Abstract

Carbon nano-dots (C-Dots) possess great benign properties, making them ideal for use in biomedical fields, especially due to their high aqueous solubility, outstanding photoluminescence (PL), favorable biocompatibility, low toxicity, chemical inertness, and easy functionalization properties. Herein, the C-Dots with sizes of about 2.4 ± 0.9 nm and containing the groups –OH, CC and CO, were shown to prolong the lag phase of human insulin (HI) fibrillation following a dose-dependent manner in an in vitro study. The spontaneous growth of fibrils after a lag phase was accompanied by exothermic heat, determined by isothermal titration calorimetry (ITC), demonstrating the inhibitory effect of C-Dots. Moreover, as the dose of C-Dots was increased to 4 mg mL−1, the fibrillation process could be totally deterred for more than half a month. However, the deterrent effect of the C-Dots on HI fibrillation disappeared when just a few fibril seeds were added. This is because the association constant of HI monomers interacting with fibril seeds (K2: 1.56 × 105 M−1) is much larger than that with C-Dots (K2: 8.28 × 103 M−1), as determined by analyzing the ITC results. An ‘‘active-site targeted’’ inhibitory mechanism has also been proposed. The “active site” is mainly on the B-chain of HI, and the ITC results show that the binding between the C-Dots and HI monomers is mainly driven by the electrostatic force. This is the first time that the anti-fibrillation mechanism in the presence of C-Dots has been analyzed by ITC. These results indicate that ITC is a promising approach for comprehensively clarifying the mechanisms of protein fibrillation inhibition.