Improving polymer-based gene delivery by mechanical cell stimulation

Introduction Despite the recent success of COVID-19 vaccines, the use of non-viral carriers, namely cationic polymer and lipids, to deliver nucleic acids into cells for therapeutic purposes is still plagued by their poor gene transfer ability due to the ineffective crossing of the cell membrane1,2. To this aim, we developed a novel method to boost the uptake of polymer-based nanoparticles (NPs) by modulating the cell uptake processes through the application of an exogenous mechanical stimulus. Methodology To modulate the mechanisms underlying NPs uptake, in vitro cultured cells were mechanically stimulated through a built-in-home stimulation device able to provide nanoscale vibrations to cells. Cells were exposed to 5 min vibrational loading at 1,000 Hz and challenged with polyethyleneimine (PEI)-made NPs containing pDNA. The cell response to mechanical cues was assessed in terms of changes in cell membrane morphology and cell viability. Next, the internalization and transfection ability of pDNA/PEI NPs was studied as well. Results The cell response to the application of vibrational loading was assessed by Scanning Electron Microscopy (SEM). Compared to statically cultured cells, stimulated cells exhibited a rougher cell membrane full of blisters and protrusions that were completely reabsorbed 1 hr from stimulation release without any detrimental effect. Next, we investigated the uptake of PEI/pDNA NPs by pre-treating cells with clathrin- and caveolae inhibitors, that is, the main uptake routes for NPs. Interestingly, we found that NPs uptake in unstimulated cells only occurs by caveolae-mediated endocytosis, while in stimulated cells clathrins were responsible for NPs uptake as well. The activation of clathrin-based uptake resulted in a 15-fold increase in the amount of delivered pDNA in stimulated cells, that led to a significant boost of PEI-based NPs efficiency in terms of gene transfer ability. Conclusions The poor ability of the carriers to overcome the cell membrane is a limiting factor in the delivering of genes. Herein, we provided a straightforward strategy to improve the effectiveness of non-viral vectors by mechanically modulating the NPs uptake by cells.