Machine learning-guided fabrication of carbon dot-pepsin nano-conjugates for enhanced bioimaging, synergistic drug delivery, and visible light-induced photosensitization.

Carbon dots (CDs) are emerging as next-generation bioimaging agents due to their strong fluorescence, photobleaching resistance, and biocompatibility. However, their small size often limits efficient cell internalization, leading to unspecified cellular targets and high endosomal escape. Herein, we showcase the potential of pepsin, a simple proteolytic enzyme, to address the limitations of CDs through its covalent conjugation and subsequent creation of CD-pepsin nanoparticles (NPs). Interestingly, repeated failures in achieving CD-pepsin NPs prompted us to explore machine learning (ML), which, in a novel approach, successfully predicted the most favourable conditions for their formation. These nanoparticles unlocked two critical functionalities beyond enhanced imaging: visible light-induced reactive oxygen species (ROS) generation and sustained drug delivery, highlighting their potential for chemo-photodynamic combination therapy. The photophysical properties of the green-emissive CDs, synthesized from 3-hydroxy-2-naphthoic acid, were significantly enhanced through covalent conjugation with pepsin. This enhancement was evident in the efficient confocal imaging of human macrophage cells (THP1 cells) using both the CD-pepsin covalent conjugate and CD-pepsin NPs. Beyond enhancing the imaging potential of CDs by stabilizing their surface states through covalent conjugation with pepsin, this modification also significantly increased ROS generation, surpassing conventional photosensitizers like protoporphyrin (PpIX). Irradiation of the cells with a simple 23 W white LED induced increased oxidative stress, leading to reduced cell viability. Simultaneously, the CD-pepsin NPs functioned as a drug delivery vehicle, enabling the sustained release of the model drug, doxorubicin, over 4-5 days. These combined functionalities-bioimaging, photosensitization, and drug delivery-highlight CD-pepsin NPs as a versatile and effective multipurpose platform, overcoming key limitations of pristine CDs for advanced theranostics.