| Antimicrobial peptides(AMPs)have been characterized and isolated from a wide range of animal,plant and bacterial species and are known to play important roles in the innate immune defense system of all species.Despite the diversity in their amino acid sequences,AMPs are defined as short(10–50 amino acid residues)peptides possessing a large percentage(?30%)of hydrophobic amino acids and an overall positive charge(in general,+2 to +9).These properties permit the AMPs to fold into amphipathic conformations upon contact with the cell membranes,and bind to the cell membrane via electrostatic interaction between the positively charged hydrophilic face of peptides and the negatively charged head groups of phospholipids.Then,the hydrophobic face of the peptides facilitates insertion into the membrane through hydrophobic interactions,causing increased permeability and loss of barrier function of the target cell membrane.However,therapeutic applications of these AMPs have been impeded by their cytotoxicity.To resolve this problem,A number of structure-function relationship of both natural and synthetic AMPs have been studied so as to find the strategies to improve the bactericidal activity of AMPs against the pathogen of interest and reduce cytotoxicity at the therapeutic dosages.AMPs are commonly used to improve cationic polymer-based gene carriers mediated transfection efficiency by increasing their endosomal release.However,their applications have been impeded by their cytotoxicity in natural condition.It is necessary to increase the membrane lytic activity of AMPs in acidic condition for enhancing cationic polymer-mediated transfection efficiency.Hence,the aim of this study were increasing the bactericidal activity of AMPs against the pathogen of interest while reduce cytotoxicity and improving the membrane lytic activity of AMPs in acidic condition by amino acid substitution on natural AMPs.1.Effects of positively charged residues on the biological viability of the antimicrobial peptides.Firstly,the selective antibacterial mechanism of RV-23 was explored by comparing it to melittin and AR-23,especially the effects of the number of positively charged residues on its structural parameters and its binding and lytic activity toward the membranes of prokaryotes and eukaryotes cells.For this purpose,structural parameters such as helicity,hydrophobicity,and amphipathicity of RV-23,melittin and AR-23 were compared.The interaction of the peptides with bacterial and mammalian cells,the antibacterial activity and the cytotoxicity of the peptides were also determined.For biomedical applications,the safety of a peptide is the most important issue in vivo.Hence,the hemocompatibility of the peptides and the interactions of the peptides with the whole blood were investigated,specifically their effects on the key blood components such as erythrocyte,platelet and blood coagulation.Secondly,a series of AR-23 analogues with increased charge were designed by substitution of Ala1,Ala8 and Ile17 with positively charged residues(Arg or Lys)to study the effect of positively charged residue amount and distribution on the biological activity of the AMPs.The secondary structure of the peptides was characterized in water and membrane-mimicking environments(30?M SDS and 50% TFE,respectively).The antimicrobial activity and the anti-biofilm activity of peptides were measured.The hemolytic activity of peptides and their cytotoxicity to L929 cells were also determined.Confocal microscopy and flow cytometry were employed to explore the potential membrane destruction mechanisms of the peptides.The results showed that the all three peptides exhibited excellent lytic activity against Staphylococcus aureus and Escherichia coli,while RV-23 showing the highest potency.Moreover,RV-23 showed lower toxicity to eukaryotic cells than melittin or AR-23 at their MICs.In addition,CD experiments showed that the three peptides had typical ?-helical structure,and RV-23 possessed the lowest ?-helix content.Besides,the structural information showed that RV-23 has the lowest hydrophobicity of the three peptides.Because hydrophobicity and ?-helicity were believed to correlate with hemolysis,the results indicated that the selective bactericidal activity of RV-23 may due to its low hydrophobicity and ?-helicity,which lead to low cytotoxicity without affecting antibacterial activity.Furthermore,RV-23 did not affect the function of blood components such as red blood cells,platelets,and the blood coagulation system.The position rather than the amount of positively charged residues affects the biophysical properties and selectivity of AMPs.Substitution of Ile17 on the hydrophobic face with positively charged Lys dramatically altered the hydrophobicity,amphipathicity,helicity and the membrane-penetrating activity against eukaryocyte cells of the peptide.However,substitution on the hydrophilic face only slightly affected the peptide biophysical properties and biological activity.Substitution of Ile17 on the hydrophobic face with positively charged Lys dramatically decreased the cytotoxicity of the peptide,while slightly altered the antimicrobial activity of peptide,thus,this substitution could improve the selective toxicity of the peptide.The results denoted that the position rather than the amount of positively charged residue affects the biophysical properties and selectivity of the peptide.Of all the analogues,A8,a peptide with Ala1-Arg,Ala8-Arg and Ile17-Lys substitutions,exhibited similar bactericidal activity and anti-biofilm activity to AR-23 and much lower cytotoxicity against mammalian cells compared with AR-23.2.Effects of charge on the membrane lytic activity of the antimicrobial peptides and its enhancement ability on cationic polymer-mediated transfection efficiency.In this section,pH-sensitive peptides were designed by replacing the positively charged residues with glutamic acid residues in melittin and RV-23.To explore the molecular basis of their pH-sensitive cellular toxicity,we studied the membrane lytic activity of the derived peptides by hemolytic tests and the calcein release assays.The effects of peptides on the physicochemical characteristics of PEI/DNA polyplexes were also determined.The cellular uptake efficiency of PEI/ODN polyplexes was evaluated to determine the lytic ability on endosomal membrane of peptides.The transfection efficiency of nanoparticles containing PEI/DNA and the peptides was studied and compared with the transfection efficiency of the commercial reagent Lipofectamine 2000 in different cell lines.Furthermore,the positively charged residues of AR-23 were systematically replaced with glutamic acid residues to study the number of net charge of peptide on its pH-sensitive lytic activity.Hemolytic tests and the calcein release assays were conducted to evaluate the pH-sensitive membrane lytic activity of peptides.The transfection efficiency and cytotoxicity of nanoparticles containing PEI/DNA and the peptides were also studied.The acidic peptides(glu-replaced peptides)showed pH-sensitive lytic activity.The hemolytic activity of acidic peptides at pH 5.0 was higher than that at pH 7.4.The incorporation of acidic peptides did not affect the DNA binding ability of PEI but affected the physicochemical characteristics of the PEI/DNA polyplexes.Acidic peptides promoted FITC-ODN release from endosomes and transferred into the cytoplasm and nuclei,suggesting that the acidic peptides exhibited strong endosomal lytic activity,which may be beneficial for gene transfection.The incorporation of acidic peptides into PEI/DNA polyplexes enhanced the PEI-mediated transfection efficiency corresponding to up to 42-fold higher luciferase activity compared to that of PEI alone.Besides,the hemolytic tests and the calcein release assays indicated that analogue of AR-23 with two or three glutamic acid residues substituted had higher membrane lytic activity at acidic pH 5.0.Analogue of AR-23 with three glutamic acid residues substituted could improve PLL-mediated transfection,which was higher than that of chloroquine.Besides,analogue of AR-23 produced lower cytotoxicity than that of chloroquine.In conclusion.The position rather than the amount of positively charged residues affects the biophysical properties and selectivity of.AMPs with improved the bactericidal activity against the pathogen of interest and reduced cytotoxicity at the therapeutic dosages were designed through positively charged residue substitution,which was benefit for the its clinical application.The replacement of positively charged residues with glutamic acid residues in the AMP sequence improved its lytic ability at acidic pH condition while and decreased its cytotoxicity at neutral conditions.Acidic peptides showed higher endosomolytic activity and greatly improved the gene transfection efficiency of cationic polymer-based gene delivery vectors while maintaining low cytotoxicity. |
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