Broad-Spectrum Antibacterial Activity of Proteolytically Stable Self-Assembled αγ-Hybrid Peptide Gels

Abstract

Bacterial infections pose a serious threat to mankind, and there is immense interest in the design and development of self-assembled peptide gels using ultrashort peptides for antibacterial applications. The peptide gels containing natural amino acids suffer from poor stability against proteolytic enzymes. Therefore, there is a need to design and develop peptide gels with improved stability against proteolytic enzymes. In the present work, we report the synthesis and characterization of α/γ hybrid peptides Boc-D-Phe-γ-L-Phe-PEA (NH007) and Boc-L-Phe-γ-L-Phe-PEA (NH009) to improve the proteolytic stability. Both of the dipeptides were found to self-assemble into gels in aqueous DMSO (3-5% w/v), and the self-assembly process was studied using FTIR and CD, which indicated antiparallel β-sheet formation with random coils in NH007 gels and random or unordered conformation in NH009. The rheological studies indicated viscoelastic characteristics for both gels; the storage modulus ( G') for NH007 and NH009 gels (3% w/v) was estimated as 0.2 and 0.5 MPa, higher than the loss modulus ( G''). Also, both gels demonstrated self-healing characteristics for six consecutive cycles when subjected to varying strains of 0.1 and 30% (200 s each). The peptide gels were incubated with a mocktail of proteolytic enzymes, proteinase K, pepsin, and chymotrypsin, and stability was monitored using RP HPLC. Up to 23 and 40% degradation was observed for NH007 (3%, w/v) in 24 and 36 h, and 77 and 94% degradation was observed for NH009 (3%, w/v), within the same period. Thus α/γ hybrid peptide gels containing D-Phe exhibited higher stability than gels fabricated using L-Phe. The use of D-residue in α/γ hybrid peptide significantly enhanced the stability of peptides against proteolytic enzymes, as the stability data reported in this work are possibly the best in class. Both peptide gels exhibited broad-spectrum antibacterial activity against Gram-negative and Gram-positive bacteria, such as Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, and Staphylococcus aureus. The Pseudomonas aeruginosa and Staphylococcus aureus, in particular, are known to develop resistance. The NH007 (3%, w/v) demonstrated 65% inhibition, whereas NH009 (3%, w/v) showed 78% inhibition, with potent activity against Pseudomonas aeruginosa. Mechanistic studies, using SEM, HR-TEM, and bacterial live-dead assay, indicated entrapment of bacteria in gel networks, followed by interaction with cell membrane components and lysis. Cell viability (MTT assay) and toxicity (LDH assay) studies showed that both gels are not toxic to NIH 3T3 mouse embryonic fibroblast cells (mammalian). MTT assay showed >85% cell viability, and LDH assay exhibited not more than 15% cytotoxicity, even at higher concentrations (5%, w/v) and prolonged exposures (48 h). Overall, studies indicate the potential application of gels developed from the α/γ hybrid peptides in preventing biomaterial-related infections.

O‐Mannosylation Affords a Glycopeptide Hydrogel with Inherent Antibacterial Activities against E. coli via Multivalent Interactions between Lectins and Supramolecular Assemblies

“…In spite of their amphiphilic structures, AMPs typically cannot self‐assemble to form gel structures in water. By emulating the cationic and amphiphilic nature of AMPs, together with elegant molecular design, a number of peptides are capable of self‐assembling into mechanically rigid hydrogels that inhibit bacterial growth have been developed . In contrast to chemically cross‐linked hydrogels, self‐assembled hydrogels of peptides possessed numerous advantages, such as synthetic economy, high biocompatibility, inherent biodegradability, and unique shear‐thinning and recovery properties, which made them good candidates to prepare injectable antibacterial materials for site‐specific drug delivery or localized treatment of bacterial infection via syringes …”

Folding Behaviour and Antibacterial Activity of Ionic Complementary Peptide EAK-16

“…Ionic complementary amphiphilic peptides show broad-range antibacterial activity [19]. EAK-16 were found to have strong activity against Grampositive and Gram-negative bacteria, where it did not show the antibacterial activity preference over against Gram-positive and Gram-negative bacteria.…”

Biophysical approaches for exploring lipopeptide-lipid interactions

“…Through SEM it is possible to visualize the surface morphology of peptide gels. For example, Singh and co-workers [465] reported two a/g hybrid peptides Boc-D-Phe-g 4 -L-Phe-PEA (NH007) and Boc-L-Phe-g 4 -L-Phe-PEA (NH009) which can self-assemble into gels in DMSO solution. SEM images of dried gels of NH007 and NH009 exhibited porous morphologies (an interwoven morphology for NH007 and a flake-like network for NH009).…”