Inhibition Effect And Mechanism Of Carbon Dots On Human Insulin Protein Fibrillation

The conversion of protein from monomer to fibrils can lead to a variety of amyloid deposition diseases,such as Alzheimer’s disease(AD),type 2 diabetes(T2D).At present,varies of protein fibrillation inhibitors have been developed,such as small molecules and peptides.Nowadays,the application of nanomaterials in the biological field is favored by scientists.Carbon dots,as a new type of carbon nanomaterial,have been used in protein fibrillation because of their unique photoluminescence properties,high biocompatibility,easy surface modification,and the potential to cross the blood-brain barrier(BBB).Insulin has been found to regulate neuronal activity and is closely related to AD production.In this paper,we use human insulin protein(HI)as a model,we explored the effect of different oxygen content carbon dots and different chiral carbon dots on the inhibition of HI fibrosis by using chiral cysteine as a raw material.The differences in inhibitory effects caused by different oxygen contents and different chiralities,as well as the internal mechanism of the differences,were studied in order to provide theoretical support for a better understanding of protein fibrillation and the development of protein fibrosis-related inhibitors.The main research content of this paper are as followed:Chapter 1: Introduced the amyloid deposition disease,the relationship between HI and amyloid deposition disease,the nucleation and growth mechanism and the commonly used detection methods in the process of protein fibrillation.Moreover,we introduce strategies and methods for the treatment and diagnosis of amyloid diseases based on this mechanism.It also introduces the current research progress of protein fibrosis inhibitors,including natural products,small molecules,peptides,and carbon nanomaterials.Finally,the special geometric properties of chirality,as well as the synthesis of chiral materials and the application of nano-chiral materials in the biological field are introduced.Chapter 2: Using chiral cysteine as a raw material,with hydrothermal method,we obtain two kinds of carbon dots in alkaline environment.And detecting the spectral characteristics of these two CDs,the result indicates that they have the same spectral characteristics and chirality.In the experiments of inhibiting HI fibrillation,the results of ThT,circular dichroism and TEM show that D-CDs were better than L-CDs in prolong the lag time of fibrillation and reducing the rate of growth period.In particular,when the ratio of the mass concentration of HI to D-CDs is 2: 1,D-CDs can keep HI for a week without fibrillation.In the presence of fiber seeds,the two carbon dots still showed a highly effective inhibitory effect,which was rarely reported in previous reports.And the inhibitory effect of D-CDs was still better than L-CDs.Isothermal titration calorimetry(ITC)was used to explore the binding strength and the force form of carbon dots to protein monomers and fibril seeds in detail.The results show that the interaction between D-CDs and HI monomers and seeds is stronger than that of L-CDs,and the electrostatic interaction force is the main force between carbon dots and HI.Further characterizing the structural characteristics of the two carbon dots,we found that the main difference between the two carbon dots is the oxygen content.The surface of D-CDs contains more oxygen-containing functional groups,which led us to speculate that the different oxygens of the two carbon dots is the main reason for the different inhibitory effects.Chapter 3: Based on Chapter 2,we modified the synthesis method to obtain carbon dots L-Cys-Fru-CDs and D-Cys-Fru-CDs using chiral cysteine and fructose as raw materials.In the experiments to investigate the effect of HI fibrillation,the results of ThT,circular dichroism and TEM show that the two chiral carbon dots had no significant difference in prolonging the fibrosis lag phase,but in the exponential growth stage,Compared with D-Cys-Fru-CDs,L-Cys-Fru-CDs can reduce the growth rate better.The same conclusion can be reached in the presence of fiber seeds.Studies on the interaction between chiral carbon dots and HI protein found that the binding strength of the two with the HI protein was similar,so there are no significant difference in prolonging the HI fibrillation lag phase.And the type of force between HI and L-Cys-Fru-CDs is mainly hydrophobic interaction force,while the force between D-Cys-Fru-CDs is mainly hydrogen bond and Van der Waals force,which may be related to the different chirality between these two CDs.Chapter 4: A detailed summary of the work done,and proposed directions for further research in the future.