顺铂前药及双载药聚乙二醇化脂质体的制备、表征与体外抗肿瘤研究

Preparation,characterization and in vitro antitumor activity of cisplatin prodrug and double drug loaded PEGylated liposomes

目的合成顺铂前药顺-二氯·反-二(丁二酸氢根)·顺-二氨合铂(Ⅳ)(DSCP)。制备聚乙二醇(PEG)化脂质体,用于共递送DSCP和L-丁硫氨酸-亚砜亚胺(L-BSO),并考察其体外抗肿瘤效果。方法以顺铂为原药,经氧化和酯化反应合成顺铂前药DSCP;CCK-8实验考察不同摩尔比下DSCP溶液剂和L-BSO混合溶液剂的抗肿瘤效果,确定载药脂质体的处方;采用薄膜水化-挤出法制备PEG化双载药脂质体DSCP/L-BSO Lip和对照组DSCP Lip,通过外观、形态、粒径与电位分布、包封率、载药量、稳定性和体外释放对脂质体进行表征;考察不同制剂(DSCP溶液剂、DSCP和L-BSO混合溶液剂、DSCP Lip和DSCP/L-BSO Lip)对小鼠乳腺癌顺铂耐药(4T1/DDP)细胞和4T1细胞存活率的影响,并测定药物处理24 h后细胞内谷胱甘肽(GSH)的浓度,考察脂质体的体外抗肿瘤效果。结果成功合成了DSCP,纯度为98.26%;以1∶12(DSCP∶L-BSO)的药物摩尔比制备DSCP/L-BSO Lip;DSCP Lip和DSCP/L-BSO Lip外观为规则球型,粒径分别为(154.88±2.67)nm和(143.66±1.25)nm,Zeta电位分别为(-42.84±0.07)mV和(-42.10±0.12)mV,DSCP的包封率和载药量分别在71%和1%左右,L-BSO的包封率和载药量分别为(72.78±2.26)%和(6.37±0.19)%,DSCP Lip和DSCP/L-BSO Lip具有良好的血浆稳定性和长期稳定性,在pH 7.4 PBS释放介质中,DSCP释放较快,在4 h释放达80%;对于4T1/DDP细胞和4T1细胞,DSCP和L-BSO表现出较好的协同抗肿瘤效果,与DSCP溶液剂、DSCP和L-BSO混合溶液剂、DSCP Lip相比,DSCP/L-BSO Lip的细胞毒性较强,并具有下调GSH作用。结论制备的DSCP/L-BSO Lip包封率较高,具有较好的体外协同抗肿瘤效果。

Objective To synthesize the cisplatin prodrug cis-dichloro trans-bis(hydrogen succinate)cis-diammine platinum(Ⅳ)(DSCP),and to prepare a PEGylated liposome co-delivering DSCP and L-buthionine sulfoxide imine(L-BSO)for synergistic antitumor therapy.Methods The cisplatin prodrug DSCP was synthesized by oxidation and esterification reaction with cisplatin as the original drug.The antitumor effect of the mixed solution of DSCP and L-BSO at different molar ratios was determined by CCK-8 experiment to obtain the optimal synergistic treatment ratio.Then PEGylated L-BSO/DSCP co-encapsulated liposome(DSCP/L-BSO Lip)was formulated via film hydration-squeezing method,with single DSCP-loaded Lip as the control.The pharmaceutical characteristics(appearance,morphology,particle size and potential distribution,entrapment efficiency,drug loading,stability and in vitro release)were evaluated.Meanwhile,the therapeutic effects of different preparations such as DSCP solution,DSCP and L-BSO mixed solution,DSCP Lip and DSCP/L-BSO Lip on mouse breast cisplatin-resistant(4T1/DDP)cells and 4T1 cells were determined,as well as the intracellular concentration of GSH after 24 h treatment.Results DSCP was successfully synthesized with a purity of 98.26%.The DSCP/L-BSO Lip was prepared with the synergetic molar ratio at 1∶12(DSCP∶L-BSO).DSCP/L-BSO Lip and DSCP Lip were spherical with particle sizes of(154.88±2.67)nm and(143.66±1.25)nm,and Zeta potential of(-42.84±0.07)mV and(-42.10±0.12)mV,respectively.The corresponding entrapment efficiency and drug loading of DSCP were about 71%and 1%,while the entrapment efficiency and drug loading of L-BSO were(72.78±2.26)%and(6.37±0.19)%,respectively.Both DSCP Lip and DSCP/L-BSO Lip were stable in the plasma and in the long term.In pH 7.4 PBS release media,80%DSCP was released within 4 h.For 4T1/DDP cells and 4T1 cells,DSCP/L-BSO Lip demonstrated the best antitumor effect and GSH-downregulated performance as compared with those DSCP solution,DSCP and L-BSO mixed solution,and DSCP Lip.Conclusion The as-prepared DSCP/L-BSO Lip possesses a synergetic antitumor effect via the downregulation of intracellular GSH,and provides a foundation for the development and translation of platinum-based formulations.