乳糖酸修饰mPEG-PLGA-PLL纳米粒靶向肝癌细胞Huh7的研究

Lactobionic acid enhances mPEG-PLGA-PLL nanoparticles targeting to hepatocellular carcinoma cell Huh7

背景与目的:去唾液酸糖蛋白受体(asialoglycoprotein receptor,ASGPR)是一种肝细胞特异性表达的膜表面蛋白,能够特异性地识别带有半乳糖残基的糖蛋白。乳糖酸含有半乳糖基团,可以作为靶向肝癌的特异性配基。该研究旨在探讨乳糖酸修饰的聚乙二醇/聚丙交酯-乙交酯/聚赖氨酸[methoxy-poly(ethylene glycol)-b-poly(D,L-lactide-co-glycolide)-b-poly(L-lysine)(mPEG-PLGA-PLL)纳米粒,mPEG-PLGA-PLL-GAL NPs)]对肝癌Huh7靶向效果,为构建新型的靶向肝癌的纳米递送系统提供实验数据。方法:MTT法确定Huh7细胞摄取mPEG-PLGA-PLL NPs与mPEG-PLGA-PLL-GAL NPs适当的浓度;通过激光共聚焦和荧光显微镜定性观察Huh7对罗丹明B标记的mPEG-PLGA-PLL NPs和mPEG-PLGA-PLL-GAL NPs的摄取;并采用流式细胞计数仪定量研究Huh7细胞对两者的摄取差别;尾静脉注射荷Huh7瘤裸鼠研究两者体内分布情况。结果:mPEG-PLGA-PLL NPs与mPEG-PLGA-PLL-GAL NPs的浓度在0.2 mg/mL时细胞存活率较高且对Huh7细胞的毒性较小。激光共聚焦断层扫描显示Huh7细胞可以较好地摄取mPEG-PLGA-PLL-GAL NPs,同时流式细胞仪定量显示mPEG-PLGA-PLL-GAL NPs在Huh7细胞的分布较mPEG-PLGA-PLL NPs高40%(P<0.05)。mPEG-PLGA-PLL NPs与mPEG-PLGA-PLL-GAL NPs在移植瘤中的分布明显多于其他脏器,并且随时间的延长mPEG-PLGA-PLL-GAL NPs体现了更好的靶向效果。结论:体外与体内实验证明乳糖酸修饰的mPEG-PLGA-PLL NPs对肝癌细胞Huh7有很好的靶向效果,可为肝癌的靶向治疗提供较好的药物载体。

Background and purpose： It is a wonderful approach to deliver drugs to hepatocellular carcinoma cell by receptor-mediated targeting. The asialoglycoprotein receptor （ASGPR） specifically recognized by galactose moiety residue is a mainly expressing membrane protein on the surface of bepatocellular carcinoma cell. Methoxy-poly （ethylene glycol）-b-poly（D, L-lactide-co-glycolide）-b-poly（L-lysine） （mPEG-PLGA-PLL） （PEAL） coupled with lactobionic acid （LA） bearing galactose group （PEAL-GAL） nanoparticles （NPs） are used as a novel drug carrier to hepatocellular carcinoma cell Huh7. This paper examined its effect on targeting Huh7 cells. Methods： Cytotoxicities of PEAL and PEAL-GAL NPs were assayed based on viability of Huh7 cells measured by MTT method. The distribution of PEAL or PEAL-GAL NPs labeled by Rhodmaine B （RB） in Huh7 cells was observed with confocal laser scanning microscope （CLSM） or fluorescence microscope. Quantitative comparisons were made between cellular uptake of RB-loaded PEAL or PEAL-GAL NPs by flow cytometer （FCM）. Finally, nude mice bearing hepatocellular carcinoma were adopted to investigate the distribution of PEAL NPs or PEAL-GAL NPs in different organs and cancer. Results： PEAL NPs and PEAL-GAL NPs did not show much cytotoxicity at the concentration of 0-0.2 mg/mL. PEAL-GAL NPs were well uptaken after being incubated with Huh7 cells for 3 h. Besides, Z-section and measurement with CLSM clearly showed that PEAL-GAL NPs were well-distributed. After the same co-incubation time, Huh7 cells uptook much more PEAL-GAL NPs than PEAL NPs （P〈0.05）. At 4-48 h after PEAL NPs and PEAL-GAL NPs were respectively injected via tail vein of nude mice, PEAL NPs and PEAL-GAL NPs were mainly distributed in cancers and livers compared to other organs. However, PEAL-GAL NPs demonstrated better cancer targeting than that of PEAL NPs. Conclusion： Compared with PEAL, LA grafted PEAL showed better targeting ability to Huh7 cells. LA promotes Huh7 cells＇ uptaking of PEAL NPs and enhances the targeting efficiency, which makes PEAL-GAL NPs a potential nano-carrier for drugs to liver cancers.