Séhédic Delphine, Roncali Loris, Djoudi Amel, Buchtova Nela, Avril Sylvie, Chérel Michel, Boury Frank, Lacoeuille Franck, Hindré François, Garcion Emmanuel
Univ Angers, Université de Nantes, Inserm, CRCINA, SFR ICAT, Angers, France.
Université de Nantes, Inserm, CNRS, CRCINA, Nantes, France.
Front Bioeng Biotechnol. 2021 Feb 25;8:602998. doi: 10.3389/fbioe.2020.602998. eCollection 2020.
Inhibition of the PI3K/Akt/mTOR signaling pathway represents a potential issue for the treatment of cancer, including glioblastoma. As such, rapamycin that inhibits the mechanistic target of rapamycin (mTOR), the downstream effector of this signaling pathway, is of great interest. However, clinical development of rapamycin has floundered due to the lack of a suitable formulation of delivery systems. In the present study, a novel method for the formulation of safe rapamycin nanocarriers is investigated. A phase inversion process was adapted to prepare lipid nanocapsules (LNCs) loaded with the lipophilic and temperature sensitive rapamycin. Rapamycin-loaded LNCs (LNC-rapa) are ~110 nm in diameter with a low polydispersity index (<0.05) and the zeta potential of about -5 mV. The encapsulation efficiency, determined by spectrophotometry conjugated with filtration/exclusion, was found to be about 69%, which represents 0.6 wt% of loading capacity. Western blot analysis showed that LNC-rapa do not act synergistically with X-ray beam radiation in U87MG glioblastoma model . Nevertheless, it demonstrated the selective inhibition of the phosphorylation of mTORC1 signaling pathway on Ser2448 at a concentration of 1 μM rapamycin in serum-free medium. Interestingly, cells cultivated in normoxia (21% O) seem to be more sensitive to mTOR inhibition by rapamycin than those cultivated in hypoxia (0.4% O). Finally, we also established that mTOR phosphorylation inhibition by LNC-rapa induced a negative feedback through the activation of Akt phosphorylation. This phenomenon was more noticeable after stabilization of HIF-1α in hypoxia.
抑制PI3K/Akt/mTOR信号通路是治疗包括胶质母细胞瘤在内的癌症的一个潜在问题。因此，抑制该信号通路下游效应器雷帕霉素靶蛋白（mTOR）的雷帕霉素备受关注。然而，由于缺乏合适的给药系统制剂，雷帕霉素的临床开发陷入困境。在本研究中，研究了一种制备安全的雷帕霉素纳米载体的新方法。采用相转化法制备负载亲脂性和温度敏感性雷帕霉素的脂质纳米囊（LNCs）。负载雷帕霉素的LNCs（LNC-rapa）直径约为110 nm，多分散指数低（<0.05），zeta电位约为-5 mV。通过分光光度法结合过滤/排阻法测定的包封率约为69%，代表0.6 wt%的载药量。蛋白质免疫印迹分析表明，在U87MG胶质母细胞瘤模型中，LNC-rapa与X射线辐射不存在协同作用。然而，在无血清培养基中，1 μM雷帕霉素浓度下，它显示出对mTORC1信号通路Ser2448位点磷酸化的选择性抑制。有趣的是，在常氧（21% O）条件下培养的细胞似乎比在低氧（0.4% O）条件下培养的细胞对雷帕霉素抑制mTOR更敏感。最后，我们还证实，LNC-rapa对mTOR磷酸化的抑制通过激活Akt磷酸化诱导了负反馈。在低氧条件下HIF-1α稳定后，这种现象更明显。
