Antibacterial Effect Of Cefoperazone Liposomes On Escherichia Coli

Avian pathogenic Escherichia coli(APEC)is an important member of Extraintestinal Pathogenic Escherichia coli(ExPEC).The genome of APEC is highly homologous with that of Neonatal Meningitis Escherichia coli(NMEC),and they share the same susceptibility model.As an important virulence repository of ExPEC,APEC can cause systemic infection,meningitis or other diseases in poultry,which triggers a potential zoonotic risk and a serious threat to human and animal health.Traditionally,antibiotics has been used to treat and prevent bacterial infection.Although some antibiotics have good antibacterial activity,the problem of antibiotic resistance is becoming increasingly serious.Besides,due to the disadvantages of limited bioavailability,low solubility,fast removal rate,non-specific toxicity to healthy organs,and so on,the clinical application of antibiotics has been limited.The use of novel antibiotic liposomes can reduce the risk of drug resistance development and toxicity to a certain extent.Liposomes are spherical nanoparticles composed of one or more phospholipid bilayers.As an antibiotic delivery vehicle,liposomes have several advantages including increased antibiotic solubility,enhanced stability,improved bioavailability,prolonged antibiotic half-lives,improved tissue targeting,and reduced adverse effects.In this study,the cefoperazone liposomes were prepared for studying antibacterial effects to APEC vitro and in vivo.First,the most sensitive antibiotic to APEC TW-XM,cefoperazone,was screened out through sensitivity test on representative antibiotics including ?-lactams,aminoglycosides,macrolides,tetracyclines and lincomycins.A spherical cefoperazone liposomes(CPZ-Vesicae)was successfully prepared using cefoperazone as the entrapment object with an entrapment efficiency of 10.3±1.5%,a small average particle size of(83±1.419 nm),a narrow and very uniform molecular weight distribution,and a relatively stable system(?-potential-55.5 mv).The antibacterial effect of cefoperazone liposomes was tested through a series of antibacterial experiments in vitro.The results of antibacterial ability test under different pH environments showed that the pH environments of 6.5,7.0 and 7.5 did not affect the stability and antibacterial ability of cefoperazone liposomes.Minimum inhibitory concentration(MIC)measurement results showed that the MIC of cefoperazone liposomes was 0.05?g/mL,which was 86%lower than that of free cefoperazone.It indicated that,as the carrier,the liposomes improved the antibacterial activity of free cefoperazone significantly.The results of time sterilization curve displayed that the antibacterial activity reached saturation when the concentration of free cefoperazone reached 1NIC.The antibacterial activity of cefoperazone liposomes was enhanced with the increase of drug concentration,showing a concentration-dependent trend.The results of biofilm formation assay indicated that cefoperazone liposomes could inhibit the formation of APEC biofilm.The interaction between cefoperazone liposomes and APEC was observed by an electron microscope.The result showed that a large numberof liposomes were attached to the APEC surface,and the interaction of liposomes with the outer membrane of bacteria caused membrane deformation.Mouse brain microvascular endothelial cells(bEnd.3)were used as an in vitro infection model to study the antibacterial ability of drugs on cells.The results of cytotoxicity test of CCK-8 showed that the dose of 32?g/mL was a safe dose and it did not produce toxicity to cells or affect cell proliferation.It indicated that cefoperazone liposomes were highly safe to cells.We compared the bactericidal ability of cefoperazone liposomes,free cefoperazone and blank liposomes against APEC on infected bEnd.3 cells.The result showed that after 2h of drug action,cefoperazone liposomes reached 99%bactericidal efficiency and had a significant bactericidal effect compared to the free cefoperazone group.The expressions of IL-6,IL-1? and TNF in b.End3 cells were detected by RT-qPCR in all treatment groups.Compared with the infection group without drug treatment,the expressions of IL-6,IL-1? and TNF decreased by 79%(p<0.0001)?82%(p<0.0001)and 60%(p<0.05)respectively in cefoperazone liposomes treatment group.Compared with the free cefoperazone liposomes-treated group,the expression levels of IL-6,IL-1? and TNF in b.End3 cells in the cefoperazone liposomes-treated group were declined by 56%(p<0.01)and 72%(p<0.001)and 55%(p<0.01),respectively.In conclusion,cefoperazone liposomes have a higher antibacterial activity in vitro than free cefoperazone.In order to study the bactericidal effect of cefoperazone liposomes on APEC TW-XM in infected mice,a mouse infection model was established.The expressions of inflammatory factors IL-6,IL-1? and TNF in the heart,liver,spleen,lung and kidney of mice were detected by RT-qPCR,so as to verify the biocompatibility and safety of cefoperazone liposomes.The results showed that the injection of 200?L(32?g/mL)cefoperazone liposomes was safe and nontoxic to mice,and the dose could be used for subsequent in vivo antibacterial test.The mice were inoculated with APEC TW-XM strain by intraperitoneal injection,and then cefoperazone liposomes were injected intravenously to treat the mice.The antibacterial effect of the drug was evaluated by bacterial colonization of tissues and organs and expression of organ inflammatory factors.The results indicated that at 106cfu challenge,cefoperazone liposomes prolonged the survival time of mice infected with APEC TW-XM,reduced the mortality of mice,bacterial colonization in organs,and the expression of inflammatory factors.The results of in vitro and in vivo experiments showed that cefoperazone liposomes could reduce the MIC while improving the antibacterial activity of free cefoperazone.After entering the blood through intravenous injection,antibiotics were slowly released and it could raise the circulating amount in the blood,slow the elimination of antibiotics,and improve bioavailability.As a result,the antibacterial effect of cefoperazone liposomes was superior to that of free cefoperazone.