目的 制備共載姜黃素及順鉑pH敏感脂質(zhì)體（cisplatin and curcumin co-loaded pH sensitive liposomes，CCL），初步考察其體外抗乳腺癌活性。 方法 半效法篩選兩藥聯(lián)用的最佳比例；薄膜分散法制備CCL；單因素考察最佳處方工藝，并用Box-Behnken設(shè)計(jì)-響應(yīng)面法優(yōu)化最優(yōu)處方；激光粒度儀、透射電子顯微鏡（transmission electron microscope，TEM）考察CCL的粒徑、ζ電位及形態(tài)；HPLC法測(cè)定藥物含量；透析袋法考察體外釋放規(guī)律；CCK-8考察4T1細(xì)胞增殖抑制作用；細(xì)胞劃痕、細(xì)胞克隆、DAPI核染細(xì)胞實(shí)驗(yàn)分別考察4T1細(xì)胞遷移能力、增殖能力以及細(xì)胞凋亡；溶血實(shí)驗(yàn)考察其生物相容性。結(jié)果 兩藥聯(lián)用的最佳比例為順鉑-姜黃素1∶6；CCL的最優(yōu)處方參數(shù)為藥脂比1∶29.83，膽脂比1∶11.23，脫氧膽酸鈉的用量14.08 mg；CCL在pH 7.0和pH 5.0條件下的粒徑分別為（156.54±2.56）、（318.92±12.82）nm，ζ電位分別為（−34.48±2.67）、（−11.07±1.05）mV；TEM下，CCL在pH 7.0條件下形態(tài)較規(guī)整，呈類球形，無(wú)明顯裂痕，在pH 5.0條件下粒徑增大，形態(tài)不規(guī)則，出現(xiàn)裂痕；HPLC法測(cè)定CCL中姜黃素和順鉑的包封率和載藥量分別為（96.45±0.53）%、（79.85±0.20）%和（1.43±0.13）%、（0.30±0.05）%；體外釋放結(jié)果顯示，CCL具有一定的緩釋作用，符合一級(jí)釋放模型；CCK-8實(shí)驗(yàn)結(jié)果顯示，CCL對(duì)乳腺癌4T1細(xì)胞具有增殖抑制作用，并與藥物質(zhì)量濃度呈正相關(guān)；細(xì)胞劃痕實(shí)驗(yàn)結(jié)果表明，同一時(shí)間下與對(duì)照組和其他給藥組比較，CCL抑制細(xì)胞遷移能力顯著增強(qiáng)（P＜0.001）；DAPI核染實(shí)驗(yàn)結(jié)果表明，CCL組細(xì)胞核固縮、細(xì)胞數(shù)目少于其他給藥組；細(xì)胞克隆形成實(shí)驗(yàn)結(jié)果表明，CCL組抑制細(xì)胞克隆的能力比其他組顯著增強(qiáng)（P＜0.001）；溶血實(shí)驗(yàn)結(jié)果表明，CCL生物相容性良好。結(jié)論 成功制備CCL，且姜黃素和順鉑兩藥聯(lián)用比其單用的體外抗乳腺癌效果好，為藥物聯(lián)用治療乳腺癌的研究提供新思路。

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.

R283.6

國(guó)家自然科學(xué)基金資助項(xiàng)目(82060723);云南省科技廳-云南中醫(yī)藥大學(xué)應(yīng)用基礎(chǔ)研究聯(lián)合專項(xiàng)重點(diǎn)項(xiàng)目(202001AZ070001-008);云南省科技廳生物醫(yī)藥重大科技專項(xiàng)(202002AA100007);云南省科技人才平臺(tái)計(jì)劃(202105AG070012);云南省傣醫(yī)藥與彝醫(yī)藥重點(diǎn)實(shí)驗(yàn)室開放課題(202210SS2205);云南省傣醫(yī)藥與彝醫(yī)藥重點(diǎn)實(shí)驗(yàn)室開放課題(202210SS2206);云南省傣醫(yī)藥與彝醫(yī)藥重點(diǎn)實(shí)驗(yàn)室開放課題(202210ZD2207);云南省教育廳科學(xué)研究基金項(xiàng)目(2024Y345);國(guó)家中醫(yī)藥管理局"十二五"重點(diǎn)學(xué)科——傣藥學(xué)