射线光电子能谱(XPS))以及原子力显微镜(AFM)的测试结果证明聚合物成功接枝在基材表面.利用紫外-可见吸收光谱(UV-Vis)对表面固定的3种多肽进行了定量表征.体外内皮细胞和平滑肌细胞黏附结果表明,3种不同多肽修饰的共聚物表面均能够有效阻抗平滑肌细胞的黏附,同时不同程度地促进内皮细胞的黏附,从而实现了基材表面内皮细胞的选择性黏附.其中与RGD和YIGSR多肽修饰的表面相比,REDV多肽修饰的表面呈现出更优异的内皮细胞选择性.这种具有内皮细胞特异选择性的界面在心血管支架涂层原位内皮化方面具有良好的应用前景.     

REDV/雷帕霉素复合涂层构建内皮细胞选择性功能界面

运用复合涂层的概念构建了兼具药物洗脱和内皮促进作用的载药涂层. 以载雷帕霉素(Rapamycin, RAP)的聚乙二醇甲基丙烯酸酯(PEGMA)-甲基丙烯酸丁酯(BMA)(PEGMA-BMA, PEGB)为内层, Arg-Glu-Asp-Val(REDV)多肽修饰的PEGBN为外层包裹载药涂层. 体外药物释放结果表明, 雷帕霉素可以维持缓慢稳定的长效释放, 释放过程中没有出现暴释现象. 表面细胞生长行为表明, 雷帕霉素可以有效地阻抗内皮细胞和平滑肌细胞的黏附, 抑制细胞活性; 随着药物释放的进行, 雷帕霉素浓度逐渐减低, 但涂层依然维持对平滑肌细胞的非特异性阻抗; 而REDV修饰的外涂层开始呈现内皮细胞的选择性黏附, 随着释放时间延长, 内皮细胞特异选择性也逐渐加强. 雷帕霉素和REDV多肽协同构建的复合涂层能够有效抑制平滑肌细胞的增殖, 获得内皮细胞选择性黏附.     

仿细胞外层膜结构修饰交联壳聚糖的研究

采用溶液自由基聚合,合成甲基丙烯酰氧乙基磷酰胆碱(MPC)-甲基丙烯酰氧丙基三甲氧基硅(TSMA)二元共聚物(PMT82),将其涂覆在戊二醛交联壳聚糖(CS-GA)表面,通过三乙胺蒸汽催化处理获得具有仿细胞外层膜结构(CS-GA-PMT82b)的表面.用动态接触角(DCA)、X-射线光电子能谱(XPS)对改性后交联壳聚糖表面的亲疏水性、元素组成等进行表征,并通过血小板黏附实验对其抗凝血性进行评价.研究结果表明,这种利用涂覆催化交联的方式将含有三甲氧基硅可交联基团的磷酰胆碱聚合物交联固定在壳聚糖表面,获得了较为稳定的仿细胞外层膜结构的CS-GA-PMT82b涂层表面.与壳聚糖相比,改性后壳聚糖的血小板黏附显著减少,抗凝血性能显著提高.这种改善材料的方式有望成为生物医用材料表面改性领域的有效的新手段.     

钛表面固定特异性识别内皮祖细胞的多肽适配子

在钛表面固定可与循环血液中的内皮祖细胞(EPC)特异性结合的多肽适配子,构建内皮祖细胞的特异性识别表面,用于心血管材料的表面改性.首先,采用固相合成法合成可与EPC特异性结合的多肽适配子,其序列为TPSLEQRTVYAK,并在羧基端进行生物素修饰;然后,采用磷酸处理钛表面,在钛表面获得化学键合的羟基,该羟基化表面与3-...     

MMA/MPC共聚物膜的制备及其抗凝血性能研究

采用自由基共聚制得了甲基丙烯酸甲酯与2-甲基丙烯酰氧基乙基磷酰胆碱(MPC)的共聚物, 在模板中将溶剂蒸发得到了共聚物膜. 用差示扫描量热仪(DSC)、凝胶渗透色谱(GPC)、元素分析仪(EA)、氢核磁共振(¹H NMR)及扫描电子显微镜(SEM)对共聚物及其膜的结构与形态进行了表征, 并测定了膜的溶胀度与表面的亲水性, 结合对牛血清蛋白(BSA)吸附研究结果表明: 共聚物膜的溶胀度随着MPC含量的增加而逐渐上升, 并且随着温度的升高而逐渐增大; 由动态接触角(DCA)结果可知共聚物膜表面的链段可随着环境的变化而发生重排, MPC链段向膜表面的迁移提高了膜表面的亲水性, 降低了对蛋白质的吸附. 并通过体外血小板粘附试验对膜材料的抗凝血性能进行了评价. 结果表明, 当共聚物膜中w(MPC)＝0.25时, 膜表面吸附的血小板数量明显减少, 共聚物膜表现出良好的抗凝血性能.     

反应性梳状聚乙二醇改性聚酯薄膜表面及其内皮细胞相容性的研究

以高密度梳状PEG(CPEG)作为表面改性材料, 将PEG末端羟基转化为醛基, 将梳状PEG和线形PEG固定在氨基化的PET膜表面, 并利用表面的反应性醛基进一步固定了氨基酸和整合素配体多肽片段RGD多肽. 红外光谱、 接触角和X射线光电子能谱(XPS)测定结果表明, 该法可有效地固定氨基酸和多肽, 获得模拟细胞膜中多糖-蛋白质复合物结构的特异性功能表面. 对两种不同结构的PEG细胞培养实验结果表明, CPEG比线形PEG(LPEG)具有更好的抗非特异粘附性. 此外, CPEG比LPEG具有更多的活性反应基团, 用PEG末端活性的醛基固定整合素配体多肽片段RGD, 可有效地诱导材料表面的内皮细胞化, 改善材料的细胞相容性.     

仿细胞膜结构共聚物的合成及涂层性能

采用饥饿法将2-甲基丙烯酰氧乙基磷酰胆碱(MPC)分别与甲基丙烯酸十八烷基酯(SMA)、 甲基丙烯酸十二烷基酯(LMA)及甲基丙烯酸正丁酯(BMA)聚合, 通过改变投料比例和沉淀剂种类, 合成了一系列含磷酰胆碱基团的仿细胞膜结构的两亲性二元随机共聚物. ¹H NMR和元素分析结果表明, 合成的两亲性二元随机共聚物的组成与投料比相近. DSC结果表明, 聚合物具有较低的玻璃化转变温度. 表面张力及水的动态接触角(DCA)研究发现, 聚合物涂层表面具有明显的两亲性及表面结构易变性, 在空气中憎水基团在表面取向, 在水环境中亲水的磷酰胆碱基团则迁移取向到涂层表面形成仿细胞外层膜结构界面, 最终形成不溶于水的仿细胞膜结构涂层.     

磷酰胆碱色谱固定相的制备及其在分离生物大分子中的应用

以三氯氧磷、氯化胆碱和甲基丙烯酸-2-羟乙酯(HEMA)为原料合成了含磷酰胆碱的单体2-甲基丙烯酰氧乙基磷酰胆碱(MPC). 在硅胶表面嫁接聚合MPC, 得到磷酰胆碱两性离子交换色谱填料. 研究了该填料对标准蛋白的分离性能及pH对蛋白质保留的影响. 结果表明, 该填料对溶菌酶和牛血清白蛋白的动态吸附容量分别为13.8和18.7 mg/g, 其基质磷酰胆碱色谱固定相可同时基线分离两种酸性和两种碱性蛋白.     

基于电聚多巴胺技术一锅法快速构建生物活性界面的研究

通过精确控制电化学参数采用循环伏安法在中性无氧水环境中制备得到膜厚可控的电聚多巴胺膜,并将这种电聚多巴胺技术与生物活性分子的负载相结合,通过一锅法电聚得到含REDV活性短肽的聚多巴胺活性膜,快速便捷地构建了具有促内皮细胞粘附的生物活性界面.椭圆偏振仪、扫描电子显微镜证实了材料界面上形成了均一的聚多巴胺膜;X射线光电子能谱以及荧光分析结果证实了REDV短肽已负载于电聚多巴胺涂层中.内皮细胞体外黏附实验证实REDV短肽保持了良好活性,可有效促进内皮细胞黏附、铺展及粘着斑的形成.这种一锅法快速制备具有生物活性的电聚多巴胺涂层技术有望为复杂的导电生物材料和装置的多功能界面修饰提供新的途径.     

基于树状高分子固定DNA的电化学生物传感器的研究

用电化学氧化法使玻碳电极表面氧化生成羧基,利用偶联活化试剂将1.0G树状高分子(PAMAM)固定在玻碳电极表面,并通过共价结合固定ssDNA。以亚甲基蓝为指示剂,采用循环伏安法、示差脉冲伏安法等电化学方法对DNA电化学生物传感器进行了表征。结果发现,通过亚甲基蓝与双链dsDNA作用的氧化还原电流的变化,可以识别和定量检测溶液中互补的ssDNA片段。经过条件优化,本法测定DNA的浓度线性范围为2×10-9～2×10-7mol/L,检出限为1×10-9mol/L。     

An enzyme-immobilization method for integration of biofunctions on a microchip using a water-soluble amphiphilic phospholipid polymer having a reacting group

A water-soluble phospholipid polymer having an active ester group in the side chain, poly[2-methacryloyloxyethyl phosphorylcholine (MPC)-co-n-butyl methacrylate (BMA)-co-p-nitrophenyloxycarbonyl polyethyleneglycol methacrylate (MEONP)(PMBN), was used for the immobilization of an enzyme on a plastic microchip. The MPC polymers with BMA units were adsorbed onto the poly(methyl methacrylate)(PMMA) microchip, and the active ester group in the MEONP unit reacted with the amino groups of the proteolytic enzyme, trypsin. Trypsin was immobilized on the sample reservoir, and catalyzed the hydrolysis of the fluorescently labeled ArgOEt to Arg. The consequent separation of product from the substrate, and their detection, were integrated on the microchip and this meant that all procedures from the enzymatic activity to product detection were completed in less than three minutes.     

Different complex surfaces of polyethyleneglycol (PEG) and REDV ligand to enhance the endothelial cells selectivity over smooth muscle cells

Arg-Glu-Asp-Val (REDV) peptide with endothelial cells (ECs) selectivity was immobilized onto PEG based polymeric coating via the active p-nitrophenyloxycarbonyl group. The adhesion and proliferation of human umbilical vein endothelial cells (HUVECs) and human aortic smooth muscle cells (HASMCs) onto surface modified either by REDV end-tethered polyethylene glycol (PEG) or by the complex of free PEG and REDV were investigated to understand the synergic action of nonspecific resistance of PEG and specific recognitions of REDV. Cell culture results indicated that the surfaces end tethered by REDV peptide via PEG "spacer" (n=1, 6, 10) exhibited slight EC selectivity and showed small difference between different lengths of PEG chain. Both separate-culture and co-culture of HUVECs and HASMCs indicated that the introducing of free PEG into REDV tethered surface inhibited HASMCs adhesion significantly and remained a high level of HUVECs growth. Furthermore, the surface with short free PEG chain (n=6) was much more effective to enhance ECs selectivity than long EG chain (n=23). The combination of nonspecific resistance of short free PEG and the ECs selectivity of REDV peptide presents much better ability to enhance the competitive adhesion of HUVECs over HASMCs.     

Stabilization of phospholipid polymer surface with three-dimensional nanometer-scaled structure for highly sensitive immunoassay

A phospholipid polymer platform and an antibody as a bioaffinity ligand were used to construct a biointerface for a highly sensitive immunoassay. The platform had a nanometer-scaled particle deposition surface and it was constructed with poly[2-methacryloyloxyethyl phosphorylcholine (MPC)-co-n-butyl methacrylate (BMA)-co-p-nitrophenyloxycarbonyl poly(ethylene glycol) methacrylate (MEONP)] (PMBN) by an electrospray deposition (ESD) method. The PMBN surface could immobilize specific antibodies through covalent chemical bonding by the reaction between MEONP units and amino groups in the antibody. In addition, the PMBN could prevent nonspecific protein adsorption from an analyte. However, the nanometer-scaled structure of the PMBN lost its shape after immersion in an aqueous medium. To stabilize the nanometer-scaled structure in an aqueous medium, the PMBN was cross-linked with 1,4-butylenediamine and then heat-treated. These treatments effectively improved the stability of the nanometer-scaled structure, that is, the structure had a high porosity even after immersing in an aqueous medium. The stabilization affected the specific signal in the enzyme-linked immunosorbent assay (ELISA), that is, the specific signal in ELISA was enhanced.     

磷酸胆碱基细胞膜仿生药物缓释涂层材料的研究

随着现代医学的飞速发展 ,各种微创介入医疗装置如医用导管 (Catheter)、手术导引线(Guidewires)、金属支架 (Stents)等已广泛地应用到各种医疗技术中 ,极大地丰富了现代医学诊疗手段 .然而在临床应用中 ,现有的装置依然不同程度地存在感染、凝血和术后组织增生等问题[1 ] .设计生物相容性聚合物基载药涂层和实现药物在预定部位的定向释放 ,为解决这些问题提供了有效途径 [2～ 5] .血红细胞膜具有由磷脂分子自组装形成的双层膜 .细胞生物学研究表明 ,细胞膜外层带有等量正电荷和负电荷的卵磷脂不会激活内源性凝血途径 .因此人们设计了磷酸…     

Surface modification on microfluidic devices with 2-methacryloyloxyethyl phosphorylcholine polymers for reducing unfavorable protein adsorption

Surface modification of polymer materials for preparing microfluidic devices including poly(dimethyl siloxane) (PDMS) was investigated with phospholipids polymers such as poly(2-methacryloyloxylethyl phosphorylcholine(MPC)-co-n-butyl methacrylate) (PMB) and poly(MPC-co-2-ethylhexyl methacrylate-co-2-(N,N-dimethylamino)ethyl methacrylate) (PMED). The hydrophilicity of every surface on the polymer materials modified with these MPC polymers increased and the value of zeta-potential became close to zero. The protein adsorption on the polymer materials with and without the surface modification was evaluated using a protein mixture of human plasma fibrinogen and serum albumin. Amount of proteins adsorbed on these polymeric materials showed significant reduction by the surface modification with the MPC polymers compared to the uncoated surfaces ranging from 56 to 90%. Furthermore, we successfully prepared PDMS-based microchannel which was modified by simple coating with the PMB and PMED. The modified microchannel also revealed a significant reduction of adsorption of serum albumin. We conclude that the MPC polymers are useful for reducing unfavorable protein adsorption on microfluidic devices.     

Rapid development of hydrophilicity and protein adsorption resistance by polymer surfaces bearing phosphorylcholine and naphthalene groups

In order to provide a protein adsorption resistant surface even when the surface was in contact with a protein solution under completely dry conditions, a new phospholipid copolymer, poly (2-methacryloyloxyethyl phosphorylcholine (MPC)- co-2-vinylnaphthalene (vN)) (PMvN), was synthesized. Poly(ethylene terephthalate) (PET) could be readily coated with PMvN by a solvent evaporation method. Dynamic contact angle measurements with water revealed that the surface was wetted very rapidly and had strong hydrophilic characteristics; moreover, molecular mobility at the surface was extremely low. When the surface came in contact with a plasma protein solution containing bovine serum albumin (BSA), the amounts of the plasma protein adsorbed on the dry surface coated with PMvN and that adsorbed on a dry surface coated with poly(MPC-co-n-butyl methacrylate) (PMB) were compared. Substantially lower protein adsorption was observed with PMvN coating. This is due to the rapid hydration behavior of PMvN. We concluded that PMvN can be used as a functional coating material for medical devices without any wetting pretreatment.     

Molecular-Integrated Phospholipid Polymer Nanoparticles with Highly Biofunctionality

Surface modifications of nanoparticles with phospholipid polymers composed of 2-methacryloyloxyethyl phosphorylcholine (MPC), are summarized. The MPC can be available for various polymerization methods such as conventional radical polymerization and living radical polymerization, and easily copolymerized with other vinyl compounds. The MPC polymers have been widely used as biocompatible coating and stabilizer for nanoparticles even when they are under biological environment. Additionally, for immobilization of biomolecules, such as antibody and enzyme, the MPC polymers having active ester group are applicable. These MPC polymers coated on the nanoparticles immobilize protein under mild condition and the protein maintained bioactivity well. Moreover, introduction of functional inorganic nanocrystals inside of the nanoparticles is effective to obtain good imaging tool for specific cells. The potential of molecular integration on nanoparticles based on MPC polymer chemistry will be expanded nanobiosensing, nanoimaging and nanodiagnostic system.     

Measurement of the electrophoretic mobility of sheep erythrocytes using microcapillary chips

Cell electrophoretic mobility (EPM) can be used to characterize individual cells. The purpose of this study is to establish reproducible and reliable cell EPM values obtained using microcapillary electrophoresis (microCE) chips. We studied cell electrophoresis on microCE chips through the comprehensive measurement of EPM and zeta potential. The inner wall of microchannels in microCE chips was coated with three kinds of reagents, namely bovine serum albumin (BSA), gelatin, and 2-methacryloyloxyethylphosphorylcholine (MPC) polymer to prevent nonspecific adhesion and interaction between cells and the inner wall. Electrophoresis was conducted in phosphate-buffered saline (pH 4-9) using erythrocytes extracted from sheep whole blood. Electroosmotic flow (EOF) mobility was measured using noncharged particles, and then the true EPM was calculated by subtracting the EOF mobility from the electromigration. MPC polymer coatings in microCE chips reduced the zeta potential of the inner wall and fully prevented nonspecific adhesion. EPM data obtained using microCE chips were almost the same and reproducible over a wide range of pH irrespective of the coating reagent used. In conclusion, reliability in the measurement of cell EPM using microCE chips was realized.