目的 制备共载姜黄素及顺铂pH敏感脂质体(cisplatin and curcumin co-loaded pH sensitive liposomes，CCL)，初步考察其体外抗乳腺癌活性。方法 半效法筛选两药联用的最佳比例;薄膜分散法制备CCL;单因素考察最佳处方工艺，并用Box-Behnken设计-响应面法优化最优处方;激光粒度仪、透射电子显微镜(transmission electron microscope，TEM)考察CCL的粒径、ζ电位及形态;HPLC法测定药物含量;透析袋法考察体外释放规律;CCK-8考察4T1细胞增殖抑制作用;细胞划痕、细胞克隆、DAPI核染细胞实验分别考察4T1细胞迁移能力、增殖能力以及细胞凋亡;溶血实验考察其生物相容性。结果 两药联用的最佳比例为顺铂-姜黄素1∶6;CCL的最优处方参数为药脂比1∶29。83，胆脂比1∶11。23，脱氧胆酸钠的用量14。08 mg;CCL在pH 7。0和pH 5。0条件下的粒径分别为(156。54±2。56)、(318。92±12。82)nm，ζ电位分别为(−34。48±2。67)、(−11。07±1。05)mV;TEM下，CCL在pH 7。0条件下形态较规整，呈类球形，无明显裂痕，在pH 5。0条件下粒径增大，形态不规则，出现裂痕;HPLC法测定CCL中姜黄素和顺铂的包封率和载药量分别为(96。45±0。53)％、(79。85±0。20)％和(1。43±0。13)％、(0。30±0。05)％;体外释放结果显示，CCL具有一定的缓释作用，符合一级释放模型;CCK-8实验结果显示，CCL对乳腺癌4T1细胞具有增殖抑制作用，并与药物质量浓度呈正相关;细胞划痕实验结果表明，同一时间下与对照组和其他给药组比较，CCL抑制细胞迁移能力显著增强(P<0。001);DAPI核染实验结果表明，CCL组细胞核固缩、细胞数目少于其他给药组;细胞克隆形成实验结果表明，CCL组抑制细胞克隆的能力比其他组显著增强(P<0。001);溶血实验结果表明，CCL生物相容性良好。结论 成功制备CCL，且姜黄素和顺铂两药联用比其单用的体外抗乳腺癌效果好，为药物联用治疗乳腺癌的研究提供新思路。
Preparation of pH sensitive liposomes co-loaded with curcumin and cisplatin and evaluation of their anti-breast cancer activity in vitro
Objective To prepare cisplatin and curcumin co-loaded pH sensitive liposome (CCL) and investigate its anti-breast cancer activity in vitro. Methods Semi-model screening of the optimal ratio of two drugs combined;CCL was prepared by a thin film dispersion method. Box-Behnken design optimization optimal prescription;The particle size,ζ potential and morphology were investigated by laser particle size analyzer and transmission electron microscope. HPLC was used to determine the drug content. The release of 4T1 cells in vitro was investigated by dialysis bag method,and the proliferation inhibition of 4T1 cells was investigated by CCK-8. The migration,proliferation and apoptosis of 4T1 cells were investigated by cell scratch,cell clone and DAPI staining assay. The biocompatibility was investigated by hemolysis test. Results The optimal ratio of the two drugs was cisplatin-curcumin 1∶6;The optimal prescription parameters of CCL were:drug to lipid ratio 1∶29.83,bile to lipid ratio 1∶11.23,the dosage of sodium deoxycholate 14.08 mg;The particle size of CCL at pH 7.0 and pH 5.0 were (156.54±2.56) nm and (318.92±12.82) nm,and the ζ potential was (−34.48±2.67) mV and (−11.07±1.05) mV,respectively. Under transmission electron microscope (TEM),CCL was more regular and spheroidal at pH 7.0,without obvious cracks. Under pH 5.0,CCL had larger particle size,irregular shape and cracks. The encapsulation rate and drug loading of Cur and cisplatin were (96.45±0.53)％,(79.85±0.20)％,(1.43±0.13)％,(0.30±0.05)％,respectively,determined by HPLC. In vitro release results showed that CCL had a sustained release effect,which was consistent with the first-order release model. The results of CCK-8 experiment showed that CCL inhibited the proliferation of breast cancer 4T1 cells and was positively correlated with drug concentration. The results of cell scratch test showed that CCL inhibited cell migration significantly (P<0.001) compared with the control group and other groups at the same time. The results of DAPI staining showed that in CCL group the number of cell was less than that in other groups and nucleus pyknosis. The results showed that the inhibition of cell cloning in CCL group was significantly higher than that in other groups (P<0.001). The results of hemolysis test showed that CCL had good biocompatibility. Conclusion The pH sensitive liposomes of CCL were successfully prepared,and the anti-breast cancer effect of the two drugs combined was better than that of the single drug,which provided a new idea for the study of drug combined treatment of breast cancer.
curcumincisplatinpH sensitive liposomesthin film dispersion methodbreast cancerBox-Behnken design-response surface method
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