Stiffness Directs Intracellular Fate and Intercellular Transport of Nanoparticles

Abstract Nanoparticles have been developed as delivery vehicles to achieve uniform distribution of drugs in the tumor, but they are still not optimal for deep tumor penetration due to the hindrance by the dense extracellular matrix. Enabling active transport via tumor cells has been explored as a solution, which has been achieved by tuning the size and surface chemistry of nanoparticles. Here, we show that particle stiffness is another key determinant. Using positively charged polyglycerol nanogels with matched size, charge, and composition, we found that stiff nanogels preferentially enter cells via clathrin-mediated endocytosis (CME), undergo lysosomal processing, and are efficiently exocytosed, enabling transcellular transport. In contrast, soft nanogels are taken up mainly by macropinocytosis, accumulate in the Golgi apparatus, and exhibit prolonged intracellular retention. Finally, the efficient CME and subsequent exocytosis of stiff nanoparticles contribute to the enhanced transcytosis efficiency, allowing them to penetrate 3D tumor spheroids. This stiffness-dependent cellular itinerary operates without additional surface modifications, revealing a general design principle whereby intrinsic mechanical properties can control intracellular and intercellular transport behavior of nanoparticles.