Decoding Endothelial Glycocalyx‐Nanoparticle Interactions via a Systematic Polymeric Nanoparticle Library and High‐Content Characterization

ABSTRACT Understanding how nanoparticle physicochemical properties influence cellular interactions is critical for optimizing drug delivery systems. Here, we report the high‐throughput synthesis and biological characterization of a 42‐member library of self‐assembled polymeric nanoparticles that systematically vary in hydrodynamic diameter and surface charge. Using reversible addition‐fragmentation chain‐transfer (RAFT) polymerization and polymerization‐induced self‐assembly (PISA), nanoparticles were synthesized in microplate formats and characterized for cytotoxicity and uptake by endothelial cells expressing either immature or mature glycocalyces. We found that surface charge had a greater impact on cytotoxicity and cellular uptake than hydrodynamic diameter, with some cationic nanoparticles exhibiting higher toxicity and uptake. Notably, cells with a mature glycocalyx showed increased tolerance and uptake of cationic nanoparticles, suggesting a protective and regulatory role of the glycocalyx. A semi‐automated imaging workflow incorporating machine learning‐based cell segmentation enabled single‐cell quantification of nanoparticle uptake, revealing population‐level variability across the library. Protein corona formation in serum conditions further modulated nanoparticle surface charge and interactions. This study highlights the importance of integrating nanoparticle design with biological context and scalable analytical tools, offering a framework for optimizing nanoparticle formulations for targeted delivery across diverse cell types.