Univ. Paris-Sud, UMR CNRS 8612, Faculté de Pharmacie, Châtenay-Malabry, France.
Eur J Pharm Biopharm. 2011 Nov;79(3):612-20. doi: 10.1016/j.ejpb.2011.07.003. Epub 2011 Jul 19.
We have designed an amphiphilic prodrug of the anticancer agent gemcitabine (dFdC), by covalent coupling to squalene. This bioconjugate, which self-assembled into nanoparticles (NPs) in water, was previously found to display an impressive anticancer activity both in vitro and in vivo. The present study aims to investigate the impact of SQdFdC nanoparticles on cellular membranes. MTT assays showed that, in the nanomolar range, squalenoyl gemcitabine (SQdFdC) was slightly less active than dFdC on a panel of human cancer cell lines, in vitro. However, above 10 μmol L(-1) SQdFdC was considerably more cytotoxic than dFdC. Contrarily to its parent drug, SQdFdC also induced cell lysis in a few hours, as evidenced by LDH release assays. Erythrocytes were used as an experimental model insensitive to the antimetabolic activity of dFdC to further investigate the putative membrane-related cytotoxic activity of SQdFdC. The bioconjugate also induced hemolysis in a time- and dose-dependent fashion, unlike squalene or dFdC, which clearly proved that SQdFdC could permeabilize cellular membranes. Structural X-ray diffraction and calorimetry studies were conducted in order to elucidate the mechanism accounting for these observations. They confirmed that SQdFdC could be transferred from NPs to phospholipid bilayers and that the insertion of the prodrug within model membranes resulted in the formation of nonlamellar structures, which are known to promote membrane leakage. As a whole, our results suggested that due to its amphiphilic nature, the cell uptake of SQdFdC resulted in its insertion into cellular membranes, which could lead to the formation of nonlamellar structures and to membrane permeation. Whether this mechanism could be the source of toxicity in vivo, however, remains to be established, since preclinical studies have clearly proven that squalenoyl gemcitabine displayed a good toxicity profile.
我们设计了一种抗癌药物吉西他滨（dFdC）的两亲前药，通过与角鲨烯共价偶联。这种生物缀合物在水中自组装成纳米颗粒（NPs），先前已被发现具有令人印象深刻的体外和体内抗癌活性。本研究旨在研究 SQdFdC 纳米颗粒对细胞膜的影响。MTT 测定表明，在纳摩尔范围内，角鲨烯酰基吉西他滨（SQdFdC）在体外对一系列人类癌细胞系的活性略低于 dFdC。然而，在 10 μmol L(-1)以上，SQdFdC 的细胞毒性明显高于 dFdC。与母体药物不同，SQdFdC 还在数小时内诱导细胞裂解，这可以通过 LDH 释放测定来证明。红细胞被用作对 dFdC 代谢活性不敏感的实验模型，以进一步研究 SQdFdC 潜在的与膜相关的细胞毒性活性。与角鲨烯或 dFdC 不同，该生物缀合物还以时间和剂量依赖的方式诱导溶血，这清楚地证明 SQdFdC 可以渗透细胞膜。进行了结构 X 射线衍射和量热法研究，以阐明解释这些观察结果的机制。它们证实 SQdFdC 可以从 NPs 转移到磷脂双层中，并且前药在模型膜中的插入导致形成非层状结构，众所周知，这种结构促进膜渗漏。总的来说，我们的结果表明，由于其两亲性，SQdFdC 的细胞摄取导致其插入细胞膜，这可能导致非层状结构的形成和膜渗透。然而，由于临床前研究清楚地证明了角鲨烯酰基吉西他滨具有良好的毒性特征，因此这种机制是否是体内毒性的来源仍有待确定。
