Impact of Nanoparticle Stiffness on Endosomal Escape and Signaling Pathways in Cytosolic Delivery

Viruses utilize stiffness tuning to enhance cell entry and uncoating, as cells can regulate the uptake process by sensing the mechanical stimulation of particles. The improved cytosolic delivery efficiency enhances the nanoparticle (NPs) accumulation in target sites, which is a prerequisite for achieving efficient treatment performance. However, the preparation of NPs with similar physicochemical properties but distinct stiffnesses is relatively limited, and the role of NP stiffness in intracellular distribution remains elusive. In this study, using two silica precursors at different molar ratios, silica nanocapsules (SNCs) are synthesized with a stiffness range (1.37 MPa to 1.72 GPa) spanning orders of magnitude. Additionally, an endosomal escape assay (EEA) is developed to enable rapid quantification of NP intracellular distribution based on cell fractionation. SNCs with lower stiffness exhibit superior cellular uptake efficiency. The hard SNCs however, demonstrate ≈1.8 fold-enhanced endosomal escape efficiency compared to soft SNCs. Sequencing results reveal that SNCs with higher stiffness activate the reactive oxygen species (ROS)-mediated mechanism, which facilitates rapid endosomal escape by inducing moderate oxidative stress. This work highlights the critical role of NP stiffness in regulating cytosolic delivery and the trade-off between nanotoxicity and delivery efficiency.