一种小粒径壳聚糖纳米粒作为基因载体的研究

发布时间：2018-07-10 03:28

本文选题：壳聚糖 + TPP　；参考：《功能材料》2017年12期

【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。
[Abstract]:The nanoparticles prepared by cross-linking chitosan with TPP ions with different molecular weights were systematically compared to be used as gene transport vectors to evaluate their translocation effects on plasmids and siRNA. The particle size, polydispersity coefficient and Zeta potential of chitosan nanoparticles (CSNPs) were measured by particle size analyzer. The biocompatibility of CSNPs in mouse muscle was observed by tissue section. CSNPs were encapsulated with plasmid and siRNA respectively. The encapsulation ability of CSNPs was determined by gel electrophoresis and spectrophotometry. Laser confocal or flow cytometry was used to analyze the transfection of tumor cells, and hemolysis and hemagglutination were used to analyze the blood compatibility of tumor cells. The results showed that CSNPs prepared by 1 mg / mL CS (160kDa) and TPP (10:1 by mass ratio) were about 100 nm in size, uniform in distribution, stable in distribution, regular in morphology under transmission electron microscope, and their cytotoxicity was in grade 0 ~ 1, which met the toxicity standard of biomaterials. No obvious inflammatory reaction was found in the tissue section. The biocompatibility of CSNPs was higher than that of the plasmid and siRNA, and could be successfully transported into the cells, but the fluorescence of the plasmid transfected with the small size nanoparticles was weak. The results of flow cytometry showed that the transfection efficiency of CSNPs carrying siRNA was higher than that of commercial transfection reagents, and the hemolysis and hemagglutination experiments also showed that the materials had good blood compatibility. Therefore, the prepared CSNPs are more suitable as a safe and efficient siRNA transport vector, and can be modified for gene targeting therapy.
【作者单位】：青岛大学医学部检验学系;青岛大学附属医院泌尿外科与男科学重点实验室;青岛大学附属医院中心实验室;青岛大学附属医院检验科;
【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)
【分类号】：O636.1;R450

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