Controllable Self-assembly And Antibacterial Activity Of Short Peptides

Bacterial resistance has caused serious threat and great economic burden to the social public health and medical care system.It has become a major problem threatening the human health in the 21^st century.Existing antibiotics can hardly meet this demand,and we urgently needed a series of new antibiotics to solve this problem.Antimicrobial peptides（AMPs）are the first line of life defense of many organisms and play a key role in the antibacterial infections.Most of the natural and artificial AMPs in the past were long-chain peptides,which were not advantageous in terms of production cost.Short peptide is a kind of molecule,which is easy to synthesize,modify and have a good biocompatibility.But the short peptide faces the disadvantage of insufficient antibacterial activity.Therefore,we should spare no effort to improve the antibacterial efficiency of short peptides,in order to develop a practical application material.In recent years,researches have shown that the self-assembly can effectively improve the antibacterial efficiency of cationic short peptides,which brings opportunity for the short peptide.However,how to design more efficient,broad spectrum and good biocompatible antibacterial materials is still a major challenge for researchers.In this dissertation,a series of cationic antibacterial peptides were obtained by optimizing the sequence of short peptides,and the effects of interaction forces during self-assembly on the antimicrobial activity and biocompatibility were investigated.Based on the excellent photo-responsive characteristics of azobenzene,we constructed a short peptide assembly with unchanged morphology,particle size and surface potential after UV irradiation.However,the antibacterial activity was significantly improved after irradiation,which realized the environment-friendly antibacterial concept of self-inactivation in the natural environment.The detailed research contents are as follows:First,self-assembly has been identified as an innovative strategy for improving the antimicrobial efficacy and bioavailability of short peptides.We construct two constitutional isomeric peptides,which contained the same serine,alkaline,and phenylalanine residues but in a different order.Transmission electron microscopy（TEM）,cryogenic transmission electron microscopy（cryo-TEM）and atomic force microscopy（AFM）revealed that the constitutional isomers self-assembled into different morphologies in an aqueous solution.The sequence with alkaline residues located at both termini of the peptide favored the formation ofβ-sheet conformation and nanofibers,while irregular nanospheres were observed when positioning the alkaline residues at the center of the isomeric peptide.Theζ-potential measurements showed that the nanofibers had a net potential of+17.40 m V,whereas the apparent potential of nanospheres dropped steeply to+1.00 m V.These differences of the constitutional isomeric peptides were directly reflected in their antimicrobial activities.In comparison with the nanofibers,the constitutional isomer nanospheres exhibited much higher antimicrobial efficacy against Staphylococcus aureus,Bacillus subtilis,Escherichia coli,and Pseudomonas aeruginosa.Moreover,several pairs of constitutional isomeric peptides with a similar sequence layout yielded the same outcome.These collective results not only highlight the importance of the isomeric sequence on the antimicrobial efficacy of short peptides but also increase further potential in optimizing the design of self-assembled nano-antimicrobial peptides.Second,according to the results of the first chapter,we replaced serine（S）with asparagine（N）and glutamine（Q）in the peptide sequence to make it easier to self-assemble into aβ-sheet structure.Circular dichroism（CD）spectroscopy,thioflavin T（Th T）titration and fourier transform infrared（FTIR）spectroscopy showed that the substituted peptides with cationic amino acids at both ends showed theβ-sheet secondary structure in aqueous solution.TEM and zeta potential results showed that the assembled nanofibers have the similar surface potential,which stimulated us to study their antibacterial activity.Surprisingly,the short peptides easily assembled intoβ-sheet secondary structures demonstrated relatively low antimicrobial activity.We suspect that this may be due to the over-tight accumulation of peptide chains,which resulted in the hydrophobic residues being deeply hidden in the molecular layer and largely limited the ability to destroy the cell membrane.The NMR results show that Mn²⁺can effectively influence the signal peak of short peptide.It was found that the benzene signal peak in the substituted short peptide needed more Mn²⁺to shield it,indicating that the benzene ring did form a relatively tight accumulation in the self-assemblies.We also learned that,reducing the hydrogen bond in the short peptide assembly by urea or replacing Asn with Ser can improve the antibacterial activity of the assembly.Unilaterally reducing the hydrophobicity of the peptide sequence would destroy the balance of multiple forces in the assemblies,resulting in random coil spheres and low antibacterial activity,suggesting that the balance of self-assembly forces should be considered during the optimization of short peptide sequences.It is worth noting that,the dense accumulated assembly exhibited low cytotoxicity and low hemolysis rates in a wide range of concentrations（1050μM）,indicating that the over-tight accumulation can greatly improve the biocompatibility of peptides.Third,we have known that the assemblies can also show toxic to normal cells at the antibacterial times.In this chapter,we hoped to obtain a stimulus-responsive antimicrobial peptide to effectively regulate the balance of activity and toxicity in the antimicrobial system.Light is an ideal means of non-invasive,non-polluting device with adjustable intensity and position.We designed and synthesized a short peptide with azobenzene group in the side chain and self-assembled in aqueous solution by utilizing hydrophobic,hydrogen bond and electrostatic force.TEM,cryo-TEM and zeta potential results indicated that the peptide can self-assembled into micrometer-length fibers with high surface potentials（+52 m V）.AFM further indicated that the resulting nanofibers was represented the bilayer stacks of the peptides,which extended along the direction of the long axis of nanofibers.The peptide was photo-responsive in aqueous solution,allowing a reversible transition between trans and cis conformations in a relatively short period of time.Moreover,no obvious light fatigue was found during the multiple repetitions.Since the azobenzene group is located in the side chain of the short peptide sequence,its own conformational transition during UV irradiation could hardly disrupt the balance of multiple forces between the short peptide main chains,resulting in no significant changes in the morphology,surface potential and particle size of the short peptide assemblies before and after light irradiation.However,light could greatly affect the antibacterial activity of the assemblies.In the antibacterial experiments,the UV light irradiated assemblies can increase the antibacterial activity by four times with the same antibacterial mechanism of nature antimicrobial peptides,achieving the goal of light-regulated antibacterial activity of short peptides.In addition,a series of other short peptides with side chains containing azobenzene groups also showed the same results.Due to the higher surface potential and the controllable accumulation of hydrophobic residues,the short peptide showed good selectivity to normal cells before and after irradiation in the range of 1.50 times the minimum inhibitory concentration,indicating that this peptide has higher potential application values.In conclusion,we made full use of the peptide self-assembly,and optimized the sequence design of antibacterial short peptides by investigating the location and type of amino acid residues in peptide sequence.And we have deeply analyzed the mechanism and driving force of peptide assemblies during the antimicrobial activity.Finally,we constructed an environmentally friendly antimicrobial peptide with UV-activated antibacterial activity,which realized the concept of self-inactivation under nature environmental conditions.It is envisioned that this design principle is expected to solve the development of antibiotic resistance and provide a new idea for designing the new antibiotics.