Study On The Antibacterial Effect Of Near Infrared Light Triggered Dual-valence Platinum Nanoparticles

Objective:To prepare near-infrared light responsive dual-valent platinum nanoparticles and evaluate their antibacterial effect in vitro and in vivo and then explore their antibacterial mechanism and provide theoretical support for the use of platinum nanoparticles as a new antibacterial drug,thus providing a practical model for multi-mode accurate simultaneous treatment.Materials and methods:Pt(IV)nanogels were prepared through chloroplatic acid induced polyacrylic acid-b-polypropylacrylamide-b-polyacrylic acid(p AA100-b-p NIPAM200-b-p AA100,PNA)crosslinking.With the addition of Na BH₄,the uncoordinated Pt⁴⁺ions were reduced to Pt⁰ nucleus while the coordinated Pt⁴⁺ions formed the Pt²⁺ions shell due to the protection of ligands.Through the metallic interaction,Pt²⁺ions shell was anchored on the surface of Pt⁰ nucleus and finally forms dual value platinum nanoparticles(dv Pt NPs).In addition,zero-value platinum nanoparticles(Pt(0)-NPs)and divalent platinum nanoparticles(Pt(II)-NPs)were also synthesized according to similar procedures.In addition,Pt(0)-NPs and Pt(II)-NPs were mixed according to the proportion of Pt⁰ and Pt²⁺in dv Pt NPs to form the physical mixed Pt(0)-Pt(II)-NPs as the control.In vitro experiments,Gram-negative bacteria Escherichia coli and its persisters,together with Gram-positive bacteria Methicillin-resistant Staphylococcus aureus were co-cultured with various platinum nanoparticles and then stimulated by 808 nm near-infrared light.Before and after infrared light excitation,the antibacterial effect of various platinum nanoparticles was analyzed by measuring colony forming units(CFU),bacterial dying and their growth curves.In order to further understand the specific mechanism on bacteria killing,the morphology of bacteria was observed by scanning electron microscope.The integrity of bacterial wall membrane structure was confirmed by nucleic acid leakage experiment.Furthermore,the expression levels of DNA damage repair related genes were detected by q PCR while the rhodamine degradation experiment and reactive oxygen fluorescent staining was taken to confirm the effects of platinum materials on the redox homeostasis of bacteria.Finally,the effect of platinum materials on bacterial metabolism and status was tested according to the bacterial ATP levels and their membrane potentials.In order to further verify the antibacterial properties of the dv Pt NPs in vivo,mice subcutaneous abscess models were utilized.The model was constructed by subcutaneous injection of bacteria and then the suspension of platinum nanoparticles was injected to the affected area,which was stimulated by near-infrared light,and the abscess and skin lesions were observed after 5 days.At the same time,the mice in each group were killed.The tissue of the affected area was taken and CFU of each group were observed to evaluate the antibacterial effect of each group of platinum nanoparticles in vivo.Besides,the tissue of the affected area was taken,and the healing effect of every platinum nanoparticles on the tissue was determined by tissue sectioning and HE staining.Results:H₂Pt Cl₆ induced the crosslinking of(p AA100-b-p NIPAM200-b-p AA100)(PNA)and thus forming the Pt(IV)nanogels which were used as templates for the synthesis of dv Pt NPs.Then dv Pt NPs used in the experiments and the control groups Pt(0)-NPs and Pt(II)-NPs were synthesized by two-step reduction method.The hydrodynamic diameters of Pt(0)-NPs,Pt(II)-NPs and dv Pt NPs were 74.2±3.5 nm,90.1±2.7 nm and 69.7±2.4 nm,respectively.Their zeta potentials were-13.96±0.41m V,-8.12±0.27 m V and-8.90±0.31 m V,respectively.Furthermore,the absorption peaks of dv Pt NPs were at 200 nm and near infrared spectrum.In the experiments of time-temperature curves and the temperature cycle curves,dv Pt NPs showed excellent photo-thermal conversion efficiency and photo-thermal stability.Pt²⁺ions in dv Pt NPs were released only under the activation of near infrared excitation.Next,the antibacterial properties of dv Pt NPs were tested in vitro and in vivo.According to the results of CFU experiments,bacterial growth curves,bacterial dying and mice abscess models,it could be observed that dv Pt NPs exerted powerful antibacterial effect on Gram-positive bacteria MRSA and Gram-negative bacteria E.coli and the antibacterial efficiency reached more than 90%.In addition,in CCK8 experiments,dv Pt NPs showed no obvious inhibition of the proliferation of L929 and NIH3T3 cells and the nano-particles could metabolized through liver and kidney.At last,the various antibacterial mechanisms of dv Pt NPs were confirmed:dv Pt NPs caused the shrinkage and perforation of bacterial surface and induced a filamentous morphology in E.coli.;RNA leakage was observed followed the perforation of bacterial cells;dv Pt NPs induced the increase of ROS level in bacteria;dv Pt NPs could also affect the ATP level and the membrane potentials in bacteria.Conclusion:In vivo and vitro experiments,dv Pt NPs showed excellent antibacterial property when activated by near-infrared light,which achieved precise and cooperative multi-mode antibacterial effect.Besides,dv Pt NPs possessed good biocompatibility.This study built a theoretical foundation for the future application of platinum nanoparticles in the field of anti-infection.