本文旨在制备T7肽和穿膜肽TAT双修饰的脂质体（T7 and TAT dual modified liposomes,T7-TAT-LIP）用于血脑屏障和脑肿瘤细胞双级靶向药物递送。研究以CFPE为荧光探针,T7修饰的PEG-DSPE、TAT修饰的PEG-DSPE、卵磷脂、PEG-DSPE和胆固醇为材料,采用成膜水化法制备脂质体,对T7浓度、TAT浓度、连接T7和TAT的PEG长度进行优化,表征其粒径、zeta电位、形态和稳定性。以b End.3细胞和C6细胞为模型,考察T7-TAT-LIP的细胞摄取能力,表征其穿过血脑屏障和脑肿瘤细胞靶向能力。结果表明,T7用量为脂质的6%、修饰T7所用PEG链长为2000、TAT用量为脂质的0.5%、修饰TAT所用PEG链长为1000时所得到的双修饰脂质体被C6细胞摄取能力最强。优化后T7-TAT-LIP粒径为118 nm,zeta电位为-6.32 m V,透射电镜下形态圆整。脂质体在PBS中较为稳定,37℃放置24 h,浊度和粒径无明显变化;4~8℃放置1个月,粒径和PDI无明显变化。在不同时间点,b End.3和C6细胞摄取T7-TAT-LIP的强度均高于单配体修饰脂质体,且随着孵育时间提高,摄取浓度逐渐提高。这些结果说明,双修饰脂质体具有血脑屏障和脑肿瘤细胞双级靶向能力,且效果优于单配体修饰脂质体。
Cell-penetrating peptides(CPPs)have been widely used to enhance the membrane transloca-tion of various carriers for many years,but the non-specificity of CPPs seriously limits their utility in vivo.In this study,cholesterol-anchored,reduction-sensitive PEG(first synthesized by our laboratory)was applied to develop a co-modified liposome with improved tumor targeting.Following optimization of the formulation,the in vitro and in vivo properties of the co-modified liposome were evaluated.The co-modified liposome had a much lower cellular uptake and tumor spheroid uptake,but a much higher tumor accumulation compared to CPP-modified liposome,indicating the non-specific penetration of CPPs could be attenuated by the outer PEG coating.With the addition of exogenous reducing agent,both the in vitro and in vivo cellular uptake was markedly increased,demonstrating that the reduction-sensitive PEG coating achieved a controllable detachment from the surface of liposomes and did not affect the penetrating abilities of CPPs.The present results demonstrate that the combination of cholestervsitive PEG and CPPs is an ideal alternative for the application of CPP-modified carriers in vivo.
Jie TangLi ZhangHan FuQifang KuangHuile GaoZhirong ZhangQin Hen
本文旨在构建穿膜肽R8(RRRRRRRR)和p H敏感型PEG(聚乙二醇,polyethylene glycols)共修饰的脂质体(R8 peptide and p H sensitive PEG co-modified liposomes,Cl-Lip)用于乳腺癌的靶向药物传递。通过卵磷脂、胆固醇、DSPE-PEG2K-R8和PEG5K-Hz-PE(p H敏感型PEG)为材料制备共修饰脂质体,对其粒径和zeta电位进行表征。采用4T1细胞考察不同预孵育条件下细胞对Cl-Lip的摄取。同时对其入胞途径、溶酶体逃逸能力及肿瘤球穿透能力进行考察。结果表明,Cl-Lip粒径为(110.4±5.2)nm,PDI为0.207±0.039,zeta电位为(-3.46±0.05)m V。体外实验证明,Cl-Lip具有良好的血清稳定性。在不同的预孵育时间点,4T1细胞对Cl-Lip的摄取均呈p H依赖性,在p H 6.0条件下的摄取均高于p H 7.4条件下的摄取。Cl-Lip的入胞机制主要为网格蛋白介导的内吞途径和能量依赖型的内吞途径。在p H 6.0条件下,Cl-Lip具有较强的溶酶体逃逸能力和肿瘤球穿透能力。这些结果表明,共修饰脂质体具有较好的p H响应性,是一种潜在的靶向药物传递系统。
