目的 探索坎地沙坦（CD）与雷公藤红素（CEL）共载还原敏感型胶束的体内外协同抗肿瘤作用。方法 以透明质酸-1,6-己二胺-坎地沙坦（HHCD，非氧化还原敏感性对照）、透明质酸-胱氨-坎地沙坦（HCCD，氧化还原敏感性）作为前药聚合物材料，通过溶剂挥发法分别制备HHCD、HCCD及负载CEL的HHCD/CEL、HCCD/CEL聚合物胶束。采用MTT法考察CD与CEL的抗肿瘤作用联合指数，并比较4种聚合物胶束（HHCD、HCCD、HHCD/CEL、HCCD/CEL，CEL与CD质量比为1∶ 2）溶液和游离药CD、CEL（0.3、0.6、1.2、2.5、5.0 μg·mL^-1）对人肝癌细胞HepG-2及正常肝细胞HL-7702的细胞毒性；利用阿霉素（DOX）的自发荧光特性，以DOX为阳性对照，制备HHCD/DOX和HCCD/DOX胶束，通过HepG-2细胞摄取实验评价肿瘤细胞对胶束的摄取能力；利用小鼠成纤维细胞NIH-3T3，考察游离CD、HHCD、HHCD/CEL、HCCD、HCCD/CEL（药物质量浓度均为3 μg·mL^-1）对上清液中胶原蛋白I（Col-I）、转化生长因子-β1（TGF-β1）含量的影响。建立H22荷瘤小鼠肝癌模型，以小鼠的体质量变化、存活率以及肿瘤体积为依据，对CD、CEL、CD/CEL、HHCD、HHCD/CEL、HCCD、HCCD/CEL（2 mg·kg^-1）的抗肿瘤活性以及安全性进行评价； ELISA法检测肿瘤组织Col-I、TGF-β1含量。结果 细胞实验结果显示，游离药物CEL与CD的质量比为1∶ 2时，联合指数最小，为0.736（＜1），表明2药具有协同作用。与对照组比较，游离CEL使正常肝细胞HL-7702存活率显著降低（P＜0.05、0.001），HHCD和HCCD具有较好的生物相容性；与其他给药组相比，HCCD/CEL聚合物胶束表现出最佳HepG-2细胞毒性（P＜0.05、0.01、0.001），其中HHCD/CEL胶束在1.2、2.5、5.0 μg·mL^-1质量浓度与HCCD/CEL胶束有显著性差异（P＜0.001）； HCCD胶束能有效被肿瘤细胞摄取；与对照组比较，游离CD组、HCCD/CEL组Col-I表达显著降低（P＜0.05、0.01）；与HHCD/CEL组相比，HCCD/CEL组Col-I表达显著降低（P＜0.05）；与对照组比较，CD、HHCD、HCCD、HHCD/CEL、HCCD/CEL组的TGF-β1含量显著下降（P＜0.05），HHCD/CEL和HCCD/CEL聚合物胶束呈更强作用趋势。体内抗肿瘤实验结果表明，与模型比较，给药组都体现出一定的抗肿瘤活性（P＜0.001），HCCD/CEL胶束与HHCD/CEL胶束相比有更好的抗肿瘤活性（P＜0.05）；除坎地沙坦药组外，其余各给药组小鼠的存活率均为100%；在整个实验周期中，各给药组小鼠体质量之间均未出现无显著差异；与模型组比较，HCCD/CEL组Col-I、TGF-β1含量显著降低（P＜0.001）；且HCCD/CEL组Col-I、TGF-β1含量显著低于HHCD/CEL组（P＜0.01），HHCD组Col-I、TGF-β1含量显著低于HCCD组（P＜0.05）。结论 CD与CEL共载还原敏感型胶束具有较好的细胞选择性毒性和生物安全性，可通过调控细胞外微环境实现良好的协同抗肿瘤作用。

Objective To explore the synergistic anti-tumor effects of co-loaded candesartan and celastrol in redox-sensitive micelles in vitro and in vivo. Methods Using hyaluronic acid-1,6-hexanediamine-candesartan (HHCD, non-redox-sensitive control) and hyaluronic acid-cystamine-candesartan (HCCD, redox-sensitive) as prodrug polymer materials, HHCD, HCCD, and their drug-loaded counterparts HHCD/CEL and HCCD/CEL polymeric micelles were prepared via solvent evaporation. The combination index of the antitumor effects of CD and CEL was evaluated using the MTT method, and the cytotoxicity of four types of polymer micelle solutions (HHCD, HCCD, HHCD/CEL, HCCD/CEL, with a mass ratio of CEL to CD of 1∶ 2), as well as free drugs CD and CEL (0.3, 0.6, 1.2, 2.5, 5.0 μg·mL^-1), was compared against human hepatocellular carcinoma HepG-2 cells and normal liver HL-7702 cells. Taking advantage of the spontaneous fluorescence of DOX, DOX was used as a positive control to prepare HHCD/DOX and HCCD/DOX micelles, and the cellular uptake of these micelles by HepG-2 cells was assessed. Using NIH-3T3 mouse fibroblasts, the effects of free CD, HHCD, HHCD/CEL, HCCD, and HCCD/CEL (all at a drug concentration of 3 μg·mL^-1) on the levels of collagen I (Col-I) and transforming growth factor-β1 (TGF-β1) in the supernatant were investigated. A murine H22 tumor-bearing model of hepatocellular carcinoma was established, and the antitumor activity and safety of CD, CEL, CD/CEL, HHCD, HHCD/CEL, HCCD, and HCCD/CEL (2 mg·kg^-1) were evaluated based on changes in body weight, survival rate, and tumor volume. The levels of Col-I and TGF-β1 in tumor tissues were determined by ELISA. Results Cellular experiments showed that the lowest combination index (0.736 < 1), indicating synergistic effects, was achieved when the mass ratio of free drug CEL to CD was 1∶ 2. Compared with the control group, free CEL significantly reduced the survival rate of normal liver cells HL-7702 (P<0.05, 0.001), whereas HHCD and HCCD exhibited better biocompatibility. Among all treatment groups, HCCD/CEL polymer micelles demonstrated the highest cytotoxicity against HepG-2 cells (P<0.05, 0.01, 0.001). Notably, HHCD/CEL micelles showed significant differences from HCCD/CEL micelles at concentrations of 1.2, 2.5, and 5.0 μg·mL^-1 (P<0.001). HCCD micelles were effectively internalized by tumor cells. Compared with the control group, both the free CD group and the HCCD/CEL group showed significantly reduced Col-I expression (P<0.05, 0.01); compared with the HHCD/CEL group, the HCCD/CEL group also exhibited significantly lower Col-I expression (P<0.05). Compared with the control group, TGF-β1 levels were significantly decreased in the CD, HHCD, HCCD, HHCD/CEL, and HCCD/CEL groups (P<0.05), with HHCD/CEL and HCCD/CEL polymer micelles showing a stronger inhibitory trend. In vivo anti-tumor experiments revealed that all treated groups exhibited significant anti-tumor activity compared with the model group (P<0.001), and HCCD/CEL micelles showed superior anti-tumor efficacy compared with HHCD/CEL micelles (P<0.05). Except for the candesartan-treated group, the survival rates of mice in all other treatment groups reached 100%. Throughout the entire experimental period, no significant differences in body weight were observed among the treated groups. Compared with the model group, the HCCD/CEL group showed significantly reduced levels of Col-I and TGF-β1 (P<0.001). Moreover, the levels of Col-I and TGF-β1 in the HCCD/CEL group were significantly lower than those in the HHCD/CEL group (P<0.01), and the HHCD group had significantly lower levels than the HCCD group (P<0.05). Conclusion The candesartan-celastrol co-loaded reduction-sensitive prodrug micelles showed favorable selective cytotoxicity and biological safety. By modulating the extracellular microenvironment, they achieve effective synergistic antitumor effects.

R965

河南省科技攻关项目（252102310435，252102311282，）；河南省高等学校重点科研项目（25B350003，26A350004）