Liu Chunxi, Liu Tingxian, Yu Xiaoyue, Gu Yizhu
Department of Pharmacy, Qilu Hospital, Shandong University, Ji'nan 250012, China.
School of Pharmaceutical Science, Shandong University, Ji'nan 250012, China.
Acta Pharm Sin B. 2017 May;7(3):361-372. doi: 10.1016/j.apsb.2017.02.003. Epub 2017 Apr 1.
Previously developed Asn-Gly-Arg (NGR) peptide-modified multifunctional poly(ethyleneimine)-poly(ethylene glycol) (PEI-PEG)-based nanoparticles (TPIC) have been considered to be promising carriers for the co-delivery of DNA and doxorubicin (DOX). As a continued effort, the aim of the present study was to further evaluate the interaction between TPIC and human umbilical vein endothelial cells (HUVEC) to better understand the cellular entry mechanism. In the present investigation, experiments relevant to co-localization, endocytosis inhibitors and factors influencing the internalization were performed. Without any treatment, there was no co-localization between aminopeptidase N/CD13 (APN/CD13) and caveolin 1 (CAV1). However, co-localization between CD13 and CAV1 was observed when cells were incubated with an anti-CD13 antibody or TPIC. As compared with antibody treatment, TPIC accelerated the speed and enhanced the degree of co-localization. TPIC entered HUVEC not only together with CD13 but also together with CAV1. However, this internalization was not dependent on the enzyme activity of CD13 but could be inhibited by methyl--eyclodextfin (MCD), further identifying the involvement of caveolae-mediated endocytosis (CvME). This conclusion was also verified by endocytosis inhibitor experiments.
先前开发的天冬酰胺-甘氨酸-精氨酸（NGR）肽修饰的基于多功能聚（乙烯亚胺）-聚（乙二醇）（PEI-PEG）的纳米颗粒（TPIC）被认为是用于共递送DNA和阿霉素（DOX）的有前景的载体。作为持续的研究工作，本研究的目的是进一步评估TPIC与人脐静脉内皮细胞（HUVEC）之间的相互作用，以更好地了解细胞内吞机制。在本研究中，进行了与共定位、内吞抑制剂以及影响内化的因素相关的实验。未经任何处理时，氨肽酶N/CD13（APN/CD13）和小窝蛋白1（CAV1）之间没有共定位。然而，当细胞与抗CD13抗体或TPIC孵育时，观察到CD13和CAV1之间存在共定位。与抗体处理相比，TPIC加快了共定位速度并增强了共定位程度。TPIC进入HUVEC不仅与CD13一起，还与CAV1一起。然而，这种内化不依赖于CD13的酶活性，但可被甲基-β-环糊精（MCD）抑制，进一步证实了小窝介导的内吞作用（CvME）的参与。内吞抑制剂实验也验证了这一结论。
