Clathrin-Mediated Endocytosis for Enhanced Apoptosis Induction and Cellular Imaging in Triple-Negative Breast Cancer...

The development of multifunctional nanoplatforms capable of drug delivery and real-time cellular imaging remains a key challenge in cancer theranostics. Herein, we report the development of a casein-protected maleic acid-derived nitrogen-doped carbon dot-based luminescent nanoplatform (MNCD@Cas NPs) for efficient delivery of the anticancer drug doxorubicin hydrochloride (DOX) to triple-negative breast cancer cells. Synthesized via a facile two-step method, the MNCD@Cas NPs exhibit bright blue fluorescence (λ_em = 390 nm), high water dispersibility, excellent colloidal stability, and substantial DOX loading capacity (∼84%) driven by electrostatic interactions. The drug-loaded MNCD@Cas NPs (DOX@MNCD@Cas NPs) exhibited clathrin-mediated endocytosis in MDA-MB-231 cells and pH-dependent drug release at higher levels under mild acidic conditions. Clathrin-mediated endocytosis was validated by confocal microscopic imaging of early endosomal antigen 1 expression, which showed a significant reduction in red fluorescent vesicles upon pretreatment with monodansyl cadaverine, a clathrin-mediated endocytosis inhibitor, compared to nystatin, an inhibitor of caveolin-mediated uptake. Further, the flow-cytometry-based quantification of DOX fluorescence confirmed the reduced intracellular uptake in MDC-pretreated cells, supporting the predominant involvement of clathrin-mediated internalization. The system induced pronounced cell cycle arrest at sub-G1 and G2-M phases, indicating apoptosis induction, as evident by confocal microscopy imaging and flow cytometric analysis. The intrinsic fluorescence of a biocompatible carbon dot enables real-time cellular imaging without using any external dye. This dual functionality such as anticancer drug delivery and imaging capability positions DOX-loaded MNCD@Cas NPs as a highly promising nanotheranostic platform.