In Vitro Antibacterial Study Of Baicalein-iron Nanomaterials

Background:The problem of drug-resistant bacterial infections has become one of the most serious challenges in healthcare.As bacteria become resistant,more and more antibiotics are becoming less effective or even ineffective,putting human lives at risk.While antibiotics are being used more carefully to prevent the development of resistance,there is also a need to find alternative antibacterial agents to antibiotics as soon as possible.Baicalein is a polyphenolic compound with antibacterial properties,but the poor water solubility of baicalein results in low bioavailability.The ligand chelation of polyphenols with various metal ions such as copper,iron,zinc and manganese to form a metal polyphenol network can improve the bioavailability of polyphenols.In addition,transition state metal ions,such as Fe²⁺,can catalyse the production of highly toxic hydroxyl radicals from H₂O₂ via the Fenton reaction,which can further enhance the biological functions of polyphenols.Based on this,baicalein-iron nanomaterials were investigated for their antibacterial properties,and the Fenton reaction properties of baicalein-iron nanomaterials were used to enhance the antibacterial effect of the nanomaterials for efficient antibacterial activity.Methods:The in vitro antibacterial properties of baicalein-iron were determined by the inhibition circle method,plate coating method,turbidity method,MTT method and LDH assay.The in vitro antibacterial properties of baicalein-iron combined with H₂O₂ were determined by TMB colorimetric assay and the in vitro antibacterial properties of baicalein-iron nanomaterials were determined by plate coating method,turbidimetric method and ROS assay.The biosafety of baicalin-iron nanomaterials was examined by cytotoxicity assay and hemolysis test.Results:（1）Characterization of baicalein-iron nanomaterialsThe baicalein-iron nanomaterials have a particle size of 135 nm,a polydispersity index of 0.001 and a negatively charged surface.The results by Fourier transform infrared spectroscopy showed that the hydroxyl group of baicalin complexed with iron ions to form baicalein-iron nanomaterials.（2）In vitro antibacterial study of baicalein-iron nanomaterialsBaicalein-iron nanomaterials have excellent resistance to sensitive strains of Gram-negative bacteria such as Escherichia coli and resistant strains of Gram-negative bacteria such as ampicillin-resistant Escherichia coli and carbapenem-resistant Klebsiella pneumoniae.35μg/m L baicalein-iron completely kills Gram-negative bacteria in a concentration-and time-dependent manner.Up to 400μg/m L baicalein-iron nanomaterials had no antibacterial properties against Gram-positive bacteria,including Staphylococcus aureus.（3）In vitro antibacterial study of baicalein-iron combined with H₂O₂Baicalein-iron nanomaterials in combination with H₂O₂ have highly effective anti-Gram-negative properties.4μg/m L baicalein-iron nanomaterials killed about 15%of Escherichia coli and ampicillin-resistant Escherichia coli,and 8μg/m L baicalein-iron nanomaterials killed about 10%of carbapenem-resistant Klebsiella pneumoniae.The commonly used disinfectant dose of H₂O₂ was 0.5%-3%（147-882 mmol/L）.The combination of baicalein-iron nanomaterials with H₂O₂ revealed that 4μg/m L baicalein-iron nanomaterials combined with 100μmol/L H₂O₂ could completely kill Escherichia coli and ampicillin-resistant Escherichia coli,and 8μg/m L baicalein-iron nanomaterials combined with 200μmol/L H₂O₂ could completely kill carbapenem-resistant Klebsiella pneumoniae.Baicalein-iron nanomaterials combined with H₂O₂ have excellent anti-Gram-positive properties.30μg/m L baicalein-iron nanomaterials combined with 5 mmol/L H₂O₂ can completely kill Staphylococcus aureus and 40μg/m L baicalein-iron nanomaterials combined with 5 mmol/L H₂O₂ can completely kill methicillin-resistant Staphylococcus aureus.The antibacterial mechanism is the Fenton reaction property of baicalein-iron nanomaterials producing large amounts of ROS,which leads to bacterial death.（4）Study on biological safety of baicalein-iron nanomaterialsL02 cells were treated with baicalein-iron nanomaterials at concentrations much higher than the antibacterial concentration,and the cytotoxicity of the nanomaterials was measured by MTT.The results showed that the 200μg/m L baicalein-iron nanomaterials group did not show significant cytotoxicity to human normal hepatocytes L02 cells.The hemolysis rate of160μg/m L baicalein-iron nanomaterials was 1%when applied to red blood cells using more than 4 times the antibacterial concentration,which is lower than the international standard of 5%,indicating that baicalein-iron nanomaterials have good hemocompatibility.Conclusion:Baicalein-iron nanomaterials can effectively kill Gram-negative bacteria and have excellent antibacterial effect against both Gram-negative and Gram-positive bacteria when combined with H₂O₂.The baicalin-iron nanomaterials have no significant cytotoxicity and good hemocompatibility,and have good potential for antibacterial applications.