Modulating PhpC bioavailability and cellular activity in lung cancer cells via gold nanoparticle functionalization

RNA G-quadruplexes (G4s) embedded within precursor microRNAs (pre-miRs) can hinder Dicer processing and reduce mature miR output. In lung cancer (LC), a G4 in pre-miR-3196 suppresses miR-3196 maturation. Particularly, the small molecule phenylpyrrolocytosine (PhpC) destabilizes this G4 and restores miR-3196, however, its intracellular bioavailability and persistence may be limited by efflux and metabolic turnover. We therefore hypothesized that gold nanoparticle (AuNPs) delivery could preserve PhpC activity by improving its intracellular availability. We developed pegylated AuNPs covalently coupled to G4 destabilizing PhpC via EDC/sulfo-NHS chemistry (AuNP@PhpC) to restore the expression of miR-3196 in LC cells. The conjugation and colloidal integrity were assessed by UV–vis spectroscopy, DLS, ζ-potential, TEM, and FTIR-ATR. Photophysics and redox-responsive release were evaluated by fluorescence titration/quenching and dithiothreitol (DTT) challenge assays. In A549 cells, we quantified uptake (by flow cytometry and confocal microscopy), acute cytotoxicity (by MTS and trypan blue assays), and miR-3196 expression (by RT-qPCR). The Au-nanoconjugates preserved PhpC´s biological activity, while displaying colloidal stability, predictable photophysical behavior, and low acute cytotoxicity. Although free PhpC exhibits faster cellular uptake, AuNP@PhpC achieves comparable intracellular levels after 48 h of incubation, highlighting that our strategy has changed the way the molecular effectors enter cells, from passive entry for the PhpC to endocytosis for AuNP@PhpC, without affecting the global activity of the small molecule. Collectively, our strategy allows for gaining control over the bioavailability of PhpC, without affecting its unique ability to fight against G4-associated, cancer-specific genetic dysregulations.