Synergetic Antibacterial Effect Of Ultrasound Microbubbles Combined With Chitosan- Modified Polymyxin B Liposomes On Biofilm-producing Acinetobacter Baumannii

PART?PREPARATION AND CHARACTERIZATION OF C-PMB-LIP AND MICROBUBBLESObjective: To prepare chitosan-modified polymyxin B liposomes(c-PMB-Lips)and microbubbles(MBs)and investigate their general properties.Methods: c-PMB-Lip was prepared by an injection method and detected its particle size,Zeta potential,encapsulation efficiency,drug loading and in vitro release rate,its morphology was observed by the light microscope.MBs were prepared by a mechanical shaking method and their morphology,Zeta potential and particle size were detected.MB-c-PMB-Lip was then prepared by electrostatic adsorption and its attachment was examined by fluorescence microscopy.Results: The prepared c-PMB-Lip had a uniform particle size distribution with an average particle size of(215.2±5.03)nm,a Zeta potential of(12.64±1.44)m V,an encapsulation efficiency of(90.31±2.84)%,and a drug loading of(15.62±1.97)%.And the full antibacterial drug could be completely released within 24 h,with a certain degree of sustained release.The particle size of MB was(4.54 ± 0.8)?m,the Zeta potential was(-2.42 ± 0.43)m V.The fluorescence microscope observation of the connection showd that the c-PMB-Lips carrying a positive charge were evenly distributed around the negatively charged MBs,forming a garland-like structure with red fluorescence.Conclusion: In this study,we successfully prepared c-PMB-Lip and connected with MBs to form MB-c-PMB-Lips.Fluorescence microscopy showed that the complexs had a uniform particle size distribution and high binding rate,suggesting that it can be used as the basis for drug research in vitro and in vivo experiments.PART ? STUDY OF ANTIBACTERIAL EFFECT OF ULTRASOUND MICROBUBBLES COMBINED WITH CHITOSAN-MODIFIED POLYMYXIN B LIPOSOMES ON BIOFILM-PRODUCING ACINETOBACTER BAUMANNIIObjective: To investigate the synergistic antibacterial effect of ultrasound microbubbles combined with chitosan-modified polymyxin B liposome on biofilm-producing Acinetobacter baumannii and explore the effect of the combinations on biofilm structure.Methods: Bacterial biofilms were prepared using 96-well plates.The minimal biofilm inhibitory concentration(MBIC)of clinical isolates of Acinetobacter baumannii(AB)was determined by micro-broth dilution method.The antibacterial effects and dose-effect relationship of chitosan-modified polymyxin B liposomes(c-PMB-Lips),ultrasound microbubble + polymyxin B(USMB+PMB)and ultrasound microbubble + chitosan modified polymyxin B liposomes(USMB+ c-PMB-Lip)were further revealed by a crystal violet staining assay and a resazurin assay.Then 24-well plates were used.to cultivated bacterial biofilms and biofilm morphology following treatment was determined by scanning electron microscopy.Results: The MBIC of c-PMB-Lip was(8.0±2.0)?g·m L-1 against biofilm-producing AB.USMB could enhance the inhibitory effects of polymyxin B on biofilm-producing AB.Moreover,USMB combined with lipid agents could obtain the most significant inhibitory effects and even completely remove the biofilm-producing AB.With the concentration of drugs increasing,the antibacterial effects of each group showed a dose-effect relationship within a certain range.Compared with the sterile blank group,USMB+c-PMB-Lip could completely remove the bacterial biofilm and achieve a maximum antibacterial effects at 2?g/m L(P > 0.05).From the scanning electron microscopy,it showed that scattered pores formed by cavitation on the surface of AB biofilms after treatment with ultrasound microbubbles,a large number of biofilm structures remained after treatment with polymyxin B,and the biofilm scavenging was very obvious,only existed in individual bacteria after USMB+ c-PMB-Lip treatment.Conclusion: c-PMB-Lip combined with USMB have the significant synergistic antibacterial effects on biofilm-producing AB.