Synthesis Of Novel Semiconductor Copper Iron Selenide Nanomaterial And Study On Antimicrobial Properties And Mechanisms

With the severe shortage of antimicrobial-related drugs and multidrug resistance due to antibiotic abuse,it become increasingly urgent for us to develop novel antimicrobial drugs.Nanomaterials have already been studied as anti-tumor drugs because of their unique size,strong transmembrane ability,inhibition of efflux pump function and less prone to resistance,but their antifungal applications are a new topic research in recent years.Copper iron selenide nanomaterials have hitherto not been used in antifungal research.In this thesis,the copper iron selenide nanomaterial is facilely synthesized by a mild,green and non-polluting aqueous precipitation method.Its physical and chemical properties such as elemental content and valence,chemical composition,morphology and crystal forms were tested by ICP-OES（Inductively Coupled Plasma Optical Emission Spectrometer）,XPS（X-Ray Photoelectron Spectroscopy）,XRD（X-ray Diffraction）,FTIR（Fourier Transform Infrared Spectrometer）,TEM（Transmission Electron Microscope）,SEM（Scanning Electron Microscope）,UPS（Ultraviolet Photoelectron Spectroscopy）and so forth.Eventually,the structural formula was determined to be CuFeSe₂-PVP.This unique semiconductor CuFeSe₂-PVP demonstrates superior antifungal activity than the positive control drugs（terbinafine,fluconazole,5-fluorocytosine）and traditional antimicrobial nanoparticles（Ag NPs,Au NPs,Ti O₂NPs）in both planktonic fungi and biofilm.The antifungal effect of CuFeSe₂-PVP on Candida albicans（ATCC10231,ATCC14053,BNCC359501）was determined by inhibition rate test in microplate with MIC90 as low as 1.56μg/m L and was verified by the spread-plate experiment.Then,crystal violet assay and live/dead staining were usually used to quantify the anti-biofilm efficacy,and CuFeSe₂-PVP with the concentration of 1.56μg/m L attained a nearly complete biofilm eradication even facing the pre-formed Candida albicans biofilm.Animal experiments in vivo have strongly demonstrated that the CuFeSe₂-PVP nanomaterial had higher therapeutic efficacy than 5-fluorocytosine,and mice treated with the CuFeSe₂-PVP nanomaterial had completely recovered to healthy levels by analyzing H&E and Masson staining of skin wound.In the study of the fungicidal mechanism,the fact that massive fungi were killed in a short period was demonstrated by making time-dependent fungal inhibition assay,detecting the leakage of nucleic acid and protein,and measuring the leakage of K⁺which implied the leakage of intracellular components.It was observed by SEM and TEM that the fungal death was directly bound up with the fact that the nano-knives derived from the action of sharp edges itself could be inserted into the fungal body.ROS generation is also extensively reported as bactericidal mechanisms.Encouragingly,CuFeSe₂-PVP could generate ROS（Reactive Oxygen Species）detected by ESR（Electron Spin Resonance）experiments and DCFH-DA staining assay.In conclusion,the ability of CuFeSe₂-PVP to effectively killing fungi in a short duration is achieved through combining aforementioned mechanisms.Furthermore,biocompatibility is a prerequisite for the application of all materials in vivo.At first,from the perspective of selected materials,elements in this nanomaterial are the essential metals for humans and PVP is widely used for stabilizing and shape-directing agent,thus the obtained nanomaterial is inherently safe.In vitro experiments,the survival rate of H9C2 and the rate of hemolysis in rabbit erythrocyte were examined.And the animal toxicity of the nanomaterial was tested by H&E staining of the heart,liver,spleen,lung and kidney organs in vivo experiments.All of the above experiments have preliminary demonstrated the biocompatibility of the CuFeSe₂-PVP nanomaterial.At the same time,the bactericidal effects of CuFeSe₂-PVP in vitro on Staphylococcus aureus（ATCC29213）and Staphylococcus epidermidis（ATCC12228）were determined by inhibition rate test in microplate with MIC90 as low as 6.25μg/m L and 1.56μg/m L,respectively.The antibacterial effect was also verified by spread-plate experiment.The above results revealed that the antibacterial effect exceeded that of conventional antibacterial nanoparticles（Ag NPs,Au NPs,Ti O₂NPs）and was equivalent to that of antibiotics（vancomycin,ciprofloxacin,thiosporine）.Then,crystal violet assay and live/dead staining were often used to quantify the anti-biofilm efficacy.Intriguingly,CuFeSe₂-PVP with low concentrations could attain a nearly complete biofilm eradication even facing the pre-formed bacterial biofilms.In vivo studies,the therapeutic efficacy of CuFeSe₂-PVP has robustly been demonstrated,where H&E and Masson staining of skin samples from wounds showed that mice cured with CuFeSe₂-PVP nanomaterial had recovered to healthy levels.In addition,On the research of the bactericidal mechanism,the fact that large number of bacteria were killed in a short period was illustrated by detecting the leakage of nucleic acid and protein and measuring the leakage of K⁺.Besides,the body of bacteria was found by SEM and TEM to be obscured or even invisible,while nanomaterials were observed surrounding the bacterial“corpse”.Last but not least,CuFeSe₂-PVP can generate massive ROS,which is primarily associated with the release of Cu⁺.Generally speaking,the superior bactericidal properties of CuFeSe₂-PVP nanomaterial are achieved by combining above mechanisms.At last,a novel nanomaterial CuFeSe₂-PVP is obtained which has simple synthesis method,high intrinsic anti-fungal and anti-bacterial ability,excellent stabilization,and outstanding biocompatibility,making it easier to be further transformed.This study not only provides a novel fungicidal and bactericidal drug candidate,but also inspires the future design of anti-fungal and antibacterial nanomedicines.