藻蓝蛋白/羧甲基壳聚糖纳米球的制备及其对人宫颈癌HeLa细胞增殖的抑制

Optimized preparation of novel C-phycocyanin-carboxymethyl chitosan nanoparticles and its inhibitory effects on human cervical carcinoma He La cells proliferation

目的:制备新型负载藻蓝蛋白(C-phycocyanin,C-PC)的羧甲基壳聚糖(carboxymethyl chitosan,CMC)纳米微球(nanoparticle,NP),探讨藻蓝蛋白羧甲基壳聚糖纳米微球(C-PC/CMCNP)对人宫颈癌He La细胞生长的影响及其分子机制。方法:采用正交分析方法,以CMC和C-PC的质量比、CMC浓度、Ca Cl2浓度为考察因素,通过检测C-PC/CMCNP的粒径和CMC的包封率,优选制备C-PC/CMCNP的最佳条件,并制备C-PC/CMCNP。CCK-8法检测C-PC、CMC和C-PC/CMCNP对He La细胞增殖的影响,流式细胞术检测He La细胞凋亡情况,Westren blotting检测He La细胞中caspase-3蛋白的表达。结果:CMC与C-PC的质量比为1∶2、CMC质量浓度为1 mg/ml,Ca Cl2质量浓度为1 mg/ml为最佳制备条件,最终制备的C-PC/CMCNP的平均粒径为(118.4±2.07)nm,并具有较高的包封率(63.2%),其缓释12 h的累积缓释率超过60%。C-PC、CMC和C-PC/CMCNP均能抑制He La细胞的增殖,诱导细胞凋亡,并促进caspase-3蛋白的表达,但C-PC/CMCNP的作用效果最为显著。结论:优化制备条件得到具有高效缓释性能的C-PC/CMCNP,其对He La细胞显著的抑制作用可能是通过促进caspase-3蛋白的表达进而诱导肿瘤细胞凋亡来实现的。

Objective: To develop a methodology of preparing novel C-phycocyanin-carboxymethyl chitosan nanoparticles( C-PC / CMCNPs) and determine the effect of C-PC / CMCNPs on the growth of He La cells. Methods: An orthogonal experiment was designed with the particle diameter and entrapment efficiency as index and CMC: C-PC mass ratio,CMC concentration,and Ca Cl2 concentration as factors to determine the best preparing condition of C-PC / CMCNPs. The effects of the generated C-PC / CMCNPs on the growth and apoptosis of He La cells were assessed by CCK-8 assay and flow cytometry respectively. Caspase-3 protein expression in He La cells was quantified by Western blotting. Results: The optimal condition for C-PC / CMCNPs preparations were as follows: C-PC to CMC ratio of 1∶ 2,CMC concentration of 1 mg / ml and Ca Cl2 concentration of 1 mg / ml. The C-PC / CMCNPs prepared in these optimal conditions had a high entrapment efficiency with an average particle diameter of 118. 4 nm. C-PC,CMC and C-PC / CMCNPs were all capable of inhibiting proliferation and inducing apoptosis in He La cells by up-regulating the expression of the caspase-3 protein,but the effect of CPC / CMCNPs was significantly more pronounced( P < 0. 05). Conclusions: We have optimized the conditions of preparing C-PC / CMCNPs. The nanoparticles prepared under these conditions have an acceptable safety profile of sustained C-PC release and possess a caspase-3-dependent anti-tumor activity,suggesting potential clinical implications.