Study On The Synthesis And Antibacterial Properties Of Graphene-based Nanocomposite

Although we live in an era of highly developed medical technology and diversified health promotion related industries,the infection caused by pathogen is still one of the greatest threats to human health,causing millions of infection-related morbidities annually.The abuse of antibiotics has caused serious antimicrobial resistance,while the increasing antibiotic-resistant pathogens are compromising the therapeutic effects of traditional antimicrobials,especially the “superbugs” with multidrug resistance are invalidating even the most powerful antibiotics.Because of the lagging development of new drugs and the lack of ideal antibiotic alternatives,antibiotic resistance is still enforcing the increasing doses of antibiotic consumption,while the excessive use of antibiotics in turn causes the rapid emergence of new resistance mechanism,adverse drug reaction and environmental contamination.It seems to have fallen into a vicious circle.Bacterial resistance has become a public health problem around the world that urgently needs to be addressed.Given the plight of the serious threat to human health caused by bacterial resistance could not be efficiently resolved by traditional antibiotics,we have to reconsider the future development of anti-infective treatment.It is urgently important to explore new,low-cost and effective antimicrobial substances or alternative treatment strategies.In recent years,non-antibiotic antimicrobial substances have attracted a wide interest,among which nano-antibiotic material is considered to be the most promising strategy to overcome bacterial resistance and available for practical application because of its unique physical and chemical characteristics and inherently or obtained antibacterial properties through modification,which are rarely expressed in their bulk form.Some scholars thus have even defined these nanomaterials as “nanoantibiotics”.As an emerging nanomaterial,graphene oxide(GO)is an ideal carrier for antimicrobial active agents due to its high surface volume ratio,excellent flatness,rich and available oxygen-containing groups for further modification,and good biocompatibility.Silver particle(AgNPs)of good bactericidal activity is one of the first materials to be loaded onto GO.The prepared GO-Ag antibacterial nanocomposite shows an enhanced bactericidal activity due to the synergistic effect between GO and AgNPs.As a result,there appears a lot of research and reports on various types of GO-Ag.We found that most of the previously reported GO-Ag nanocomposites could maintain stable in water,but few studies investigated the stability of the entire nanocomposites in physiological solutions as well as their long-term effectiveness,however,which is is essential for its practical application.Moreover,the data on the inherent antibacterial property of bare GO are still contradictory at present.Therefore,we first investigated the antibacterial activity of bare GO in this work.Then two kinds of GO-based antibacterial nanocomposites were prepared via modification of Polyethylene glycol(PEG)and polyethyleneimine(PEI)respectively and further decoration of AgNPs.The systematic characterization and function evaluation of the synthesized nanocomposites were also performed.First,in view of the controversy about the inherent antibacterial activity of GO,we prepared GO in lab which was fully purified and systematically characterized.The antibacterial activity of GO was examined by colony counting method and TEM.The results showed that GO had no bactericidal activity,and the bacterial morphology after treatment of GO was the same as that of the control group under TEM,meanwhile,it was observed that GO came into obvious aggregation and stacking,leading to the increase of layer height and the loss of its sharp edges.GO thus lost the capacity of physical cutting,which might be an important reason for its failure to exhibit bactericidal activity.Second,a PEGylated and AgNPs loaded GO antibacterial nanocomposite(GO-PEG-Ag)was prepared.According to the structure-function relationship,the effects of different experimental factors on the preparation of the materials were analyzed.The modification order was determined that the Pegylation priors to the loading of AgNPs.We further screened the synthetic precursors of AgNPs and optimized the reaction conditions.The morphology,crystal structure,functional groups and composition of nanomaterials were systematically characterized by AFM,UV-vis absorption spectroscopy,XRD,FTIR,TGA,dynamic light scattering and zeta potential instrument,TEM / HRTEM,AAS and etc.The results showed that the in-situ reduction and loading of AgNPs on GO-PEG could be completed without addition of extra reducing agents within 3 min.The AgNPs with the small size of ~7nm and well-formed polycrystalline structure are uniformly attached on the entire surface of the nanocomposite in a high density(~27wt%).Stability tests showed that the synthesized GO-PEG-Ag nanocomposite could disperse stably over one month in a series of media even resist centrifugation at 10,000 ×g for 5 min.The time-kinetic experiment was conducted to investigate the relationship between the bactericidal effect and the treatment time,meanwhile,the time required for the stable bactericidal effect of GO-PEG-Ag was determined(2.5 h).The antibacterial activity and biotoxicity of GO-PEG-Ag nanomaterials was investigated via the minimum inhibitory concentration(MIC)test,instant and long-term bactericidal experiment and cytotoxicity analysis in comparison with GO-Ag nanomaterial.The results indicated that GO-PEG-Ag showed the same effective antibacterial activity against normal and resistant bacteria.The reduction rates of Gram-negative E.coli,including the drug-resistant bacteria carrying blaNDM-1 and mcr-1,were >99.5% after treatment with 5 μg/ml GO-PEG-Ag while that of Gram-positive normal S.aureus and MRSA were >95% after treatment with 10 μg/ml GO-PEG-Ag.The cell viability of HeLa cells remained ~80% after 24 h incubation with GO-PEG-Ag at a quite high concentration of 50 μg/ml.The results of the comparison tests confirmed that the ternary GO-PEG-Ag composite was superior to the binary GO-Ag composites both in bactericidal ability and biocompatibility.Importantly,GO-PEG-Ag presented stable and long-term antibacterial effectiveness,remaining ~95% antibacterial activity after one week in saline solution versus <35% for GO-Ag.We also investigated the bactericidal mechanism of GO-PEG-Ag nanomaterial by TEM,fluorescence confocal microscopy,the detection of protein leakage,ATP level and intracellular reactive oxygen species(ROS).The antibacterial mechanisms of GO-PEG-Ag were evidenced as damage to the bacterial structure and stimulation of oxidative stress injury,causing cytoplasm leakage and metabolism decrease.Third,the GO-PEI-Ag nanocomposite was successfully prepared by introducing branched PEI,and the systematic characterization and function evaluation of GO-PEI-Ag were carried out.The results showed that the properties of GO-PEI-Ag nanocomposite were similar to that of GO-PEG-Ag.GO-PEI-Ag also possessed the capacity of stable dispersion in various physiological solutions.In addition,GO-PEI-Ag may be more susceptible to effective contact with negatively charged bacteria because of the higher positive electricity,resulting in the slightly stronger bactericidal ability of GO-PEI-Ag than that of GO-PEG-Ag.After treatment of 10 μg/ml GO-PEI-Ag,the reduction rate of E.coli and A.baumannii reached ~100% while that of S.aureus and MRSA were 99.1% and 98.0% respectively.After storage in saline for one week,the bactericidal rates of 10 μg/ml GO-PEI-Ag against E.coli and A.baumannii were all > 99% while that against S.aureus and MRSA were all > 96%.The cytotoxicity evaluation showed that the survival rate of HeLa cells was ~ 72% after 24 h incubation with 50 μg/ml GO-PEI-Ag,indicating GO-PEI-Ag had low biotoxicity.In summary,we have prepared two kinds of GO-based antibacterial nanocomposites with the excellent stability in various physiological solutions.GO-PEG-Ag and GO-PEI-Ag nanocomposites,as antibacterial materials,have the advantages of high efficiency bactericidal property,good biocompatibility and long-term effectiveness,which indicates their good application prospects in biomedical and public health as the promising alternatives to antibiotic therapy for pathogens,especially drug-resistant infections.Meanwhile,we hope to provide a practical reference via our work for the preparation of similar antimicrobial materials.