Nitrogen-doped carbon dots as biocompatible fluorescent agents for labelling human red blood cells

Evaluating the biocompatibility of nanoparticles with blood is essential to demonstrate their biosafety, reduce potential adverse effects, and enable their application in nanomedicine. Although many studies have explored interactions between blood and nanomaterials, only limited number have specifically addressed the compatibility of nitrogen-doped carbon dots (N-CD) with red blood cells (RBC), the most abundant cells in blood, that are essential for healthy functioning of all vertebrates through their role in oxygen transport. This study investigated the biological properties of several concentrations (25, 50, 100, 200 μg/mL) of negatively charged, green fluorescent N-CD, synthesized using environmentally friendly precursors through a hydrothermal method, on healthy human RBC in vitro. Scanning electron microscopy and atomic force microscopy revealed that the treatment with N-CD, even at the highest concentration, did not significantly affect RBC morphology. Interfacial interaction between N-CD and RBC was demonstrated by photoluminescence spectroscopy, fluorescence microscopy, and synchronous fluorescence spectroscopy analysis. The treatment with N-CD at the highest concentration had no effects on the RBC osmotic fragility, slightly increased the RBC deformability, and demonstrated a noticeable protective effect on the RBC hemolysis after 24 h. Flow cytometry analysis confirmed that N-CD did not alter the RBC morphology and did not result in an increase in the production of reactive oxygen species or reactive nitrogen species, indicating that their interaction did not lead to oxidative stress induction in human RBC. These findings suggest that fluorescent N-CD are biocompatible, fluorescent RBC imaging agents and hold promise as candidates for developing novel RBC-based drug delivery systems.