Modulation of nanoparticles uptake by mechanical stimuli

Introduction: In the past decades, the rise of nanotechnology has brought new hope for the development of novel therapeutics (1). However, the complex biological environment in which such kinds of nano-agents operate greatly hinders their ultimate efficiency. The first barrier to an efficient delivery of nanotherapeutics into cells is represented by the cell membrane, that constitutes a highly organized barrier between the extra- and the intracellular environment (2). In this context, we herein provide a novel strategy to boost the uptake of nanoparticles (NPs) by mechanically modulating the cell processes enabling intracellular trafficking. Such strategy really improved the effectiveness of nanomedicines such as gene delivery vectors. Methods: To modulate the mechanisms underpinning the uptake of NPs, cells were mechanically stimulated through a built-in-home device able to provide vertical nanoscale vibrations to cells. Cells were thus exposed to 5 min vibrational loading at 1,000 Hz and challenged with polymer NPs containing luciferase-encoding pDNA. Next, the modulation of i) the activation of target endocytic processes, ii) the internalization of NPs and iii) the transfection efficiency by means of the mechanical loading was assessed. Results: The application of nanoscale cues to 2D-cultured cells triggered cell membrane responses, as shown by Scanning Electron microscopy (SEM) images in Figure 1a. The contribution of mechanical-driven membrane remodeling on the ultimate activation of endocytosis mechanisms was assessed by pre-treating cells with clathrin or caveolae inhibitors before transfection. Interestingly, clathrin-mediated endocytosis (CME) was affected by the mechanical stimulation of cells (Figure 1b). Specifically, clathrin inhibition significantly impaired the transgene expression of luciferase in stimulated cells, as opposed to statically cultured cells, in which the transfection was not affected by the inhibition of CME. Moreover, we found that the mechano-induced CME increase improved NPs uptake in stimulated cells, as shown in Figure 1c. Such positive cell responses ultimately translated into a significant boost of NPs efficiency. As depicted in Figure 1d, the luciferase expression in stimulated cells was 40 times higher than in statically cultured cells. Conclusions: Overall, we herein provide a straightforward strategy to improve the effectiveness of nanomedicines by mechanically modulating their intracellular delivery.