载紫杉醇的pH敏感型热休克蛋白纳米载体的肿瘤细胞摄取特性及药理学评估

Tumor cellular uptake properties and pharmacological evaluation of paclitaxel-loaded pH-sensitive heat shock protein nano-carriers

为探究载紫杉醇(PTX)的pH敏感型热休克蛋白纳米载体(PT-HSP)的肿瘤细胞摄取特性、药理学活性及生物安全性,采用荧光显微技术和分光光度法分析不同相对分子质量和聚磺胺嘧啶/聚乙二醇丙烯酸共聚物(PSPA)比例制备所得的PT-HSP对细胞摄取的影响,采用台盼蓝染色法分析载PTX的PT-HSP对肿瘤细胞的选择性杀伤能力,通过树脂天青法分析其细胞毒性并进行溶血试验,最后利用不同的内吞抑制剂对肿瘤细胞摄取PT-HSP的机制进行探讨.结果显示:当PT-HSP中PEG的相对分子质量达到6000且与HSP亚基的摩尔比为2∶1时,可以显著抑制正常细胞对PT-HSP的摄取,而不影响肿瘤细胞对其摄取,说明其具有良好的肿瘤细胞靶向性;同时,与PTX相比,载PTX的PT-HSP对正常细胞伤害小,对肿瘤细胞杀伤力强,并具有良好的生物安全性;摄取机制分析结果表明PT-HSP可能通过依赖能量的巨胞饮方式以及小窝介导的内吞方式进入肿瘤细胞.综上所述,PT-HSP能选择性进入肿瘤细胞释放PTX并发挥抗肿瘤药效,有望成为一种安全、有效的靶向药物载体用于抗肿瘤治疗.

In this paper,we investigated the tumor cellular uptake properties,pharmacological activity and bio-safety of paclitaxel(PTX)-loaded pH-sensitive heat shock protein nano-carriers(PT-HSP).The cellular uptake of PT-HSP was observed through fluorescence microscopy and spectrofluorimetry to explore the influences of molecular mass and ratio of poly sulfadiazine/polyethylene glycol acrylate copolymer(PSPA)on cellular uptake.The lethal selectivity to tumor cells was evaluated by trypan blue staining,the cytotoxicity was assessed with resazurin method,and the hemolysis assay was performed.We also treated tumor cells with various endocytosis inhibitors to analyze the mechanism of cellular uptake.The results showed that the uptake of PT-HSP by normal cells was significantly inhibited but not the uptake by tumor cells when the relative molecular weight of PSPA amounted to 6000 and the molar ratio of HSP subunit to PSPA was 1∶2,indicating the good tumor cellular targeting property.In addition,compared with PTX,PTX-loaded PT-HSP was less lethal to normal cells while highly lethal to tumor cells,which performed good biological safety.We speculated that PT-HSP might be taken through the energy-dependent endocytosis and caveolin-mediated endocytosis by uptake mechanism analysis.Taken together,these results indicated that PT-HSP was able to enter tumor cells selectively and release PTX to perform the anti-tumor effect.Thus,PT-HSP may be a very potential safe and efficient targeting nano-carrier in the anti-tumor therapy.