High-Oxygen-Content Carbon Dots as a High-Efficiency Inhibitor of Human Insulin Aggregation

Neurodegenerative diseases consist of Alzheimer's disease and type 2 diabetes which pose serious health effects. Carbon dots are widely used in the treatment of these types of diseases with their excellent biocompatibility and low toxicity. Herein, we use l- or d-cysteine to synthesize nontoxic high-oxygen carbon dots with a size around 3 nm and full of highly oxygenic functional groups. Through a ThT fluorescence assay, far-UV CD spectroscopy, and TEM, we find that carbon dots (CDs) can inhibit the nucleation process and elongate the lag phase by a large degree, and the higher-oxygen content one (d-CDs) exhibits a more significant inhibitory effect. When the dose of d-CDs reached 0.5 mg mL^-1, it can impede the protofibril formation for nearly 1 week. Through the isothermal titration calorimetry (ITC) experiment, we obtain this result because d-CDs interact with the human insulin (HI) monomer (K_a1 = 2.46 × 10⁴ L mol^-1) stronger than l-CDs do (K_a2 = 1.62 × 10³ L mol^-1). More importantly, these two high-oxygen CDs still have a gorgeous inhibitory effect; even human insulin was nucleated, which has not been reported previously. This is because the association constant of fibril seeds interacting with d-CDs (K_a3 = 2.91 × 10⁴ L mol^-1) is large and competes against the interaction with the HI monomer so it can slow the fibrillation rate. Moreover, the interaction is mainly driven by electrostatic force. This is the first time results have been found of highly oxygenic CDs having a great inhibitory effect when they already have the growth sites and are illustrated by ITC experiments, which suggest further protein fibrillation inhibition.