一种小粒径壳聚糖纳米粒作为基因载体的研究  

Optimizing small size nanoparticles based on chitosan as gene delivery systems

作　　者：张文华 梁晔[2] 周泉[3] 李彤[1] 李明钰 苗玉 徐文华 王丽萍 ZHANG Wenhua;LIANG Ye;ZHOU Quan;LI Tong;LI Mingyu;MIAO Yu;XU Wenhua;WANG Liping(Department of Inspection, Qingdao University, Qingdao 266003, China;Key Laboratory, Department of Urology and Andrology, The Affiliated Hospital of Qingdao University, Qingdao 266003, China;Central Laboratory, The Affiliated Hospital of Qingdao University, Qingdao 266003, China;Clinical Laboratory, The Affiliated Hospital of Qingdao University, Qingdao 266003, China)

机构地区：[1]青岛大学医学部检验学系,山东青岛266003 [2]青岛大学附属医院泌尿外科与男科学重点实验室,山东青岛266003 [3]青岛大学附属医院中心实验室,山东青岛266003 [4]青岛大学附属医院检验科,山东青岛266003

出　　处：《功能材料》2017年第12期12084-12093,共10页Journal of Functional Materials

基　　金：国家自然科学基金资助项目(81401899);山东省自然科学基金资助项目(ZR2014HP011);青岛青年科学家应用基础研究基金资助项目(15-9-1-51-jch);青岛大学附属医院青年基金会资助项目(2417)

摘　　要：系统比较了不同分子量的壳聚糖与TPP离子交联制备的纳米粒,用于基因转运载体,评价其对质粒和siRNA的转运效果。通过粒度仪测定壳聚糖纳米粒(CSNPs)的粒径、多分散系数以及Zeta电位;透射电镜观察CSNPs的形态;MTT法测定其细胞毒性;通过CSNPs在小鼠肌肉内的组织切片观察其生物相容性。CSNPs分别包载质粒和siRNA,凝胶电泳和分光光度法测定其包载能力,荧光显微镜、激光共聚焦或流式细胞术分析其对肿瘤细胞的转染效果;溶血实验和血凝实验分析其血液相容性。结果表明,1 mg/mL的CS(160 k Da)和TPP(质量比为10∶1)制备的CSNPs粒径约100 nm,且分布均一、稳定,透射电镜下为形态规则的类球型粒子;其细胞毒性均在0~1级,符合生物医用材料毒性标准;组织切片没有发现明显的炎症反应,生物相容性良好;CSNPs对质粒和siRNA的包载率较高,并能成功将其转运至细胞内,但小粒径的纳米粒转染质粒后荧光较弱,而携带siRNA的CSNPs转染后荧光较强,且流式细胞术结果表明其与商品化转染试剂相比转染率较高;溶血及血凝实验也发现材料具有良好的血液相容性。所以,制备的小粒径CSNPs更适合作为一种安全高效的siRNA转运载体,后续可以加以修饰应用于肿瘤的基因靶向治疗中。Chitosan is a natural cationic polysaccharide,which is often used for preparing biomedical materials because of its high biocompatibility.In this study,chitosan with different molecular weights was chosen to prepare chitosan nanoparticles(CSNPs)as gene vector by ionic cross linking with tripolyphosphate(TPP).CSNPs were characterized in terms of particle size,zeta potential,polydispersity index(PDI)by the Zetasizer,and morphology was detected with transmission electron microscopy(TEM).Furthermore,the cytotoxicity and biocompatibility of CSNPs were correspondingly examined by MTT assay and the muscle slices of mice.The transfer effect on plasmids and siRNA was evaluated.Agarose gel electrophoresis method and spectrophotometry were used to detect the loading efficiency.The cell transfection efficiency of plasmids or siRNA loaded by CSNPs was analyzed by the fluorescence microscopy,laser scanning confocal microscopy or flow cytometry.The blood compatibility was analyzed by hemolytic experiment and full automatic blood coagulator.Results showed that when CS(160kDa)was1mg/mL with TTP(mass ratio was10∶1),CSNPs were prepared successfully by ionic gelation method,which had smaller particle size(about100nm),stable dispersibility,high gene loading efficiency and spheroidal particles with morphological rules under TEM.Their cytotoxicity is at the01level,which is in line with the toxicity standard of biomedical materials.No obvious inflammatory reaction was found in the tissue section,showing good tissue biocompatibility.CSNPs could transfer the plasmids or siRNA to cells with high loading rate.However,CSNPs might have much higher transfecting efficiency for siRNA than plasmids,which implied that CSNPs might be more suitable for being a safe and efficient vector for delivering siRNA than plasmids.The results of flow cytometry showed that the transfection rate was higher than that of commercial transfection reagents.Results also showed that CSNPs had good blood compatibility,so it can be applied as a safe and efficient siRNA

关 键 词：壳聚糖 TPP 纳米粒 基因转运 

分 类 号：R318.08[医药卫生—生物医学工程]