Red-emitting manganese-doped carbon dots as competitive trypsin inhibitors: Mechanistic insights from spectroscopy, molecular docking, and biological assays

Herein, we have pioneered a hydrothermal synthesis approach to fabricate red-emitting manganese-doped carbon dots (Mn-CDs), which exhibit a pronounced fluorescence peak at 665 nm under 525 nm excitation. This significant red-shift in emission wavelength not only minimizes autofluorescence interference but also enhances tissue penetration, making Mn-CDs highly promising for biological applications. Capitalizing on these optical properties, our study delves into the interactions between Mn-CD and trypsin, employing sophisticated spectral analysis and molecular docking techniques. Our findings reveal that Mn-CDs non-covalently bind to the catalytic center and microenvironment of trypsin through hydrogen bonds and van der Waals forces, inducing conformational changes and forming a stable complex that impedes enzymatic activity. In vitro assays further substantiate the competitive inhibitory effect of Mn-CDs on the trypsin's catalytic activity, with enzyme kinetics demonstrating a reduced Michaelis-Menten constant without altering the maximum catalytic velocity. Notably, gel electrophoresis, human cervical carcinoma cells digestion micro-imaging, and clone formation experiments collectively demonstrate that this inhibitory effect effectively shields human serum albumin and Hela cells from trypsin-mediated degradation. These insights into nanomaterial-protease interactions pave the way for innovative enzyme-regulated tumor therapies, harnessing the inhibitory properties of Mn-CDs to modulate protease activity within cancerous microenvironments. This research not only deepens our fundamental understanding of nano-bio interfaces but also propels the practical designs of diagnostic/therapeutic nanoplatforms for biomedical applications.