Synthesis And Antimicrobial Activities Of Hyperbranched Polylysine And Its Derivatives

Drug-resistant microbial infections have become a global problem and pose a severe threat to the public health.Compared with small molecule antibiotics,polymeric antimicrobials kill the pathogens via a membrane disruption mechanism with a low propensity for developing resistance and are expected to become the next-generation of antimicrobial agents.Although polymeric antimicrobials have been extensively studied,there are few in clinical practice.It is still challenging to design polymeric antimicrobials with broad-spectrum antimicrobial activities and biocompatibility.Poly(amino acid)s are one of the most promising antimicrobial polymers due to their good biodegradability and biocompatibility.Starting from lysine,four different types of polylysines,e.g.α-polylysine(α-PL),ε-polylysine(ε-PL),dendritic polylysine(DPL)and hyperbranched polylysine(HPL),can be obtained by different polymerization techniques.Although HPL has a less regular structure than the other three counterparts,it has still attracted more and more attention in recent years because of the simplest synthesis process,lower cytotoxicity and high gene transfection efficiency.So far,there has no report on antimicrobial activities of HPLs.This thesis focuses on the synthesis,modification and antimicrobial activities of HPLs.The main contents and conclusions are summarized as follows:1)HPL was synthesized by the "one-pot" method and six fractions(HPL1-6)with number average molecular weights(Mn)from 2800 to 12000 g/mol were obtained by fractionation through dialysis.The effect of the molecular weight(MW)of HPLs on their antibacterial activities and toxicities was studied.The antibacterial activities of HPL 1-6 were evaluated by the broth microdilution method,and the results showed that higher MW HPLs,e.g.HPL3-6,had better antibacterial activities,and the MIC values for both Gram-negative and Gram-positive bacteria were less than 50 μg/mL.The MTT assay showed that the cytotoxicity of HPLs increased with the increase of MWs.The resistance assay results demonstrated that S.aureus and E.coli did not develop resistance to HPL3 while bacteria were exposed to HPL3 at 1/8 MIC for 12 passages.The live/dead bacteria viability assay,membrane integrity study and morphological characterization were performed and these results demonstrated that HPL3 killed bacteria through the disruption of cell membranes.In addition,in vivo experiments showed that HPL3 was non-irritating to the mice skin and had low systemic toxicities to organs.The acute oral toxicity test showed that the median lethal dose(LD50)of HPL3 was higher than 5000 mg/kg.2)Guanylation was performed on HPLs to regulate their antibacterial activities and toxicities.For HPL1-6,all amino groups were completely converted into guanidinium groups to obtain HPL1-6-G100,while partial guanylation of amino groups was performed on HPL3 to obtain HPL3-Gx,where x represents the percentage of guanidinium groups.For HPL1-6-G100 series,the higher MW polymres had higher antibacterial activities.For HPL3-Gx series,the antibacterial activities depended on x and HPL3-Gx with x=40-60 showed the highest antibacterial activities.In vitro hemolysis assay and the MTT test showed that guanylated HPLs had very low hemolytic activity and low cytotoxicity.The resistance assay results showed that S.aureus and E.coli did not develop drug resistance to HPL3-G60 and HPL3-G100 when being exposed to these two polymers at 1/8 MIC for 12 passages.The live/dead bacteria viability assay,membrane integrity study and morphological characterization proved that HPL3-G60 and HPL3-G100 kill bacteria through the cell membrane disruption mechanism.In vivo experiments showed that HPL3-G60 and HPL3-G100 were non-irritating to the skin of mice and had low systemic toxicity to organs.The acute oral toxicity test showed that the LD50 of HPL3-G60 and HPL3-G100 were higher than 5000 mg/kg.In addition,HPL3,HPL3-G60 and HPL3-G100 have comparable in vivo efficacy to vancomycin in the MRS A-infected mouse peritonitis model.3)The antifungal activities of HPL1-6,HPL1-6-G100 and HPL3-G20-80 were systematically studied by the broth microdilution method.The antifungal activities of HPL1-6 showed strong dependence on MWs and the trend was in contrary to the antibacterial activities,i.e.lower MW HPLs had better antifungal activities.For HPL1-6-6100,the antifungal activities gradually increased with the increase of MWs.The antifungal activities of HPL3-Gxs against four Candidas and A.niger were vastly enhanced compared to the starting HPL3,and were dependent on the degree of guanylation.HPL3-Gxs with x=40-60 were more potent for all tested fungi.The resistance assay results showed that C.albicans did not develop drug resistance to HPL3,HPL3-G60 and HPL3-G100 when it was exposed to these polymer solutions at 1/8 MIC for 12 passages.The fungal staining assay,membrane integrity study and morphological characterization demonstrated that HPL3,HPL3-G100 and HPL3-G60 kill fungi through the cell membrane disruption mechanism.In addition,HPL1-6,HPL1-6-G100 and HPL3-G20-80 had the same MIC values for both clinically isolated multidrug resistant(MDR)C.albicans and the drug-sensitive C.albicans,indicating that HPLs and its guanylated derivatives could be potentially used to combat the growing threat of MDR fungi.4)A simple pH-responsive reversible amino group-shielding strategy was designed to reduce the toxicity of high MW HPLs.5-Hydroxymethylfurfural(HMF)was used to react with HPL5 to obtain HPL5-HMFx(x represents the grafting percentage of HMF)in which the aldehyde group of HMF and the amino group of HPL5 form the Schiff bases.Under physiological pH conditions,HMF and HPL5 were linked by the imine bonds,which could shield the positive charge of amino groups.In the slightly acidic environment where bacteria multiply,the imine bonds are cleavable,exposing the amino groups of HPL5.The antibacterial assay results showed that HPL5-HMF5 and HPL5-HMF10 had the same MIC value as unmodified HPL5 and the antibacterial activities were not reduced,but the cytotoxicities of HPL5-HMFx were significantly reduced and the IC50 values gradually increased from 78 μg/mL for HPL5 to 140 μg/mL for HPL5-HMF10.In addition,HPL5-HMF5 and HPL5-HMF10 showed negligible hemolytic activity up to 1500 μg/mL and the hemolytic percentage of red blood cells was less than 2%.5)A water-insoluble PL-DOSS complex was prepared by the electrostatic interaction between ε-polylysine(ε-PL)and docusate sodium(DOSS),and the antimicrobial TPU/PL-DOSS composite films(TPU-x,where x is the percentage of PL-DOSS in the composite film,w/w%)were prepared through blending the medical grade thermoplastic polyurethane(TPU)and the PL-DOSS complex.>99%reduction of colony forming unit(CFU)was obtained in TPU-x composite films even at relatively low content of PL-DOSS,e.g.0.13%for Methicillin resistant S.aureus(MRSA)and 0.5%for E.coli.The excellent antibacterial activities were attributed to the formation of PL-DOSS nanoparticles that provided high concentration of antibacterial agents upon contact with the bacterial membrane.TPU-0.5 composite films showed long-term stability and their antibacterial activities against MRS A did not change even after they were stored in saline for 21 days.Compared to neat TPU,the thermal and mechanical properties of TPU-x composites were only slightly changed.In addition,TPU-x composite films exhibited low hemolytic activity and cytotoxicity.In conclusion,we hope that this study can provide new ideas and new strategies for the design and development of branched antimicrobial polymers,and provide theoretical basis for further clinical applications.