目的：制备一种新型的C-藻蓝蛋白（C-phycocyanin，C-PC）靶向纳米微球（nanometer microspheres，NPs），并探究其对HeLa细胞的靶向治疗作用。方法：采用离子交联法制备靶向羧甲基壳聚糖（carboxymethyl chitosan，CMC ）纳米载药颗粒C-PC/CMC-NPs；响应面优化法筛选出最佳制备条件，并用透射电镜、激光粒度仪观察纳米载药颗粒的表征；MTT法检测靶向纳米颗粒C-PC/CMC-CD59sp-NPs对人宫颈癌HeLa细胞生长的影响；溶血实验检测C-PC/CMC-CD59sp-NPs的组织相容性及安全性；Western blotting和免疫组化法分别检测C-PC/CMC-CD59sp-NPs 对HeLa细胞内活化的Caspase-3/PARP蛋白和Bcl-2蛋白表达的影响。结果： 成功制备C-PC/CMC纳米微球，透射电镜观察到纳米微球呈现出规则的球形，分散均匀，其粒径约为160 nm。最佳制备条件：羧甲基壳聚糖浓度为2.0 mg/ml，CaCl2浓度为1.0 mg/ml，粒径约为120 nm的球形，包封率为(62±5)%；CMC与C-PC投药量为3∶1，载药量为(20±3)%；C-PC/CMC-NPs体外表现出缓释特征，在120 h内pH=5.4和74条件下，释放率分别达到80%和60%；同时未出现溶血现象。C-PC/CMC-CD59sp-NPs对HeLa细胞增殖有明显的抑制作用，促进活化的Caspase-3/PARP蛋白的表达，抑制Bcl-2蛋白的表达（均P＜0.05）。结论：新型靶向纳米药物C-PC/CMC-CD59sp-NPs能在体外抑制HeLa细胞的生长，诱导其凋亡。为海洋药物的研发提供了新的思路，对靶向纳米药物的进一步研究提供理论基础。

Objective:To prepare C-phycocyanin (C-PC) contained polymeric nanaparticles with targeting ligand and study its target treatment effect on HeLa cells. Methods: The targeting drug-loading carboxymethyl chitosan (CMC) nanoparticles were synthesized by ionic-gelation method, and the particles with the most optimal condition were selected by orthogonal analysis. The surface morphology and particles size were observed by transmission electron microscopy (TEM) and dynamic light scattering (DLS). The effects of nanoparticles on the proliferation of HeLa cells were assessed by MTT assay. The tissue compatibility and safety of nanoparticles were studied by hemolysis assay. The expression of cleaved Caspase-3 and Bcl-2 was determined by Western blotting and immunofluorescent staining, respectively. Results: The optimal condition for the preparation of the nanoparticles was: CMC at 2.0 mg/ml, CaCl2 at 1.0 mg/ml, and the nanoparticles size of about 120 nm with spherical morphology; the entrapment efficiency was (62±5)%; the ratio of CMC:C-PC was 3∶1, and drug-loading amount was (20±3)%. The drug-loading nanoparticles showed slow release characteristic in vitro and without hemolysis. The nanoparticles significantly inhibited the proliferation of HeLa cells and induced the cell apoptosis; in addition, it promoted the expressions of cleaved Caspase-3 protein but suppressed the expression of Bcl-2 protein. Conclusion: The novel targeting CPC nanoparticles could inhibit HeLa cell growth, with a superior apoptosis inducing effect over the other drugs, which provided a new idea for the research on marine drugs and an important theoretical value for further study on targeting drug-loading nanoparticles.

国家自然科学基金资助项目（No.81471546,No.81001346)