Final Report Summary - PEPTIDEBASEDHYDROGEL (Toward the development of peptide-based hydrogels for tissue regeneration applications with inherent antibacterial activity)
Bacterial infections are a common problem associated with dermal wounds as well as implanted materials. These infections can prolong or impair wound healing, contributing to tissue morbidity and in extreme cases, result in sepsis. With regard to biomaterial implants, infections at the tissue-implant interface can lead to implant failure. Antibacterial hydrogels can be used to coat implant surfaces or directly treat accessible wounds to prevent or kill existing infection. The basis of this project was the development and study of antibacterial hydrogels prepared from self-assembling peptides. This project aimed to provide a multidisciplinary approach to design, prepare and study inherently antibacterial gels capable of delivering mammalian cells to wound sites. The material and antibacterial properties of the gels made from self-assembly peptides can be modulated by making changes to the peptide's sequence. Structure-based peptide design, biophysical and materials properties analysis as well as mammalian and bacterial cell culturing were used to formulate peptide-based hydrogels that meets the desired requirements. Importantly, the mechanism of action that defines the antibacterial property of the gels was investigated.
The project started with the use of structure-based design to generate a family of novel gelating peptides. These peptides were designed to assemble into a fibrillar network where the surface chemistry of the fibrils can be controlled by simply modulating the amino acid composition of the peptide monomer used for self-assembly. A total of ten peptides were then synthesized and purified. To assess and characterise hydrogels formation, hydrogels were prepared from the purified peptides. The peptides secondary structure as well as kinetics of peptides folding and self-assembly were analysed by circular dichroism. Oscillatory rheology was used to study the hydrogels mechanical properties. The peptides prepared were shown to fold into an amphiphilic ?-hairpin conformation, containing distinct hydrophobic and cationic faces, that rapidly self-assembles into a network of fibrils that define self-supporting, rigid hydrogels. The cationic face of each folded peptide in every fibril remains exposed to solvent and serves as a potential warhead for bacterial engagement. Since the gel is composed of individual self-assembling peptides, structure-activity relationships were used to relate amino acid composition to material properties. Selected hydrogels were tested for cytotoxicity towards mammalian cells and the ability to encapsulate cells homogenously. It was shown that the gels are cytocompatible towards mammalian cells. Additionally it was shown that the gels with a fast kinetics of gel formation have the ability to encapsulate cells homogenously. The hydrogels surface was also tested for the antibacterial activity against both gram-positive and gram-negative bacteria. The antibacterial activity results show that the arginine-containing peptide hydrogels were the most effective at killing both gram-positive and gram-negative bacteria. Together with the rheological properties, the results show that these gels are the ones displaying the best material and biological properties. These studies culminated in an optimised gel, composed of a peptide containing six arginine residues, PEP6R. PEP6R gels demonstrate high potency against bacteria but are cytocompatible towards human erythrocytes as well as mammalian mesenchymal stem cells. The mechanism of action of these hydrogels against E. coli, P. aeruginosa, and S. aureus was investigated using PEP6R as a representative gel. The mechanistic studies of the gels suggest an antibacterial mode of action that involves membrane disruption initiated by the dissociation of essential divalent metal ions from the bacterium's cell wall as it come in contact with the material's surface.
The hydrogels developed in this work have relevance to the wound healing and tissue regeneration fields. Hydrogel materials with antibacterial activity can be used to coat implant surfaces or directly treat accessible wounds to prevent or kill existing infection. This way the use of these gels can significantly decrease the occurrence of wound-and biomaterial-centered infections. In the gels here described and developed no added antibacterial agents are necessary, since the nanostructure of the gels, itself, is the active agent. This study also contributes to a more in-depth understanding of how peptide composition influences the self-assembled material properties. We show the ease by which the bulk properties of a self-assembled material can be modulated by making simple structural changes of its monomeric building block. Additionally, this study also makes a significant contribution on the understanding of the mechanism by which material surfaces exert antibacterial action
The project started with the use of structure-based design to generate a family of novel gelating peptides. These peptides were designed to assemble into a fibrillar network where the surface chemistry of the fibrils can be controlled by simply modulating the amino acid composition of the peptide monomer used for self-assembly. A total of ten peptides were then synthesized and purified. To assess and characterise hydrogels formation, hydrogels were prepared from the purified peptides. The peptides secondary structure as well as kinetics of peptides folding and self-assembly were analysed by circular dichroism. Oscillatory rheology was used to study the hydrogels mechanical properties. The peptides prepared were shown to fold into an amphiphilic ?-hairpin conformation, containing distinct hydrophobic and cationic faces, that rapidly self-assembles into a network of fibrils that define self-supporting, rigid hydrogels. The cationic face of each folded peptide in every fibril remains exposed to solvent and serves as a potential warhead for bacterial engagement. Since the gel is composed of individual self-assembling peptides, structure-activity relationships were used to relate amino acid composition to material properties. Selected hydrogels were tested for cytotoxicity towards mammalian cells and the ability to encapsulate cells homogenously. It was shown that the gels are cytocompatible towards mammalian cells. Additionally it was shown that the gels with a fast kinetics of gel formation have the ability to encapsulate cells homogenously. The hydrogels surface was also tested for the antibacterial activity against both gram-positive and gram-negative bacteria. The antibacterial activity results show that the arginine-containing peptide hydrogels were the most effective at killing both gram-positive and gram-negative bacteria. Together with the rheological properties, the results show that these gels are the ones displaying the best material and biological properties. These studies culminated in an optimised gel, composed of a peptide containing six arginine residues, PEP6R. PEP6R gels demonstrate high potency against bacteria but are cytocompatible towards human erythrocytes as well as mammalian mesenchymal stem cells. The mechanism of action of these hydrogels against E. coli, P. aeruginosa, and S. aureus was investigated using PEP6R as a representative gel. The mechanistic studies of the gels suggest an antibacterial mode of action that involves membrane disruption initiated by the dissociation of essential divalent metal ions from the bacterium's cell wall as it come in contact with the material's surface.
The hydrogels developed in this work have relevance to the wound healing and tissue regeneration fields. Hydrogel materials with antibacterial activity can be used to coat implant surfaces or directly treat accessible wounds to prevent or kill existing infection. This way the use of these gels can significantly decrease the occurrence of wound-and biomaterial-centered infections. In the gels here described and developed no added antibacterial agents are necessary, since the nanostructure of the gels, itself, is the active agent. This study also contributes to a more in-depth understanding of how peptide composition influences the self-assembled material properties. We show the ease by which the bulk properties of a self-assembled material can be modulated by making simple structural changes of its monomeric building block. Additionally, this study also makes a significant contribution on the understanding of the mechanism by which material surfaces exert antibacterial action