Endocytosis of PEGylated polymeric mesoscale nanoparticles is dynamin- and macropinocytosis-dependent

Abstract Nanotechnology is rapidly transforming medicine by enabling versatile platforms for targeted delivery, controlled release, and intracellular transport of therapeutic payloads. Polymeric mesoscale nanoparticles (MNPs) are 300 to 500 nm in diameter with a PEGylated surface that exhibit unique renal tropism, specifically toward renal tubular epithelial cells. Despite their well-described therapeutic applications and route of localization to the tubules, we do not yet understand their physicochemical stability and cellular internalization mechanisms. In this study, we investigated the stability of MNPs under stress conditions by subjecting them to repeated freeze-thaw cycles and varying storage conditions to evaluate the effects on particle size and polydispersity index. MNPs demonstrated negligible changes in size and PDI up to 4 freeze-thaw cycles. We found that both empty and dye-loaded MNPs demonstrated negligible change in size under standard −20°C storage conditions. While empty MNPs were only stable at room temperature for one day, and not at 37°C, dye-loaded nanoparticles were stable for at least eight days under both storage conditions. We then performed in vitro studies to evaluate MNP cellular uptake mechanisms using the human renal cell carcinoma cell line 786-O treated with pharmacological inhibitors of uptake pathways. We found that MNP internalization is almost entirely prevented by dynamin inhibitors, while macropinocytosis inhibition also reduced uptake, suggesting that such standard nanoparticle uptake pathways are robust to the mesoscale size range. These findings provide key insights into the stability profile and endocytosis mechanisms of MNPs, which are critical for materials scale-up and translation of novel kidney-targeted drug and gene therapies. Competing Interest Statement AA is an employee of Eli Lilly and Company. RMW is the Founder of Zipcode Therapeutics, Inc. Footnotes The author list has been updated to include P. Ghosh.