一种靶向肿瘤新生血管的新型长循环纳米脂质体的制备

Preparation of a novel neovessel-targeted PEGylated liposome formulation

目的:构建一种靶向肿瘤新生血管的长循环纳米脂质体,以期成为一种新型抗肿瘤药物载体。方法:采用9-芴甲氧羰基(fluorenylmethyloxycarbonyl,FMOC)固相合成法合成丙氨酸-脯氨酸-精氨酸-脯氨酸-甘氨酸(alanyl-prolyl-arginyl-prolyl-glycine,APRPG)肽—赖氨酸-甘氨酸-甘氨酸(lysine-glycine-glycine,KGG)臂—棕榈酸(palmitate,Pal)复合物;应用薄膜超声分散法制备靶向血管的长循环纳米脂质体(neovessel targeted-polyethylene glycol-liposomes,T-PEG-LP)和普通长循环纳米脂质体(polyethylene glycol-liposomes,PEG-LP),采用激光散射粒度分析仪检测其粒径分布,并在透射电子显微镜下观察其形貌特征;应用薄膜超声分散法制备罗丹明标记的血管靶向荧光长循环纳米脂质体(neovessel targeted-fluorescent-polyethylene glycol-liposomes,T-F-PEG-LP)和普通荧光长循环纳米脂质体(fluorescent-polyethylene glycol-liposomes,F-PEG-LP),通过荧光分光光度计和FCM检测其与人脐静脉内皮细胞(human umbilical vein endothelial cell,HUVEC)和人肺癌A549细胞的特异性结合力;同时,将该载体包封抗肿瘤药物紫杉醇(paclitaxel,PTX)制备紫杉醇长循环纳米脂质体(polyethylene glycol-paclitaxel liposomes,PEG-PTX-LP)和血管靶向的紫杉醇长循环纳米脂质体(neovessel targeted-polyethylene glycol-paclitaxel-liposomes,T-PEG-PTX-LP),应用荧光分光光度计和FCM检测HUVEC和A549细胞对其的摄取能力。结果:透射电子显微镜下观察到T-PEG-LP和PEG-LP均呈规则的圆形或略呈椭圆形,激光散射粒度分析仪检测其平均粒径<100nm;T-F-PEG-LP与HUVEC和A549细胞的结合能力均优于F-PEG-LP(P<0.05),2种细胞对T-PTG-PTX-LP的摄取量均高于市售紫杉醇溶液(Taxol)和PEG-PTX-LP(P<0.05)。结论:构建的靶向血管的长循环纳米脂质体有望成为一种新型有效的肿瘤靶向药物载体。

Objective： To construct a neovessel-targeted PEGylated liposome formulation to promote the efficiency of the intracellular delivery of autitumor reagents. Methods： The complex of （ alanyl-prolyl-arginyl-prolyl-glycine, APRPG） and （ lysine-glycine- glycine, KGG） were synthesized by using fluorenylmethyloxycarbonyl（FMOC） solid-phase synthesis method. The neovessel-targeted polyethylene glycol liposomes （T-PEG-LP） and polyethylene glycol-liposomes （PEG-LP）were prepared by thin film sonication-disper- sion method. Mean particle size and particle size distribution of targeted PEGylated liposomes were determined using laser particle sizer. The morphology of liposomes was observed under transmission electron microscope （TEM）. To evaluate the affinity between targeted li- posomes and tumor ceils or vessel endothelial cells, rhodamine-labelled neovessel targeted fluorescent polyethylene glycol liposomes （T- F-PEG-LP） and fluorescent polyethylene glycol liposomes （F-PEG-LP） were synthesized by using thin film sonication-dispersion me- thod. The specific binding of T-F-PEG-LP and F-PEG-LP with human umbilical vein endothelial cell （HUVEC） and human lung cancer A549 cells was detected by fluorospectrophotometer exanfiuation and flow cytometry analysis. Polyethylene-glycol paclitaxel liposomes （PEG-PTX-LP） and neovessel-targeted polyethylene glycol paclitaxel liposomes （T-PEG-PTX-LP） were also prepared. The cellular up- takes of PEG-PTX-LP and T-PEG-PTX-LP by HUVEC and A549 cells were detected by fluorospectrophotometer examination and flow cytometry analysis. Results：TEM showed that both the T-PEG-LP ~ PEG-LP shaped regular round or a little elliptic. Laser particle sizer indicated the average size of particles was less than 100 nm. T-F-PEG-LP had stronger affinity to HUVEC and A549 cells compared with F-PEG-LP（P 〈 0.05 ）. The uptakes of T-PTG-PTX-LP by HUVEC and A549 cells were higher than that of Taxol solusion and PEG-PTX-LP（P 〈 0.05）. Conclusion：The constructed formulation of neovessel-targeted PEGylated liposomes might be a novel effective carrier for anti-tumor reagents.