Casting Light on Intracellular Tracking of a New Functional Graphene-Based MicroRNA Delivery System by FLIM and Raman Imaging.

Abstract

The theranostic ability of a new fluorescently labeled cationic cyclodextrin-graphene nanoplatform (GCD@Ada-Rhod) was investigated by studying its intracellular trafficking and its ability to deliver plasmid DNA and microRNA. The nanoplatform was synthesized by both covalent and supramolecular approaches, and its chemical structure, morphology, and colloidal behavior were investigated by TGA, TEM, spectroscopic analysis such as UV-vis, fluorescence emission, DLS, and ζ-potential measurements. The cellular internalization of GCD@Ada-Rhod and its perinuclear localization were assessed by FLIM, Raman imaging, and fluorescence microscopy. Biological experiments with pCMS-EGFP and miRNA-15a evidenced the excellent capability of GCD@Ada-Rhod to deliver both pDNA and microRNA without significant cytotoxicity. The biological results evidenced an unforeseen caveolae-mediated endocytosis internalization pathway (generally expected for particles <200 nm), despite the fact that the GCD@Ada-Rhod size is about 400 nm (by DLS and TEM data). We supposed that the internalization pathway was driven by physical-chemical features of GCD@Ada-Rhod, and the caveolae-mediated uptake enhanced the transfection efficiency, avoiding the lysosomal acid degradation. The cellular effects of internalized miRNA-15a on the oncogene protein BCL-2 were investigated at two different concentrations (N/P = 10 and 5), and a reduction of the BCL-2 level was detected at a low concentration (i.e., N/P = 10). miRNA-15a is considered an ideal cancer therapy molecule due to its activity on multiple transcription factors, and the elucidation of the correlation between the concentration of delivered miRNA-15a and the down-/up-regulation of the BCL-2 level, documented for the first time in this work, could be an important contribution to guide its clinical application.