Design And Properties Of Antimicrobial Oligomers With Dual-Selective Mechanisms Of Bacteria Membrane And DNA

The resistance and tolerance of antibiotics have posed great challenges to the current treatment of bacterial infection,characterized by in vivo complex infection(represented by intracellular pathogens)as the main manifestation of drug resistance and tolerance.As the structural necessity,functional diversity,and complexity of bacterial cell membrane,membrane targeted antibiotics are expected to obtain outstanding characteristics of antimicrobial agent resistance and tolerance.Membrane targeted antimicrobial agent is currently represented by cationic antibacterial polymers,with insufficient selectivity to bacterial cell membrane and host cell membrane,which resulting toxicity problems and limit its application The addition of a second antibacterial mechanism with strong bacterial cell selectivity based on retaining the targeting mechanism of antibacterial polymer membrane thus can be seen as an effective strategy to improve its therapeutic index.This project has selected DNA with accessibility differences between prokaryotic bacteria and eukaryotic host cells as the second target of cationic antibacterial polymers.As for prokaryotic bacteria,the polymer with the activity of destroying bacterial cell membrane can bind to the naked DNA after entering the bacteria,so the dual mechanisms of DNA and membrane will enable further improvement of its ability to kill bacteria;as for eukaryotic cells,the cationic antibacterial oligomers in this thesis enter the cells through endocytosis mechanism without breaking membrane,unbaling to pass through the cell nuclear membrane and mitochondrial membrane,and cannot bind to host DNA,thus forming their low cytotoxicity.The therapeutic index of antibacterial polymers can be effectively improved by the enhancement of antibacterial ability and the decrease of cytotoxicity brought by the second target of DNA.The main chain cationic antibacterial oligomers with molecular weight of about3000 Da,based on the above strategies,were synthesized by polycondensation.Such cationic antibacterial oligomers are equipped with cationic groups and hydrophobic groups composed of amidine or guanidine groups that can bind to DNA,by which we have carried out the following work:A main chain antibacterial oligomer PA-11 containing p-diamine cationic group was synthesized by polycondensation of terephthalic acid bis-imino esters hydrochloride monomer and 1,4-bis(3-aminopropyl)piperazine.PA-11 demonstrated antibacterial activity against a variety of drug-resistant bacteria,as well as good biocompatibility.Given the above facts,this study verified that PA-11 enabled its selectively damage on bacterial cell membrane,while no damage on mammalian cell membrane at effective bactericidal concentration.Provided that the in vitro binding of PA-11 and DNA grooves was confirmed,this study further verified the selectivity of PA-11 on bacterial DNA from the aspects of the distribution difference between PA-11 bacteria and cells,as well as its effect on DNA transcription and translation function.Based on the dual targeted antibacterial mechanism of PA-11,this study verified that PA-11 can be able to inhibit the formation of bacterial biofilm,remove bacteria from infected blood and selectively eliminate bacteria without posing damage on cells in the presence of mammalian cells.Above all,PA-11 also exhibited excellent therapeutic effect and biocompatibility in the in vivo infection mice model,proving the feasibility of the dual mechanism of PA-11 selective bacterial cell membrane and DNA.Considering the design concept of PA-11,PG series biguanide cationic main chain antibacterial oligomers were synthesized by polycondensation of thiomethyl isothiourea monomer and different diamine compounds.PG series demonstrated good antibacterial activity in vitro and extremely low hemolytic toxicity.Further study showed that the bactericidal effect of PG series on intracellular Staphylococcus aureus was positively correlated with its host cell accumulation.While subsequent founding showed that PG series had little bactericidal effect on intracellular Salmonella typhimurium,and its intracellular bactericidal effect was strain selective.PG-10 has the best bactericidal effect on intracellular S.aureus,it was thus selected from PG series for in-depth study on its selectivity.PG-10 could rapidly sterilize and remove bacteria from biofilm and blood infection.The analysis of the distribution and localization of PG-10 in cells infected by various intracellular pathogens was then conducted,demonstrating that PG-10 was localized in the lysosome by endocytosis and selectively killed S.aureus in the phagolysosome.Subsequently,the research proved the dual-selective antibacterial mechanism of PG-10 targeting bacterial cell membrane and DNA,and PG-10 exhibits a good therapeutic effect on mouse skin infection caused by intracellular S.aureus.This study provides a glimpse into the development and biological application of cationic antibacterial polymers in the future.