顺铂前药纳米载药系统联合丁硫氨酸亚砜亚胺增强肿瘤化疗效果的研究

Construction of cisplatin prodrug nanoparticles combined with BSO and study on their anti-cancer effect

目的 合成4种顺铂前药并考察其自组装能力,制备聚乙二醇(PEG)化顺铂前药纳米粒,联合丁硫氨酸亚砜亚胺(BSO)用于肿瘤的共同治疗。方法 以顺铂为原料药,合成4种顺铂前药(C6-Pt、C10-Pt、C12-Pt、C18-Pt);采用一步纳米沉淀法筛选出自组装能力较强的顺铂前药,制备纳米粒;完成纳米粒的外观形态、稳定性、粒径、Zeta电位及体外药物释放考察;CCK-8法考察顺铂、C12-Pt NPs对MCF-7、B16F10、LLC、4T1的细胞毒性,以B16F10为细胞模型测定BSO的无毒浓度及C12-Pt NPs和BSO联用的最佳摩尔比,考察顺铂、顺铂/BSO、C12-Pt NPs、C12-Pt NPs/BSO对B16F10细胞的生长抑制作用;试剂盒法测定顺铂、顺铂/BSO、C12-Pt NPs、C12-Pt NPs/BSO处理B16F10细胞24 h后的胞内GSH水平。结果 成功合成4种顺铂前药(C6-Pt、C10-Pt、C12-Pt、C18-Pt);顺铂前药质量浓度为1 mg·mL^(-1)时,仅有C12-Pt能自组装成为纳米粒(C12-Pt NPs);C12-Pt NPs粒径分布均匀,具有较好的长期稳定性和血浆稳定性;在含0、1 mmol·L^(-1)DTT的PBS(pH 7.4)缓冲液中纳米粒释药缓慢,在含10 mmol·L^(-1)DTT的PBS(pH 7.4)缓冲液中,72 h内药物释放率可达76%;C12-Pt NPs对4种肿瘤细胞的抑制作用强于顺铂;对于B16F10细胞,BSO未表现出明显毒性;当顺铂前药、BSO摩尔比为1∶40时对肿瘤细胞抑制作用最强,表明BSO对顺铂前药具有较好的化疗增敏作用;与顺铂相比,顺铂/BSO、C12-Pt NPs、C12-Pt NPs/BSO处理后B16F10细胞中GSH水平均显著下降(P<0.001)。结论 C12-Pt NPs制剂学性质良好,C12-Pt NPs对多种肿瘤细胞均具有较强的生长抑制作用,BSO能够有效增强C12-Pt NPs对肿瘤的杀伤效果。

Objective To synthesize four cisplatin prodrugs and examine their self-assembly ability to prepare PEGylated cisplatin prodrug nanoparticles,combined BSO for co-treatment of tumors.Methods Four cisplatin prodrug(C6-Pt,C10-Pt,C12-Pt,C18-Pt)were synthesized using cisplatin as the raw material drug;a one-step nanoprecipitation method was used to screen the cisplatin precursors with better self-assembly ability;the appearance morphology,stability,particle size,Zeta potential and in vitro drug release of the nanoparticles were investigated;The cytotoxicity of cisplatin solution and C12-Pt NPs solution on MCF-7,B16F10,LLC,and 4T1 was examined;determination of the non-toxic concentration of BSO and the optimal molar ratio for the combination of C12-Pt NPs and BSO using B16F10 as a tumor cell model;the growth inhibitory effects of cisplatin solution,cisplatin/BSO mixed solution,C12-Pt NPs solution and C12-Pt NPs/BSO mixed solution on B16F10 cells were examined;the intracellular GSH contents of B16F10 cells after 24 h treatment with cisplatin solution,cisplatin/BSO mixed solution,C12-Pt NPs solution and C12-Pt NPs/BSO mixed solution were determined.Results Four cisplatin prodrugs(C6-Pt,C10-Pt,C12-Pt,C18-Pt)were successfully synthesized;only C12-Pt could self-assemble successfully at a cisplatin prodrug concentration of 1 mg·mL^(−1);C12-Pt NPs had a uniform particle size distribution with good long-term stability and plasma stability;the drug release rate of nanoparticles was slow in PBS(pH 7.4)buffer containing 0 and 1 mM DTT,and up to 76%within 72 h in PBS(pH 7.4)buffer containing 10 mmol·L−1 DTT;the inhibitory effect of C12-Pt NPs on four tumor cells was stronger than that of cisplatin solution;for B16F10 cells,BSO did not show significant toxicity;the strongest inhibitory effect on tumor cells was observed when the molar ratio of cisplatin prodrug:BSO was 1:40,indicating that BSO has a better chemotherapy sensitization effect on cisplatin prodrugs.Compared with cisplatin,the levels of GSH in B16F10 cells significantly decreased after treatment with cisplatin/BSO,C12-Pt NPs,and C12-Pt NPs/BSO(P<0.001).Conclusion C12-Pt NPs have good formulation properties;C12-Pt NPs have strong growth inhibitory effects on a variety of tumor cells;BSO can effectively enhance the tumor-killing effect of C12-Pt NPs.