Improving Nonviral Gene Delivery by Activating Mechanosensing-Dependent Endocytic Pathways

The delivery of nucleic acids into host cells has emerged as an innovative and promising therapeutic approach for various diseases. Despite significant advances in nanoparticle delivery systems, persistent cellular barriers limit the clinical application of most existing technologies. In this study, we developed a programmable device that applies precise uniaxial cyclic stretching to cells cultured on custom polydimethylsiloxane chambers to investigate whether mechanical stimulation can enhance the transfection efficiency (TE) of gold-standard non-viral gene delivery vectors. Applying cyclic mechanical stimulation (<i>f</i> = 0.1 Hz, ε = 10% strain, <i>t</i> = 30 min) to HeLa cells and human myoblasts (hMyo) significantly increased nuclear translocation of the mechanosensitive transcription factor Yes-Associated Protein (YAP). Gene expression analysis revealed that this mechanical conditioning orchestrated a coordinated modulation of endocytic machinery, upregulating clathrin-mediated endocytosis (FCHO1) and macropinocytosis (STX1B) pathways while downregulating endocytic inhibitors (DLC1, EHD2). These mechanically induced cellular adaptations resulted in significantly enhanced TE of both plasmid DNA (pDNA)- and mRNA (mRNA)-carrying gold-standard branched polyethylenimine (<i>b</i>PEI)-based complexes in both HeLa cells and hMyo, compared to static conditions. Our findings demonstrate that mechanical stimulation is an effective complementary strategy for improving non-viral gene delivery by leveraging endogenous cellular mechanotransduction pathways. Rather than modifying vector chemistry, this mechanobiological approach enhances the performance of existing delivery systems by transiently modulating cellular uptake capacity and nuclear accessibility. This work offers mechanistic insights into how mechanotransduction regulates cellular uptake and highlights opportunities for leveraging controlled mechanical environments in applications such as ex vivo cell engineering.