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HeLa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、CaCl2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HeLa细胞增殖的影响，流式细胞术检测HeLa细胞凋亡情况，Westren blotting检测HeLa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，CaCl2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HeLa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HeLa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 HeLa 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 CaCl2 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 HeLa cells were assessed by CCK-8 assay and flow cytometry respectively. Caspase-3 protein expression in HeLa 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 CaCl2 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 HeLa cells by up-regulating the expression of the caspase-3 protein, but the effect of C-PC/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.

国家自然科学基金资助项目（No.81001346，No.81471546）；山东省医药卫生科技发展计划项目（No. 2011HZ023）；青岛市科技局公共领域科技支撑计划项目（No. 2012-1-3-5-(4)-nsh）。