T7肽修饰的硫酸长春新碱脱铁铁蛋白纳米粒制备及其对胶质瘤细胞的体外靶向性研究

Study on the Preparation of Peptide T7-modified Vincristine Sulfate Apoferritin Nanoparticles and Its in vitro Targeting to Glioma Cells

目的:制备T7肽修饰的硫酸长春新碱(VCR)-脱铁铁蛋白(APO)纳米粒,并研究其对胶质瘤细胞的体外靶向性。方法:采用解离-重组法制备VCR-APO纳米粒,以靶头(T7肽)修饰后制成T7-VCR-APO纳米粒。采用质谱分析和紫外-可见分光光度法评价靶头连接效率,并对所制纳米粒进行形态、粒径、Zeta电位、包封率等进行表征。以胶质瘤C6细胞为模型,考察T7-VCRAPO纳米粒对C6细胞及肿瘤球的靶向作用,并以低温条件和4种细胞内吞抑制剂考察C6细胞对T7-VCR-APO纳米粒的摄取机制。结果:经质谱检测证明T7肽已连接在纳米粒上,连接率为68.39%;所制得的T7-VCR-APO纳米粒形态圆整、均一,粒径为(31.14±1.26)nm,Zeta电位为(-23.30±0.42)m V,包封率为(39.49±2.84)%。体外靶向性研究显示,T7-VCR-APO纳米粒可有效地被C6细胞及肿瘤球摄取,并可到达肿瘤球核心位置;C6细胞对纳米粒的摄取是多通路共同作用的结果,主要是通过小窝蛋白介导的内吞并伴随巨胞饮途径。结论:本研究成功制得T7-VCR-APO纳米粒,其能够对胶质瘤C6细胞实现体外靶向递送药物,入胞效率大大提高。

OBJECTIVE:To prepare peptide T7-modified vincristine sulfate(VCR)-apoferritin(APO)nanoparticles,and to study its in vitro targeting to tumor cells.METHODS:VCR-APO nanoparticles were prepared by dissociation-recombination method.The targeting peptide T7 was modified to prepare T7-VCR-APO nanoparticles.Target attachment efficiency was evaluated by MS analysis and UV spectrophotometry.The morphology,particle size,Zeta potential and encapsulation efficiency of prepared nanoparticles were all characterized.Using glioma cells C6 as model,the targeting effects of T7-VCR-APO nanoparticles on C6 cells and tumor spheres were investigated.The uptake of T7-VCR-APO nanoparticles by C6 cells was investigated under low temperature and by 4 kinds of cell endocytosis inhibitors.RESULTS:T7 peptide was confirmed to be linked to nanoparticles by MS,with attachment rate of 68.39%.The prepared T7-VCR-APO nanoparticles were round and uniform.The particle size was(31.14±1.26)nm,Zeta potential was(-23.30±0.42)mV,and entrapment efficiency was(39.49±2.84)%.The in vitro targeting study of tumor cells showed that T7-VCR-APO nanoparticles could be effectively taken up by C6 cells and spheres,and can reach the sphere center.The uptake of nanoparticles by C6 cells was the result of a multi-pathway interaction mainly through caveolin-mediated endocytosis and concomitant macrophage pathways.CONCLUSIONS:T7-VCR-APO nanoparticles are prepared successfully,can achieve targeting drug delivery to glioma C6 cells in vitro,and endocytosis efficiency is greatly improved.