Charge Dependent Endocytic Pathways of Graphene Quantum Dots Revealed by a Multi Inhibitor Approach*

Graphene quantum dots (GQDs) are increasingly recognized in nano-biotechnology for their dual roles in therapeutic delivery and imaging. Successful translation of these applications to clinic depends on a clear understanding of cellular internalization routes. Yet, much of the existing literature offers conflicting conclusions, often based on a limited set of endocytosis inhibitors. In this work, we employ a comprehensive strategy using six inhibitors, accounting for their both on- and off-target effects, to examine the uptake of GQDs with different surface charges. Intracellular fluorescence tracking in HeLa and HEK-293 cells reveals that GQDs utilize multiple endocytic pathways rather than a single entry route. Pathway preference depends strongly on GQD charge and surface chemistry. Positively charged nitrogen-doped GQDs (NGQDs) are primarily internalized via fast endophilin-mediated endocytosis (FEME) in HeLa cells, with clathrin-mediated endocytosis (CME) as a secondary route, while in HEK-293 cells they favor CLIC/GEEC and FEME. By contrast, negatively charged GQDs predominantly enter HeLa cells through CME. These insights highlight how tailoring GQD surface properties can direct cellular uptake pathways, informing the rational design of nanomaterial-based delivery systems and advancing their potential further biomedical applications.