Tailoring carbon nitride nanoarchitecture and cellular interactions through hydrothermal alkaline etching

Graphitic carbon nitride (g-C₃N₄) is a versatile material for biomedical, energy, and environmental applications. Bulk g-C₃N₄ is commonly used as a precursor for nanoscale objects, yet systematic links between synthesis conditions, nanostructure morphology, surface chemistry, and biological response remain limited. Here, we fabricated a palette of carbon nitride nanostructures via hydrothermal alkaline etching. By varying the hydroxide (LiOH, NaOH, or KOH) and reaction conditions, etching yields carbon nitride quantum dots (CNQDs), carbon nitride nanowires (CNNWs) and their networks, as well as larger flakes and sponge-like objects. Automated transmission electron microscopy image analysis supported this comparison by converting heterogeneous micrographs into object-level morphology and particle-size statistics. The hydroxide type also influenced surface chemistry: LiOH and KOH promoted more extensive oxidative functionalization, whereas NaOH yielded surfaces with lower levels of carboxyl groups and a larger amount of weakly bound OH species. All nanostructured carbon nitride samples showed high biocompatibility across multiple cell lines after 24 h incubation at 200 µg mL^-1. Specifically, immortalized human cervical adenocarcinoma (HeLa) cell viability was essentially unchanged. Metabolic activity likewise remained near control levels in HeLa, immortalized human lung adenocarcinoma (A549) cells, immortalized mouse microglial (BV2) cells, human acute monocytic leukemia (THP-1)-derived macrophages, and immortalized mouse embryonic fibroblast (3T3) cells. Cellular accumulation in HeLa cells varied markedly with the preparation conditions of the nanostructured carbon nitrides. Notably, smaller nanostructures did not necessarily exhibit higher cellular accumulation, indicating that morphology and surface chemistry, in addition to particle size, influence cellular interactions. CNNW networks, with or without entangled CNQDs, may contribute more strongly to cell-associated fluorescence than isolated CNQDs. Overall, this work provides a practical route to tune carbon nitride nanostructures for bioimaging applications.