Rapamycin-Loaded Lipid Nanocapsules Induce Selective Inhibition of the mTORC1-Signaling Pathway in Glioblastoma Cells.

Delphine Séhédic,Franck Lacoeuille,Michel Chérel,François Hindré,Sylvie Avril,Nela Buchtova,Emmanuel Garcion,Frank Boury,Amel Djoudi,Loris Roncali

doi:10.3389/fbioe.2020.602998

Abstract

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 in vitro. 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% O2) seem to be more sensitive to mTOR inhibition by rapamycin than those cultivated in hypoxia (0.4% O2). 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.

Highlights

Glioblastoma (GB) is the most common and deadly primary brain tumor in adults (Ostrom et al, 2017)
The complex mTORC1 is composed of mechanistic target of rapamycin (mTOR), regulatory-associated protein of mTOR (Raptor), mammalian lethal with Sec13 protein 8, proline-rich AKT substrate 40 kDa (PRAS40), and DEP-domain-containing mTOR-interacting protein (Deptor) (Saxton and Sabatini, 2017). mTORC1 activates the eukaryotic initiation factor 4Ebinding protein, releasing the transcription factor eIF4E and the p70 ribosomal S6 kinase 1 (S6K1 or p70S6 kinase (p70S6K)) implicated in translation (Heimberger et al, 2005)
Rapamycin-loaded lipid nanocapsules for peripheral or loco-regional administration developed in this study represent a new safe nanocarrier of rapamycin capable to convey rapamycin and preserves its biological activity on cancer cells

Summary

Introduction

Glioblastoma (GB) is the most common and deadly primary brain tumor in adults (Ostrom et al, 2017). The phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mechanistic target of rapamycin (mTOR) intracellular signaling pathway plays a central role in the regulation of cell proliferation, growth, differentiation, and survival (Sonoda et al, 2001; Bjornsti and Houghton, 2004; Knobbe et al, 2005; Castellino and Durden, 2007; Jiang and Liu, 2009). Stimulation of this pathway results in the activation of a receptor tyrosin kinase (RTK) by a cytokine or a growth factor, which drive a sequential phosphorylation of PI3K, Akt, and mTOR. The complex mTORC1 is composed of mTOR, regulatory-associated protein of mTOR (Raptor), mammalian lethal with Sec protein 8 (mLST8), proline-rich AKT substrate 40 kDa (PRAS40), and DEP-domain-containing mTOR-interacting protein (Deptor) (Saxton and Sabatini, 2017). mTORC1 activates the eukaryotic initiation factor 4E (eIF4E)binding protein, releasing the transcription factor eIF4E and the p70 ribosomal S6 kinase 1 (S6K1 or p70S6K) implicated in translation (Heimberger et al, 2005)

Methods

Results

Discussion

Conclusion