肝癌干细胞niche响应型逐级靶向脂质体处方优选及体外靶向作用评价

Prescription optimization and evaluation of in vitro targeting effects of liver cancer stem cell niche responsive multistep targeting liposomes

目的制备负载多柔比星(DOX)和XAV-939的肝癌干细胞niche响应型逐级靶向脂质体(CAP@CD133-D/X-Lip),并初步评价其体外靶向作用。方法采用薄膜分散-硫酸铵梯度法制备CAP@CD133-D/X-Lip。通过Box-Behnken设计-响应面法确定CAP@CD133-D/X-Lip的最优处方制备工艺;通过加入纤维细胞活化蛋白(FAP-0x0E䥺SymbolaA@0x0F)与脂质体反应测定粒径变化考察CAP@CD133-D/X-Lip中两亲肽(CAP)的敏感性;通过细胞摄取实验考察不同制剂对肝癌干细胞(LCSCs)的体外靶向能力。结果最优处方工艺为EPC 44 mg、Chol 2 mg、XAV-9390.156 mg、DOX 1.16 mg、DSPE-PEG_(2000)2 mg、DSPE-PEG_(2000)-MAL 2 mg、DSPE-PEG_(2000)-CAP-PEG_(2000)-UAMC11102 mg,超声功率为500 W,总处方量为5 mL,两药平均包封率为79.79%±0.89%;加入重组FAP-α蛋白后CAP@CD133-D/X-Lip的平均粒径缩小至(95.56±2.32)nm,显示出CAP@CD133-D/X-Lip具有较好的微环境响应能力;细胞摄取实验显示,相比于CAP@CD133-D/X-Lip,CAP@CD133-D/X-Lip+FAP-α有着更强的摄取能力,说明其靶向LCSCs的能力明显增强。结论本研究成功制备了CAP响应型逐级靶向LCSCs的脂质体,且其具有良好的微环境响应与体外靶向能力。

Objective To prepare liver cancer stem cell niche responsive multistep targeting liposome loaded with doxorubicin(DOX)and XAV-939(CAP@CD133-D/X-Lip),and to evaluate its targeting effect in vitro.Methods Thin film dispersion-ammonium sulfate gradient method was used to prepare CAP@CD133-D/X-Lip.The optimal prescription and preparation process were optimized by Box-Behnken response surface method.The sensitivity of amphipathic peptides(CAP)in CAP@CD133-D/X-Lip was evaluated by determining the particle size changes of liposomes through adding fibroblast activation protein(FAP-0x0E䥺SymbolaA@0x0F).The in vitro targeting ability of different formulations to liver cancer stem cells(LCSCs)was investigated by cell uptake assay.Results The optimal formulation process was EPC 44 mg,Chol 2 mg,XAV-9390.156 mg,DOX 1.16 mg,DSPE-PEG_(2000)2 mg,DSPE-PEG_(2000)-MAL 2 mg,DSPE-PEG_(2000)-CAP-PEG_(2000)-UAMC11102 mg,ultrasound power was 500 W,total prescription volume was 5 mL,and the average encapsulation efficiency of the two drugs was 79.79%±0.89%.The average particle size of CAP@CD133-D/X-Lip reduced to(95.56±2.32)nm after the addition of recombinant FAP-αprotein,indicating that CAP@CD133-D/X-Lip had an excellent microenvironment response.The cell uptake experiment showed that CAP@CD133-D/X-Lip+FAP-αhad a stronger uptake ability than CAP@CD133-D/X-Lip,indicating that its ability to target LCSCs was significantly enhanced.Conclusion CAP-responsive multistep targeting liposomes to LCSCs are successfully prepared,and they have good microenvironment response and in vitro targeting ability.