Antistreptococcal Activity And Preliminary Safety Evaluation Of Levofloxacin Polyphenol-protein Nanoparticles

Objective: This study aims to evaluate the antibacterial activity and safety of levofloxacin polyphenol-protein nanoparticles（CT-L）against S.suis and S.pneumoniae in vitro and in vivo,and to provide evidence for the clinical application of CT-L.Methods: Antibacterial activity and safety of CT-L in vitro were evaluated by antimicrobials sensitivity test,time-kill curve test,red blood cell hemolysis test,cytotoxicity test,and live-dead bacterial double stain test.The antibacterial activity and safety of CT-L in vivo were evaluated through mouse blood routine and biochemical tests,tissue section HE staining,construction of pneumonia model,treatment of mouse infection model,and tissue fluorescence imaging tests.All the assays conducted with levofloxacin（L）and nano carrier（CT）as controls.Results: 1.Antibacterial activity and safety evaluation in vitro: Antimicrobial susceptibility test showed that the MICs for CT-L were distributed among 0.125μg/m L to 4 μg/m L,MICs for L were from 0.25 to 1 μg/m L,MICs for CT were greater than 32 μg/m L,indicating that CT-L was effective against S.suis and S.pneumoniae.Time-kill curve exhibited that 4 μg/m L CT-L and 8 μg/m L L could kill S.suis to the detection limit（10² CFU/m L）within 5 h.CT-L of 1.7 μg/m L and L of 5 μg/m L could kill S.pneumoniae to the detection limit（10² CFU/m L）within 8h.These results suggested that the concentration level of CT-L was lower than that of L when CT-L and L had the same bactericidal effect under the same time conditions.Live-death double staining test showed that a large amount of red fluorescence was observed in the CT-L group and the red fluorescence was more obvious than the green fluorescence after mixing.Although the red fluorescence was observed in group L,the red fluorescence was not obvious after mixing,indicating that the bactericidal effect of CT-L was stronger than that of L.The hemolysis test results showed that the hemolysis rate of CT-L was less than ₁₀% at 640 μg/m L,indicating low toxicity at high concentrations.Cytotoxicity test results showed that the survival rate of lung cell A549 was about 82% at 512 μg/m L CT-L,suggesting the high safety in spite of high concentrations of CT-L.2.Antibacterial activity and safety evaluation in vivo: Acute toxicity test showed that inhaled 70 mg/kg CT-L and L could affect the lungs of mice.HE section staining showed a large amount of hemorrhage and congestion in the lungs of mice.In addition,a large amount of red blood cells and varying number of inflammatory cells were observed in the alveolar cavity.While blood routine,blood biochemistry and HE section staining showed no abnormality compared with the control group at 50 mg/kg of CT-L,indicating that 50 mg/kg was the safe concentration of CT-L.Mouse models of S.suis and S.pneumoniae pneumonia were established respectively.The in vivo treatment experiments using these two models showed that CT-L had a significant bactericidal effect,and the bacterial load in the lung of S.suis pneumonia model was reduced by 3.9 log₁₀ CFU/g.The bacterial load of S.pneumoniae pneumonia model was reduced to the detection limit(2 log₁₀CFU/g).HE staining results showed that the alveolar wall capillaries of the infected model could recover to slight hyperemia and bleeding,without visible bacterial distribution after treatment with CT-L when compared with the control group,suggesting the treatment effect of CT-L on lung tissue was significant.In order to better characterize the activity characteristics of CT-L,the targeting of this preparation was evaluated in vivo.The fluorescence dye was used to simulate the distribution of CT-L in mice.The results showed that CT-L had strong targeting,higher content in the lungs and longer existence time than free drug L.Conclusion: This study showed that CT-L has strong antibacterial activity both in vitro and in vivo,with high safety,strong targeting,high antibiotics stability,and excellent antibacterial activity.Therefore,CT-L has high research value and broad application prospects.