【作者】 张腾霄；

【导师】 陈超； 高文运；

【作者基本信息】 西北大学 ， 微生物学， 2008， 硕士

【摘要】 本文研究了表面修饰有环氧乙烷基和羧基的功能化磁粒的制备,将对血液或血液制品中的HIV、HBV及HCV等病毒有很强的光诱导灭活作用的吩噻嗪类染料,如天青A和天青B,通过化学反应共价偶联到硅烷化的磁粒上的几种方法,制备了既可灭活血液或血液制品中的病毒、又可以用磁性分离方式将其彻底去除的血液净化材料。该材料应用于几种血液样品中指示病毒PRV的灭活,建立了用CPE实验结合Reed-Muench法评价其灭活病毒的效率的方法,鉴定了所合成的光敏剂修饰磁性微粒的光诱导的病毒灭活活性。材料合成部分中,首先用化学共沉淀法制备Fe₃O₄磁流体,磁流体经硅烷化修饰分别得到表面羧基化和环氧化的磁性载体。羧基化磁粒用EDC或DCC活化剂介导酰胺缩合反应将AA或AB固定;环氧化磁粒用采用金属三氟化物催化、离子超声催化和碱催化等3种方法,催化环氧乙烷基和芳香胺的开环加成反应,从而将AA或AB固定。初步比较各种方法,发现碱催化的方法使光敏剂偶联量效果最好,然后进一步对碱催化的合成方法优化以提高偶联量。用优化的合成方法制备该材料,并检测批内和批间差异,考察了偶联量的稳定性。UV-Vis分光光度法测定AA和AB的偶联量分别达到50nmol/mg和37 nmol/mg。研究了光敏剂的吸光度与溶液酸碱度的关系,为用盐酸溶解磁粒分光光度法测定偶联量提供了指导。研究了AA和AB的碱性稳定性,得到两者氧化分解的pH上限值分别为11.5和12.0,为合成体系优化中pH值的确定提供了指导。以DPCI为单线态氧捕捉剂,用氧化-萃取光度法测定单线态氧产率,来分析磁粒的光敏剂的偶联量,鉴定了光敏剂修饰磁粒的光敏活性。选用PRV为指示病毒,从光照时间、光敏剂修饰磁粒用量等影响光化学效应的主要因素入手,优化了光化学灭活病毒的体系,并应用于血浆、血红蛋白溶液及人血液代用品（聚合猪血红蛋白）中指示病毒的光化学灭活。结果显示该磁性材料灭活效果优良,能使被试样品中病毒滴度下降达4个LgTCID₅₀以上。光敏剂修饰磁粒把磁性微粒的优良生物相容性、磁场中的易分离性与吩噻嗪光敏剂灭活病毒的高效性相结合,将很可能是用于血液净化的新型材料。更多还原

【Abstract】 In this research, dye-containing magnetic particles were prepared by using functionalized magnetic microparticles with carboxyl group or epoxy group on its surface, and then the phenothiazinium photosensitizers, such as azure A and azure B, which have strong photo-induced virus inactivation activity were coupled onto the magnetic microparticles by several different methods. This kind of material has the super-paramagnetic feature, which makes it easy to separate the photosensitizer modified particles from blood products after photochemical virus inactivation, so the problem of dye residue is completely resolved. The material was applied for virus inactivation in blood products, using cytopathic effect experiment to titer the indicator virus infectivity and Reed-Muench method to calculate virus inactivation efficiency, and the result indicated the excellent activity of the material.In the part of the preparation of the phenothiazinium photosensitizer modified particles, super-paramagnetic Fe₃O₄ microparticles were prepared by chemical co-precipitation method firstly; Secondly, epoxy group or carboxyl group were subsequently introduced to the Fe₃O₄ particle surface as molecular anchor by silylation; Then, crosslink reagent, such as EDC and DCC, were used to catalyze the reaction for coupling AA or AB to the surface of carboxyl-terminated particles; Meanwhile, organometallic reagent Sn （II） triflate catalyzed method, FeCl₃-ultrasonic irradiation mediated method and alkali catalyzed method were tried to covalently couple AA or AB onto the epoxy-terminated particles. Comparing all the methods mentioned above, alkali-catalyzed method performed the best. Finally, the alkali-catalyzed method was optimized to improve the content of photosensitizer on the particles and the coupling amounts of AA and AB, which were determined by UV-Vis spectrometry, are up to 50 nmol/mg and 37 nmol/mg, respectively.Further more, researched on the relationship between the light absorption of phenothiazinium photosensitizers and the pH value of the solution, which can be guidance for determining the dye-coupling amount by UV-Vis method. Studied on the stability of AA/AB in different pH value PB buffer, the result of the experiment indicated that the maximum pH value borderline for oxidation-decomposition is 11.5 （AA） and 12.0 （AB）. So the experiment provided the guidance for the optimization of the pH value.In the photo-induced virus inactivation experiment, the PRV was used as indicating virus and the inactivation system was optimized by varying the irradiation time, irradiation aera and concentration of the photosensitizer modified particle, etc. The materials were applied for several blood products, such as the plasma, hemoglobin solution and poly-hemoglobin solution. The result indicated that the material performed excellently, with more than 4 LgTCID₅₀ reduction of PRV titer. In conclusion, the virus inactivation activity of photosensitizer modified particles showed strong potential in the hemopurification field.The capability of singlet oxygen yield was determined by the oxidation-extraction UV method, in which the DPCI was used as the singlet oxygen （¹O₂） capture-agent. So the dye coupling amount was detected indirectly.更多还原