Studies On The Preparation Of EGCG-CuNPs And Its Antibacterial Effect

Bacterial infection is one of the top 10 causes of death worldwide.Since the development of antibiotics in the 40s of the last century,the use of antibiotics has made great contributions to human health,but excessive use has accelerated the evolution of bacterial resistance,resulting in an increasing number and types of resistant bacteria,and gradually increasing drug resistance,posing a serious threat to human health.Different from traditional organic small molecule antibiotics,the development of nanotechnology provides the possibility for the construction of new antibacterial agents to solve the problem of bacterial resistance,and nano-antibacterial agents prepared by using nanotechnology to combine metals,polymers,natural compounds and other substances show high bacteriostatic activity,but there are still problems such as complex synthesis,harmful reagents,and low material safety.Therefore,the green rapid preparation of nano-bacteriostatic materials with high biological safety and strong bacteriostatic activity is a hot spot and difficult point in current research.Epigallocatechin gallate（EGCG）is one of the main functional components in tea,which has a variety of biological functions such as antioxidant,antibacterial,antiviral,immune regulation and prevention of cardiovascular and cerebrovascular diseases.EGCG contains a number of phenolic hydroxyl groups,which are the intrinsic basis for its biological function,but it is also prone to inactivity,resulting in low bioavailability and greatly limiting its application in various field.Therefore,improving the bioavailability of EGGC helps to give full play to its biological activity in all aspects and promote the application of EGCG in various fields.With the wide application of nanotechnology in various fields,the research focus is no longer limited to EGCG monomers,but the use of EGCG and other substances to jointly construct biomedical nanomaterials,so that it has the advantages of high bioavailability,high antibacterial activity and low toxicity.Therefore,the EGCG nanoification strategy provides a new direction to solve the problems of low bioavailability and drug-resistant bacterial infection of EGCG.Metal-EGCG nanomaterials prepared by metals and EGCG show dual advantages.In metal-EGCG-nanosystems,nanometals are considered to be potential“nanoantibiotics”,which can achieve antibacterial purposes through a variety of bacteriostatic mechanisms.Nano EGCG has the characteristics of green safety and high biological activity.Therefore,the organic combination of metal and EGCG to prepare nanomaterials is not only green and safe,but also plays an efficient synergistic antibacterial effect.Compared with other metals,copper has the advantages of low cost and easy availability,and is a trace element necessary for the human body.Because copper has the characteristics of broad-spectrum antibacterial activity,good biosafety and a variety of physiological functions,it is often used in the preparation of nano-antibacterial agents.As one of the most effective antibacterial materials,nanocopper has antagonism to a variety of pathogenic bacteria and is not easy to cause bacterial resistance.The anticancer activity of sodium diethyldithiocarbamate（DEDTC,the main active product of disulfiram）has been confirmed by cellular,in vivo and clinical studies,but there are few studies on its bacteriostatic activity,and there are a large number of gaps.Studies have shown that the combination of DEDTC and copper can improve anticancer activity,but whether it has antibacterial activity remains to be studied.In conclusion,this study successfully synthesized stable and well-dispersed nanoparticle EDCuby using EGCG,DEDTC and CuSO₄ aqueous solutions,and further characterized its chemical characterization,safety evaluation and bacteriostatic effect evaluation.The results showed that EDCuhad good biological safety,showed strong inhibitory activity against gram-positive bacteria,and could effectively inhibit the biofilm formation of methicillin-resistant Staphylococcus aureus（MRSA）,promote the healing of skin wounds in mice,and finally,the antibacterial mechanism of EDCuwas elaborated from the aspects of structural destruction,inclusion leakage,and reactive oxygen generation.The main research results of this study are as follows:（1）EDCunanoparticles were successfully prepared.In this study,EGCG,DEDTC and copper ions were self-assembled into EDCuat room temperature and pressure using a one-step green synthesis method,and the whole process only took a few seconds.The results show that EDCuparticle size is about 5 nm,and has good stability.When stored at 4°C for90 days,there was no significant change in the appearance color and UV absorption of EDCu.（2）The good biosafety of EDCuwas evaluated.The results showed that EDCuis not cytotoxic to human umbilical vein endothelial cells（HUVECs）,no significant changes in the morphology and number of HUVEC cells at high concentrations（5μM）,and does not cause hemolysis in mouse red blood cells.In addition,EDCu,at low concentrations（0.2-1μM）,can promote the migration of HUVEC cells.（3）The significant antibacterial activity of EDCuwas clarified.The result of in vitro experiments showed that EDCucould reduce the survival rate of Staphylococcus epidermidis（SE）,methicillin-resistant Staphylococcus aureus（MRSA）and Staphylococcus aureus（SA）to less than 40%at very low concentrations（2.5μM）,and inhibit the growth of MRSA biofilms.In in vivo experiments,EDCupromoted wound healing in mice,and the healing rate of skin in the EDCu-2 group（40μM）on the seventh day reached 85%.（4）The bacteriostatic mechanism of EDCuwas analyzed.The results showed that EDCucan lead to the release of inclusions such as nucleic acids and proteins in bacteria by destroying bacterial structures;After entering the bacteria,it stimulates the outbreak of reactive oxygen species（ROS）in the body,which in turn attacks other biological macromolecules,resulting in the death of the bacteria.