Preparation Of Antimicrobial Nanomedicine And Their Properties Based On Azoreductase Response

Bacteria attack various infectious diseases by attacking human tissues.Antibiotics are considered as the main solution to the infection problem.However,bacterial resistance and the emergence of biofilms pose great challenges to the application of antibiotics.The presence of biofilm exacerbates the difficulty of treating bacterial infections and induces recurrent and chronic bacterial infections in living organisms.The biofilm is usually composed of extracellular polymer（EPS）secreted by bacteria and the bacteria itself.The presence of EPS blocks the penetration and diffusion of antibiotics and reduces the therapeutic effect of antibiotics.The introduction of nanotechnology into the treatment of biofilm,that is,loading the hydrophobic antibiotics into the nanoparticles through self-assembly,so as to realize the deep penetration and uniform dispersion of drugs,and then the infection problem brought by the biofilm can be solved.As the microenvironment response group of biological membrane,azobenzene can be introduced into the structure of nanocarrier and then realize the intelligent response of nanocarrier.Therefore,a cationic polymer m-azo Poly1 containing azobenzene was designed here.By the hydrophilic and hydrophobic interaction of the polymer,rifampicin（Rifampicin）was prepared with the hydrophobic antibiotic,and its physicochemical properties and antibacterial properties were investigated.The main contents are shown as follows:（1）A multiblock polymer m-azo PTE1 was prepared by introducing the hydrophobic azobenzene（azo）into the alternating copolymer alt-PTE1 backbone by a radical polymerization reaction.Based on the structural features of m-azo PTE1,biphenyl or azobenyl groups were introduced into the backbone of the alternating copolymers alt-PTE1and alt-PTE2,respectively,and the structure-similar multiblock polymers m-azo PTE2 and m-BPTE1 were designed as experimental controls.Three cationic polymers m-azo Poly1,m-azo Poly2 and m-BPoly1 were prepared by methylation reaction.All three cationic polymers have a low critical micelle concentration that could encapsulate the hydrophobic antibiotic rifampicin to form nanomedicines m-azo Poly1_Rif NPs,m-azo Poly2_Rif NPs,and m-BPoly1_Rif NPs.In the normal physiological state,the hydration particle size of the three nano-drugs is about 200 nm,with a long-term stability.Stimulated by sodium dithionite（SDT）,m-azo Poly1_Rif NPs and m-azo Poly2_Rif NPs can release rifampicin and small fragments of cationic polymers,while m-BPoly1_Rif NPs cannot respond to SDT,that is,nanodrug degradation and drug release cannot be achieved.（2）Biocompatibility experiments showed that the hemolytic energy of all three nanodrugs was greater than 1024μg/m L and had good cytotoxicity.The antibacterial results for E.coli and Pseudomonas aeruginosa showed that the cationic polymers m-azo Poly1 and m-BPoly1 with alt-Poly1 as the structural unit had good antibacterial ability and good synergy with Rifampicin,with a graded inhibition coefficient（FIC）between 0.25and 0.5.However,there is no synergistic antibacterial interaction between the cationic polymer m-azo Poly2 and Rifampicin with alt-Poly1 as the structural unit.However,there is no synergistic antibacterial interaction between the cationic polymer m-azo Poly2 and Rifampicin with alt-Poly1 as the structural unit.Cooperative mechanism experiments show that alt-Poly1 and m-azo Poly1 can disrupt the inner and outer bacterial membranes,and that m-azo Poly1 is more damaging to the outer membrane.The alt-Poly2 and m-azo Poly2can disrupt the bacterial outer membrane,but are less able to destroy the bacterial inner membrane.Through the combination of m-azo Poly1 with Rifampicin,bacteria can effectively inhibit drug resistance.Co-incubation experiments with bacteria showed that m-azo Poly1 NPs can respond to reductase secreted by planktonic bacteria,and the hydration size of m-azo Poly1 NPs decreased from 290 nm to 240 nm,and the absorption intensity of azobenzene decreased by about 10%.The biofilm clearance experiments showed that when the Rifampicin concentration was 32μg/m L,the m-azo Poly1_Rif NPs group was able to clear 80%of the biofilm and kill the bacteria in the remaining biofilm.However,m-azo Poly1_Rif NPs and m-BPoly1_Rif NPs were less effective in biofilm clearance and failed to completely kill the bacteria in the biofilm.The results of bacterial membrane penetration experiments showed that deep penetration and uniform dispersion could be achieved after incubating the three nanodrugs with biofilms for 45 min.In conclusion,m-azo Poly1_Rif NPs can effectively remove biofilm,improve the therapeutic effect of rifampin on biofilm,improve the utilization of antibiotics,and avoid the resistance and biotoxicity caused by excessive use of antibiotics.