Effect Of Ts-TPGS Modified Calcium Phosphate Tigecycline Nano Drug Delivery System On Multidrug Resistant Klebsiella Pneumonia

Multidrug-resistant Klebsiella pneumoniae(MDR-KP)is an important pathogen causing lung infections,and its high morbidity and mortality rates pose a great challenge for clinical treatment.Tigecycline(TIG)is one of the important drugs for the treatment of MDR-KP infection,but its increased resistance rate often leads to clinical treatment failure.High expression of efflux pump gene is an important mechanism of multi-drug resistant Klebsiella pneumoniae tigecycline resistance.Nanodelivery technology is one of the effective means to overcome tigecycline resistance in Klebsiella pneumoniae.D-α-Tocopherol polyethylene glycol 1000 succinate(TPGS)can inhibit the expression of efflux pumps of multi-drug resistant bacteria,and the TPGS-modified Calcium Phosphate(Cap)nano-delivery system can overcome to a certain extent the resistance of MDR-KP resistance to tigecycline.The antimicrobial peptide(S-thanatin,Ts)can specifically bind to bacterial outer membrane lipopolysaccharide to enhance the bacterial targeting of the nano-delivery system,and the nano-delivery system modified by its functionalization can be targeted to accumulate in the bacteria and at the site of infection.Therefore,this study aims to construct a TPGS-and Ts-modified nanodelivery system that can overcome the resistance of MDR-KP to tigecycline,but also has bacterial targeting effects to further improve the therapeutic efficacy of tigecycline against clinical multi-drug resistant bacteria.The particle size,polydispersity index(PDI),zeta potential and drug release behavior of the TPGS/Cap,Ts-TPGS/Cap,TPGS/Cap/TIG(TCT)and Ts-TPGS/Cap/TIG(TTCT)nanoparticles were investigated to determine the various physicochemical properties of the formulations.The nanoparticle size was kept between25-28 nm and showed a small particle size distribution,with no significant change in particle size after Ts functionalization and TIG encapsulation.All nanoparticles showed a single-peak zeta potential distribution with negative charge.Transmission electron microscopy(TEM)imaging showed that all nanoparticles had a uniform rod-like structure.The stability of the nanopreparations was examined by dynamic light scattering(DLS)over time,which showed minimal changes at 20 days,with particle size and PDI remaining almost constant.The release of TIG from the nanoparticles showed a typical two-phase pattern,with a rapid release of about 25% of the drug during the first 0.5 h,followed by a sustained release over 72 h,with about 50% of the drug molecules released within 8 h The cumulative drug release rates of TCT and TTCT were 94.20±4.05% and 98.52±3.69%,respectively.The in vitro antibacterial activity study of TTCT against Klebsiella pneumoniae(KPN)and TIG-resistant Klebsiella pneumoniae(TRKP)revealed that TTCT had the strongest antibacterial activity and the lowest MIC value of 1 μg/m L in the KPN and TRKP groups.This may be related to the binding effect of TIG with Ts peptide,suggesting that TTCT is effective in overcoming drug resistance.The effect of different preparations on the growth curves of KPN and TRKP was further investigated.In the KPN and TRKP groups,the nanoparticles were not effective in inhibiting bacterial growth,except TTCT,which showed better antibacterial activity than TIG and TCT.Scanning electron microscopy(SEM)was used to observe the changes of bacterial morphology after the administration of different preparations.Bacteria without any treatment showed a typical rod shape with an intact and smooth surface.The bacterial morphology changed significantly after treatment with different agents.Ts-TPGS/Cap nanoparticles showed obvious holes in the cell walls of both KPN and TRKP.TTCT had a greater destructive effect on the bacterial structure in both KPN and TRKP groups,further confirming the better antibacterial effect of TTCT.Carbonyl cyanide 3-chlorophenylhydrazone(CCCP)was used as a positive control group,and Ethidium bromide(EB)was used as the efflux pump substrate,and the accumulation of efflux pump substrate was measured by fluorescence spectrophotometer under different concentrations of TPGS prescription.The EB content was measured by fluorescence spectrophotometer at different concentrations of TPGS.Quantitative analysis showed that the percentage of EB accumulation increased from22.11±3.67% to 64.75±5.91% as the concentration of TPGS increased from 20 μM to100 μM,indicating that TPGS treatment exhibited a significant increase in EB accumulation and presumably TPGS had an inhibitory effect on the efflux pump activity of TRKP.After co-culture with Ts-TPGS/Cap nanoparticles,the fluorescence signal in KPN was approximately 2-fold higher at both 2 and 6 h than that of TPGS/Cap nanoparticles.For TRKP,similar results were observed,indicating that Ts-TPGS/Cap nanoparticles exhibited better bacterial internalization activity,which may be attributed to the targeting ability of Ts antimicrobial peptides.A mouse model of acute pneumonia was established by intratracheal injection of KPN or TRKP bacteria,and indocyanine green(ICG)-labeled nanoparticles were injected intravenously 5 h after modeling.The distribution of Ts-TPGS/Cap nanoparticles in vivo was observed at 6h and 24 h after the administration,respectively.The results showed that the Ts-TPGS/Cap nanoparticles could accumulate in the lungs of KPN and TRKP infected mice,which was beneficial to increase the concentration of antibacterial drugs at the infection site and achieve better anti-infection effects.The anti-infective activity of TTCT was further investigated in vivo using the acute pneumonia model of KPN and TRKP infected mice.After 5 h of modeling,TIG,TCT,TTCT and Ts-TPGS/Cap nanoparticles were injected intravenously.The survival rates of mice in different treatment groups were recorded within 5 days.TTCT treatment group had the highest survival rate of 100% in KPN and TRKP infected mice,and its efficacy was superior to other groups.The results of inflammatory markers(white blood cell count,neutrophil count and C-reactive protein level)showed that the levels of white blood cells,neutrophils and C-reactive protein decreased substantially in the TTCT-treated group,indicating that the therapeutic effect of TTCT was superior to that of other treatment methods.Bronchoalveolar lavage fluid(BALF)was used for bacterial colony analysis showing that the TTCT-treated group had the lowest bacterial colonization in KPN-and TRKP-infected mice compared with other treatment groups.HE staining to observe the histopathological morphology of the lungs of KPN-and TRKP-infected mice showed that the TTCT-treated group had significantly better improvement in inflammatory infiltrates,edema,and alveolar wall thickening in the lungs than other treatment groups.Finally,the biosafety of nanoparticles at the cellular level and tissue level was evaluated.The cytotoxicity of TPGS/Cap and Ts-TPGS/Cap nanoparticles was evaluated by MTT method on HUVECs and EA.hy926 cells.When the concentration of nanoparticles reached 0.4 mg/m L,the cellular activity remained more than 90% after 48 hours,indicating that both have good biocompatibility.For TIG,TCT and TTCT,the percentages of hemolysis were 6.80 ± 0.11%,3.92 ± 0.52% and 4.13 ± 0.96%,respectively,which implies that the encapsulation of TIG into nanoparticles improved their biosafety.No significant pathological changes in the collecting organs were observed in the TCT,TTCT and Ts-TPGS/Cap nanoparticle groups,indicating that the adverse effects of TIG were all reduced after drug encapsulation into the nanodrug delivery system.The above findings suggest that the TTCT nanodrug delivery system constructed in this thesis can improve its tissue distribution,enhance drug efficacy,reduce bacterial resistance and decrease adverse effects through sustained drug release,good targeting and intra-bacterial accumulation in a mouse model of acute pneumonia,and these findings may provide potential applications for drug-resistant bacterial infections and the application of TPGS and Ts antimicrobial peptides in other infectious diseases These findings may provide new therapeutic strategies for the application of TPGS and Ts antimicrobial peptides in other infectious diseases.