| The abuse of antibiotics has made the problem of antibiotic resistance more and more serious all over the world.The reason is that the target of traditional antibiotics is relatively single,and bacteria could easily produce a few specific mutations and thus acquire drug resistance.In contrast,cationic antimicrobial polymers（AMPs）with the advantage of fast sterilization kinetics,not easy to induce bacterial resistance and broad-spectrum antimicrobial effect are served as solutions for the resistance problem.AMP s typically target bacterial membrane and cause physical damage to it,and the synthesis of bacterial membrane involves complicated biosynthetic pathways;thus,it is much more difficult for bacteria to generate resistance as there are too many mutations needed for such an effect.However,because of the similarity between bacterial and mammalian cell membrane structure,AMPs usually do not have enough selectivity between prokaryotic and eukaryotic cell.Consequently,AMPs typically show ineligible toxicity toward eukaryotic cells.It is difficult for established antibacterial polymer systems nowadays to be approved for clinical practice.Scientists have found that the size,ratio and spatial distribution of cationic and lipophilic structures in AMPs have a significant influence on their antibacterial activity and biocompatibility in the process of exploring their mechanism of action.In order to obtain compounds with high therapeutic index（TI）,there have been many reports on the optimization about these parameters indicated above.However,because of AMP s’poor membrane targeting selectivity,this kind of adjustments could acquire less promotion of TI,and thus the results are often unsatisfactory.To find a potential solution towards the cytotoxicity problems associated with AMPs,the strategy adopted in this thesis is to introduce a second antibacteria mechanism,namely a DNA-binding mechanism.Thus,the new mechanism can work with the traditional membrane-disruption mechanism synergistically.The introduction of the second antibacterial mechanism may bring the following advantages:（1）the DNA-binding and membrane-disruption mechanisms can work at the same time to achieve dual-targeting antimicrobial effect,and it is more difficult for bacteria to generate multiple mutations to counter more than one antimicrobial mechanism and produce drug resistance;（2）The increase in molecular weight of the oligomers after polymerization could make them unable to enter eukaryotic nucleus,while they can still bind naked bacterial DNA in the nucleoid after entering the bacteria.Thus,the introduction of this selective antibacterial mechanism can potentially be a solution to the eukaryotic toxicity problem of AMPs.Based on the above considerations,a series of imidate ester hydrochloride monomers was prepared and polymerized with different diamines to generate polyamidine-type main-chain cationic antibacterial oligomers.These oligomers have controllable molecular weights（2-4 kDa）,low dispersity,easy-to-follow preparation protocols and excellent antibacterial effect.Based on above synthesis system,studies were carried out as follows:（1）Ten oligomers（YB series）containing benzamidine group were synthesized using diethyl p-phenyldiimidates hydrochloride monomer and various diamines.The oligomers’structures and molecular weights were characterized.The antibacterial and hemolytic activity of YB series oligomers were evaluated,and YB10 as the one with the best performance was selected for further study.The bactericidal ability and killing-kinetics of YB10 were tested.（2）Dual antibacterial mechanisms of YB10,namely bacterial cell membrane disruption and DNA binding,were verified using PI staining and testing particle size experiments.Bacteremia and septicemia models were used to evaluate the bactericidal effect and biocompatibility of YB10 at the cellular level.Mice sepsis model and epidermal infection model were established to evaluate the bactericidal effect of YB10 in vivo.（3）Based on the structure of DAPI,a commonly used nucleic acid dye,four different imidate esters with DAPI-like structures were designed and synthesized.Using these imidate esters and five kinds of diamines,twenty antibacterial oligomers（PAPI series）with polyvalent DNA-binding structures were synthesized.The antibacterial activity of these PAPI oligomers were screened using the clinically important"ESKAPE"strains and M.smegmatis,a model for Mycobacterium tuberculosis.The oligomers in the PAPI1 and P PAI2 series with more hydrophobic segments showed better overall performance and were further tested towards several pathogenic mycobacteria strains.B ased on all the evaluation results mentioned above,PAPI1-2 with highest therapeutic index was selected for further studies.（4）The dual-selective antibacterial mechanism of PAPI1-2 was verified by us firstly.Then intracellular infection models of RAW264.7 cells were eatablised using M.smegmatis and M.fortuitum as the model mycobacteria to verify cellularlly biocidal activity of PAPI1-2.To further evaluate the antimicrobial effectiveness of PAPI1-2 in vivo,infected zebrafish model using Methicillin-resistant S.aureus（MRSA）and M.fortuitum as pathogens,as well as mice tail infection model using M.marinum as the pathogen were constructed.In the fish model studies,PAPI1-2 was found to significantly improve the survival rate of zebrafish compared with the control group.In the mice tail model,the oligomer also showed excellent effectiveness in infection control and inhibition of open lesion formation. |
