穿膜肽R8和pH敏感型可断裂PEG共修饰脂质体的构建

The construction of cell-penetrating peptide R8 and p H sensitive cleavable polyethylene glycols co-modified liposomes

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。

The purpose of the study is to construct R8 peptide （RRRRRRRR） and pH sensitive polyethylene glycols （PEG） co-modified liposomes （C1-Lip） and utilize them in breast cancer treatment. The co-modified liposomes were prepared with soybean phospholipid, cholesterol, DSPE-PEG 2K-R8 and PEGsK-Hz-PE （pH sensitive PEG）. The size and zeta potential of C1-Lip were also characterized. The in vitro experiment demonstrated that the Cl-Lip had high serum stability in 50% fetal bovine serum. The cellular uptake of C1-Lip under different pre-incubated conditions was evaluated on 4T1 cells. And the endocytosis pathway, lysosome escape ability and tumor spheroid penetration ability were also evaluated. The results showed the particle size of the C1-Lip was （110.4±5.2） nm, PDI of the C1-Lip was 0.207±0.039 and zeta potential of the Cl-Lip was （-3.46±0.05） mV. The cellular uptake of C1-Lip on 4T1 cells was pH sensitive, as the cellular uptake of C1-Lip pre-incubated in pH 6.0 was higher than that of pH 7.4 under each time point. The main endocytosis pathways of C1-Lip under pH 6.0 were micropinocytosis and energy-dependent pathway. At the same time, the C1-Lip with pre-incubation in pH 6.0 had high lysosome escape ability and high tumor spheroid penetration ability. All the above results demonstrated that the C1-Lip we constructed had high pH sensitivity and is a promising drug delivery system.