Modulating the self-assembly, gelation and antimicrobial properties of β-sheet forming ultra-short de novo ionic-complementary peptides via charge distribution Mohamed Ahmed Naseef Soliman 1,2 , Abdulwahhab Khedr 1,3 , Mohamed A. Elsawy 1 1 De Montfort University, UK, 2 Cairo University, Egypt, 3 Zagazig University, Egypt Bacterial infections are imposing substantial danger to the world owing to antibiotic resistance. So, we aimed to develop antimicrobial biomaterials using ultrashort sequences of ionic self-complementary peptides (4 – 5 amino acids) that self-assembled into β-sheet nanofibers upon pH adjustment, then formed 3D networks of hydrogels. These short peptides are safe, biocompatible, and easily tailored for scaling up and can be easily synthesized at low cost showing antimicrobial activity that overcome the antibiotic resistance via multi-mechanisms of bacterial killing. Phg4 peptide, previously published by Elsawy and coworkers [1] , and another two Phg4 derivatives (EPhg4 and KPhg4) designed based on varying the charge distribution and type of amino acid were investigated. The reason behind that was to explore how these modifications would affect the self-assembly of peptides into stable β-sheet fibers in aqueous medium and formation of a hydrogel and whether such modifications could impart inherent antibacterial properties for the hydrogel. All candidates succeeded to self-assemble into higher molecular structures of β-sheets and form hydrogel. Their pH titration and theoretical net charge calculations indicated that upon adjusting the medium pH, the peptide is almost neutral or slightly charged; the electrostatic repulsive force between charged sequences is greatly reduced allowing peptide chains to interact through intermolecular non- covalent interactions such as hydrogen bonding and π-π stacking forming secondary structure. The inverted vial test at pH 4.5 – 5 showed hydrogel formation at a critical gelation concentration (CGC) of 10 mg/mL for EPhg4, 20 mg/mL for Phg4 and 50 mg/mL for KPhg4. Moreover, FTIR and SEM results confirmed the self-assembly of these peptides into β-sheet arrangements and then into nanofibers. The rheological evaluations of hydrogels revealed their viscoelastic properties recording G’ 31, 106 and 111 KPa for Phg4, EPhg4 and KPhg4 at 50 mg/ mL, respectively, with thixotropic behaviour indicating their ability to be injected or sprayed into targeted area followed by structure reformation after relaxation. These selfassembled peptide-based hydrogels showed in vitro bactericidal activity towards P. aeruginosa MPAO1 planktonic population (10 5 CFU/mL) in 2h and inhibited P. aeruginosa biofilm formation without any inhibitory effect against MRSA NCTC 10442 and this result was validated by CLSM. Therefore, these ultrashort ionic complementary peptides could be beneficial to achieve cost- effective, versatile, and soft nanosystems based on stable self-assembly to be used alone or after drug loading for topical infections and tackling the antibiotic resistance. References 1. Wychowaniec, J et al., Biomacromolecules 2020,21:2670.
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