Boosting the transfection efficiency of non-viral gene delivery vectors by mechano-modulation of cells

Introduction: Non-viral gene delivery strategies emerged as a new breakthrough for clinical and research purposes (1), such as the recently developed Pfizer’s and Moderna’s COVID-19 vaccines. Unfortunately, the efficiency of such supramolecular systems is often hampered by extra- and intracellular barriers, namely exo- and endonucleases, the cell membrane, the lysosomal compartment and nuclear transport (2). Therefore, there is an urgent need to overcome the current drawbacks of non-viral vectors. In this context, we were aimed at taking advantage of the application of vibrational stimuli to mechanically tune the cell uptake of linear polyethyleneimine (lPEI)-based DNA nanoparticles, so as to boost their transfection efficiency. Methods: To modulate the cell response to the delivery of polyplexes, we developed a small, cost-effective stimulation device (Figure 1a) formed by a sinusoidal wave generator connected to a mechanical wave driver to apply micro-to-nano displacements to 2D in vitro cell cultures. To investigate the cell response to stimulation, L929 cells were morphologically inspected by SEM analysis. Besides, the overall cell viability after 5 min vibration-loading at different frequencies was assessed as well. Vibrationally-loaded L929 cells were next challenged with lPEI/pDNA polyplexes. The efficacy of the mechano-chemical transfection was evaluated in terms of cytotoxicity, transfection efficiency and polyplex uptake, and compared to that of statically-transfected cells. Results: The application of short (i.e., 5 min) vibration-based mechanical cue to cells triggered some intriguing cell responses in terms of membrane remodeling (Figure 1b) and no overt detrimental effects (Figure 1c). Besides, we tested the contribution of such mechano-induced response to the overall transfection efficiency of lPEI/pDNA polyplexes. Interestingly, the application of 500 Hz- and 1,000 Hz-vibrations to cells resulted in a 30-to-40 fold-increase in luciferase expression after the delivery of lPEI/pGLuc complexes (Figure 1d) with respect to standard transfections (static transfections). To further ascertain the contribution of such mechano-induced cell response to the ultimate transgene expression, we next quantified the amount of plasmid internalized. We found that the mechano-induced cell membrane remodeling is associated to a significant increase in polyplex uptake (Figure 1e) (3). Conclusion: We herein demonstrated that the application of a short, vibration-based mechanical cue positively affected the overall cell response to polyplex delivery with no detrimental effects. Therefore, we set up a versatile in vitro transfection strategy to boost the performances of commonly used non-viral vectors