Mechanical stimulation as a tool to boost nanoparticles uptake by cells

Introduction: In the past decades, the rise of nanotechnology and gene therapy have brought new hope for the development of novel preventatives and therapeutics, as recently demonstrated by the success of mRNA-based COVID-19 vaccines1,2. However, the complexity of the biological environment in which nano-agents operate greatly hinders their ultimate efficiency in vitro and in vivo. The cell membrane represents the first barrier to an efficient intracellular delivery of nanotherapeutics. This is a highly organized and dynamic structure between the extra- and the intracellular environment3. In this context, there is an urgent need of finding novel strategies to modulate cell permeability and deliver particles into cells. To this aim, we herein provide a novel method to boost the uptake of nanoparticles (NPs) by modulating the cell processes enabling intracellular trafficking through the application of an exogenous mechanical stimulus. Such strategy really improved the efficiency of non-viral gene delivery vectors through the mechano-modulation of uptake pathways. Materials and methods: To modulate the mechanisms underpinning the uptake of NPs, in vitro cultured L929 cells were mechanically stimulated through a built-in-home cell stimulation device (Figure 1) able to provide vertical nanoscale vibrations to cells. Cells were exposed to 5 min vibrational loading at 1,000 Hz and challenged with polyethyleneimine (PEI)-made NPs containing a luciferase-encoding pDNA. First, the cell response to the application of mechanical cues was assessed in terms of changes in cell membrane morphology and cell viability. Next, to investigate the role of nanoscale cues on the ultimate transfection efficiency of pDNA/PEI NPs, the modulation of i) the activation of target endocytic processes, ii) the internalization of NPs and iii) their transfection efficiency by means of the mechanical loading were assessed. Results: The cell response to the application of short (i.e., 5 min) nanoscale cues was first assessed by Scanning Electron Microscopy (SEM). As shown in Figure 2a, vibrational loading at high frequencies (f = 1,000 Hz) induced dramatic cell morphological changes. Specifically, stimulated cells exhibited a rough surface, densely studded with blisters, blebs, and invaginations, as opposite to unstimulated cells that were markedly smoother. Moreover, the exposure to short vibrational loading did not alter cell viability, that is, the stimulation did not trigger irreversible harmful effects on cells (Figure 2b). Next, the contribution of mechanical-driven membrane remodeling on the ultimate activation of endocytosis mechanisms occurring at the membrane level was assessed. To this aim, cells were pre-treated with either clathrin- or caveolae inhibitors before undergoing vibrational loading and NPs delivery. Interestingly, the inhibition of caveolae-mediated endocytosis significantly inhibited the uptake of nanoparticles on both unstimulated and 1,000 Hz-stimulated cells (Figure 2c). On the contrary, the inhibition of the clathrin-mediated uptake pathway significantly impaired the delivery on NPs only in stimulated cells, as opposite to unstimulated controls that were not affected by clathrin inhibition. Moreover, we found that the amount of delivered pDNA in mechanically stimulated cells was 15-times higher with respect to unstimulated counterparts (Figure 2d). The increased pDNA delivery into cells ultimately resulted in a significant boost of NPs efficiency. As depicted in Figure 2e, stimulated L929 cells showed a 40-time higher luciferase expression than statically cultured one. Discussion and Conclusion: The poor ability of the carriers to overcome the cell membrane barrier represents today a major bottleneck to effectively deliver nucleic acids into cells. Herein, we provided a straightforward strategy to improve the effectiveness of non-viral vectors by mechanically modulating the cell behavior. A short vibrational loading to cells in culture was able to activate clathrin-mediated endocytosis, which is known to be a mechano-sensitive cellular process4, without any evidence of detrimental cell responses. The overall improvement in the uptake of PEI-made NPs resulted in a higher amount of pDNA being internalized, thus in a significant boost in transgene expression.