Antimicrobial Activity of β-Sheet Forming Ultra-Short De Novo Ionic-Complementary Peptides Towards Wound Infections

Bacterial resistance is currently compromising the antibiotics activity that saved millions of people in the last 8 decades¹. Recently, peptide-based hydrogels were introduced as soft, versatile, shear-thinning and thixotropic materials that can be sprayed for topical applications. Thanks to their biocompatibility, biodegradability and physicochemical tunability, peptide hydrogels have a great potential to be used as sprayable antimicrobial biomaterials for treatment of infected wounds. They might also overcome the antibiotic resistance through multi-mechanisms of bacterial killing. So, this work aimed to utilise ultra-short <i>de novo</i> ionic-complementary peptide sequences (4 – 5 amino acids) for the development of antimicrobial bionanomaterials based on the peptide self-assembly into β-sheet nanofibers and building networks of hydrogel in response to pH change.<br/>Based on this, Phg4 peptide, previously reported by our group² was compared to 2 other Phg4 derivatives (EPhg4 and KPhg4) to investigate the role of charge type and distribution on the self-assembly into β-sheet fibers and hydrogel and whether such modifications could impart antimicrobial activity for the hydrogel. Interestingly, the three peptides self-assembled into β-sheet fibrillar assemblies and entangled to form hydrogels in response to pH change, as revealed from molecular (FTIR) and mesoscopic (SEM and TEM) characterisation. Critical gelation concentration (CGC) was significantly affected by peptide modifications, where it decreased from 37 mM for Phg4 to 15 mM for EPhg4, while KPhg4 showed to have the highest CGC at 75 mM concentration. Oscillatory rheology revealed viscoelasticity of hydrogels formed by the three peptides and ability to recover from shear stress i.e. thixotropic materials implying their sprayability. Hydrogel stiffness varied for the three peptides, with a storage moduli G’ of 10 and 93 KPa for Phg4 and EPhg4 at 75 mM, respectively, however KPhg4 appeared as viscous weak gel that did not show linear viscoelasticity.<br/>To investigate the inherent antibacterial activity of the three developed hydrogels, time killing point assays were carried out against the 2 bacterial species commonly responsible for causing wound infections (Methicillin-resistant <i>S. aureus</i>, gram positive and <i>P. aeruginosa</i>, gram negative). Strikingly, all hydrogels exhibited <i>in vitro</i> bactericidal behaviour against planktonic <i>P. aeruginosa</i> <i>MPAO1</i> (10⁵ CFU/mL) within 2 hours (hrs) besides their ability to prevent <i>P. aeruginosa</i> biofilm formation in a concentration dependent killing manner where the higher the peptide concentration, the shorter the time needed for eradication that ranged from 2--8hrs. On the other hand, hydrogels of the three peptides did not show any antimicrobial activity against <i>MRSA NCTC 10442</i>, which was also confirmed by observation of bacterial cell morphology using SEM and confocal laser scanning microscopy (CLSM). Phg4-based hydrogel (92 mM) was then selected for further investigations and showed antibacterial activity towards both planktonic and surface population of four different clinical <i>P. aeruginosa</i> strains obtained from University Hospital Southampton NHS Foundation Trust in addition to other ESKAPE pathogens (<i>A. baumannii</i> 18X50606 and <i>E. coli</i> NCTC 8003, <i>K. Pneumoniae</i> ATCC 700831).<br/>In conclusion, these ultrashort constrained ionic complementary peptides based on their stable self-assembly could be considered as a promising approach for treating wound infections as well as tackling the bacterial resistance and might be used as biomimetic ECM scaffolds for tissue regeneration at the wound site.<br/>REFERENCES<br/>[1] Ventola, C. Lee. "The antibiotic resistance crisis: part 1: causes and threats." Pharmacy and therapeutics 40.4 (2015).<br/>[2] Wychowaniec, Jacek K., et al. "Aromatic stacking facilitated self-assembly of ultrashort ionic complementary peptide sequence: β-sheet nanofibers with remarkable gelation and interfacial properties." Biomacromolecules 21.7 (2020).