The interplay between endothelial glycocalyx maturity and both the toxicity and intracellular uptake of charged nanoparticles.

Nanoparticles are widely studied for delivering treatments to target tissues, but few have reached clinical use. Most nanoparticles encounter blood vessels on their way to target tissues. The inner surface of these vessels is lined with endothelial cells covered by a glycocalyx, an extracellular matrix rich in anionic glycans. The role of the glycocalyx in nanoparticle interactions is not well understood. Here, we demonstrate that endothelial cells need extended culture times to synthesize a mature glycocalyx. Our research shows that branched polyethyleneimine functionalized gold nanoparticles bind to endothelial cells expressing either a developing or mature glycocalyx, with the interaction involving hyaluronan and heparan sulfate. These nanoparticles are subsequently internalized. Similar results were seen with poly(L-arginine). A mature glycocalyx protects cells by reducing the toxicity of these cationic nanoparticles. In contrast, lipoic acid-functionalized gold nanoparticles are internalized by cells with a developing glycocalyx, but not a mature one. Poly(L-glutamic acid) only interacts with cells when major glycans in the glycocalyx are degraded. These findings highlight the complex relationship between nanoparticle charge and structure, and their effects on toxicity, binding, and uptake by endothelial cells. This offers important insights for improving nanoparticle interactions with blood vessels in health and disease. STATEMENT OF SIGNIFICANCE: Endothelial cells lining blood vessels form a barrier through which nanoparticles must cross to reach target tissues. These cells are covered with a layer called the glycocalyx, which is rich in anionic glycans. However, the role of the glycocalyx in how nanoparticles interact with cells remains underexplored. Our research revealed that cells with a mature glycocalyx internalize cationic nanoparticles and experience reduced cytotoxicity. Conversely, a mature glycocalyx prevents anionic nanoparticles from entering cells. These results suggest that the structure of both the nanoparticles and the glycocalyx should be considered in future studies to improve the use of nanoparticles for medical applications.