Synthesis And Antibacterial Activities Of Branched Lysine-Based Copolymers

Antibiotic resistance is spreading globally and poses a significant threat to human health.Finding new classes of antibiotics is one of the key strategies to combat antibiotic resistance.Unlike antibiotics that act on specific targets,antimicrobial peptides kill the pathogens by non-specifically breaking the membrane,making it difficult to develop resistance.Polylysine,including ε-polylysine(ε-PL),α-polylysine(α-PL)and hyperbranched polylysine(HPL),have attracted attention as antimicrobial peptide mimics due to their broad-spectrum antimicrobial activity,yet they also pose various problems.ε-polylysine relies on fermentation for production and chemical synthesis is difficult and new polylysine products need to be developed.α-polylysine has poor antifungal activities and needs to be modified to improve its antifungal activities.Hyperbranched polylysine leads to toxicity due to its high charge density,which needs to be reduced by modifying it.This dissertation focuses on the improvement of several thorny issues of polylysine antimicrobial agents,including toxicity,synthesis difficulties,and antifungal activities.Hyperbranched polylysine has excellent antibacterial activity against Grampositive and Gram-negative bacteria,however,its application may be limited due to its cytotoxicity.The effect on the antimicrobial activities and cytotoxicity of hyperbranched polylysine was investigated by introducing hydrophobic amino acids.Alanine,tryptophan and phenylalanine are natural amino acids with gradually increasing hydrophobicity.we report a facile strategy to engineer the cytotoxicity of HPL by copolymerizing lysine(K)with a hydrophobic amino acid,e.g.alanine(A),tryptophan(W)or phenylalanine(F),to afford hyperbranched random copolymers.These copolymers have comparable antibacterial activities to HPL while their cytotoxicities and in vivo toxicities are lowered when the type and content of hydrophobic amino acid and the hydrodynamic diameter of copolymers are optimized.The G.mellonella infection model demonstrates that the copolymers are effective against the S.aureus infection in vivo.The copolymers kill the bacteria through the disruption of cell membranes and the bacteria do not develop resistance to the copolymers.We found that charge density has an important effect on both the antimicrobial activities and cytotoxicities of poly(amino acid)s and therefore considered reducing cytotoxicity by finding amino acids that can reduce charge density.6-aminohexanoic acid,an analogue of lysine,lacks the α-amino group compared to lysine,and copolymerization with lysine may reduce the charge density.The effect on the antimicrobial activities and toxicity of hyperbranched polylysine were investigated by introducing the unnatural amino acid 6-aminohexanoic acid.The hyperbranched random copolymer LACA was synthesized by thermal polymerization of lysine and aminohexanoic acid by one-pot method.By modulating the aminohexanoic acid content and particle size of the copolymer,copolymers with antimicrobial activities comparable to HPL and reduced cytotoxicity could be obtained.The membrane disruption mechanism of lysine-aminocaproic acid copolymers against E.coli and S.aureus was investigated by morphological analysis and content leakage assay.The antimicrobial activities of poly(amino acid)s have been investigated by copolymerizing lysine with other amino acids in Chapters 2 and 3.The copolymers have good antibacterial activities against bacteria,however,their antifungal activities are poor.The guanylated hyperbranched polylysine has excellent antimicrobial activity against fungi,therefore the effect on the antifungal activities was investigated by guanylation modification of α-PL.We synthesized four different molecular weight αpoly(L-lysine)(α-PLn,n=1-4)and further modified α-PLn to afford guanylated α-PLn(α-PLn-Gx,where x denotes the percentage of guanylation)with enhanced antifungal activities.α-PLn exhibited good antibacterial activities against E.coli and S.aureus with the MIC values of 24-96 μg/mL,but it did not show antifungal activities against C.albicans,C.parapsilosis,C.krusei and C.glabrata,except for C.tropicalis(MIC<3μg/mL).After guanylation,the antifungal activities of α-PL/n-Gx against C.albicans,C.krusei and C.parapsilosis were significantly improved when x≥80 and the MIC values of α-PL3-G100 were reduced by>32-fold,64-fold and>16-fold,respectively,compared to that of the corresponding α-PL3.The mechanistic studies demonstrated that α-PL3-G100 disrupted the integrity of cell membranes of C.albicans.