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

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.

Keywords: Akt; HIF-1α; cancer; hypoxia; mTOR; nanoparticles; radiation; rapamycin.

Figure 1

Micro-emulsion conductivity. Micro-emulsion conductivity during cycles of heating/cooling for LNCs formulation performed with 2 M or 0.5 M NaCl aqueous solution.

Figure 2

Effects of LNC-rapa assessed on mTOR phosphorylation in U87MG cells depending on oxic condition and exposure to radiation treatment. U87MG cells were treated with rapamycin, empty LNCs (LNC) or rapamycin-loaded LNCs (LNC-rapa), and maintained at two oxygenation conditions: 21 and 0.4% O₂ before proceeding to western blot analysis. Relative phosphorylation of one representative experiment was determined by volumetric ratio of p-mTOR/HSC70. (A) 0Gy. (B) 8Gy irradiation.

Figure 3

Effects of LNC-rapa assessed by clonogenic assay on U87MG cell growth depending on oxic condition and exposure to radiation treatment. (A) Photography of 6-wells plates containing U87MG cells treated with LNCs, rapamycin or LNC-rapa, radiated at 0Gy (top row) or 8Gy (bottom row) at 21 and 0.4% p0₂ and stained with crystal violet. (B,C) Cell survival was determined by measuring crystal violet staining of wells exposed to 0 and 8Gy at 21% pO₂ (B) or 0.4% pO2 (C). Data show the average values from a combination of three independent experiments and error bars display the standard deviation. Two-way ANOVA test was performed between LNC-rapa condition compared to LNC condition (*p ≤ 0.05) or between rapamycin treatment condition and untreated control condition (°p ≤ 0.05) or between rapamycin treatment condition and untreated condition (^#p ≤ 0.05).

Figure 4

Activation of alternative signaling pathways in response to exposure to LNC-rapa in U87MG. (A,B) U87MG cells were treated with free rapamycin, empty LNCs or LNC-rapa, radiated at 0Gy (A) or 8Gy (B) and maintained at two oxygenation conditions: 21 and 0.4% before proceeding to western blot analysis. Relative phosphorylation of one representative experiment was measured by volumetry ratio of p-mTOR/HSC70.

Figure 5

Rapamycin a likely double-edge sword molecular interactor in U87MG glioblastoma cells. Nanovectorized rapamycin (LNC-rapa) or rapamycin as free, solubilized in DMSO, inhibits the stabilization and transcriptional activity of HIF1α which in turn inhibits the TSC1/TSC2 inhibitor and thus promotes the activation of mTOR. On the other hand, rapamycin inhibits mTORC1 but not mTORC2, which in turn induces Akt phosphrylation. Finally, by inhibiting mTORC1, rapamycin lifts the inhibition exerted by p70s6k on IRS-1, which stabilizes this protein and induces Akt phosphorylation via the IGF-1 receptor.