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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He La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/CMCCD59sp-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 a novel C-PC-targeted nanometer microspheres (NPs) and investigate its targeted therapy on HeLa cells. Methods: Targeted carboxymethyl chitosan (CMC) nano-drug-loaded particles C-PC / CMC-NPs were prepared by ion-crosslinking method. The optimum preparation conditions were screened by response surface methodology. The effect of targeting nanoparticles C-PC / CMC-CD59sp-NPs on the growth of human cervical cancer HeLa cells was detected by MTT assay and the tissue of C-PC / CMC-CD59sp-NPs Compatibility and safety. The effects of C-PC / CMC-CD59sp-NPs on the expression of Caspase-3 / PARP and Bcl-2 in HeLa cells were detected by Western blotting and immunohistochemistry. Results: The C-PC / CMC nanospheres were prepared successfully. The transmission electron microscopy showed that the microspheres had a regular spherical shape with uniform dispersion and a diameter of about 160 nm. The optimum preparation conditions were as follows: the carboxymethyl chitosan concentration was 2.0 mg / ml, the concentration of CaCl 2 was 1.0 mg / ml and the diameter was about 120 nm. The encapsulation efficiency was (62 ± 5)%. CMC and C- PC-CMC / NP-NPs showed sustained-release characteristics in vitro with a 3: 1 dose and a drug loading of 20 ± 3%. The release rates of C-PC / CMC-NPs reached 80 % And 60%; at the same time did not appear hemolysis. C-PC / CMC-CD59sp-NPs significantly inhibited the proliferation of HeLa cells, promoted the expression of activated Caspase-3 / PARP protein and inhibited the expression of Bcl-2 protein (all P <0.05). Conclusion: The new targeted drug C-PC / CMCCD59sp-NPs can inhibit HeLa cell growth and induce apoptosis in vitro. It provides a new idea for the research and development of marine drugs and provides a theoretical basis for further research on targeting nanomedicine.