Self-assembled, peptide-based scaffolds for wound-healing and bone regeneration

Abstract

Molecular self-assembly is a process of bottom-up development in which molecular building blocks interact at the nanoscale level upwards via non-covalent interactions to allow ordering into highly organized supramolecular materials with desired functionalities. The non-covalent interactions involved are hydrogen bonding, van der Waal's forces, electrostatic, and hydrophobic interactions. At the molecular level, biomolecules like DNA, RNA, phospholipids, polysaccharides, proteins, and peptides can spontaneously form self-assembling structures. However, self-assembling peptide-based systems have emerged as the most prevalent due to the significant advantages over the other polymeric biomaterials. They are easy to synthesize with tailored functional groups, scalable, and mimic the natural nanofibrous morphology of in vivo extracellular matrix (ECM). While the polymeric biomaterials generate an immunogenic response, the biodegradation products of peptides, amino acids, are in general, non-toxic to the surrounding cells and tissues. As the structural and functional domains of naturally occurring proteins, peptides have been widely used to fabricate biomaterials to promote wound healing and bone regeneration. By introducing different amino acids, peptides can be self-assembled into nanostructures like vesicles, micelles, rods, ribbons, tapes, tubes, and nanofibers under the given physicochemical conditions, which can further be utilized for different biomedical applications. The majority of existing peptide-based biomaterials either involve the utilization of growth factors or drugs for their therapeutic responses. However, the erratic release of the drugs, stability of growth factors, and immunogenic responses limit their clinical translation. In this thesis, we have addressed these challenges by developing growth factor and drug-free peptide-based nanoarchitectures adorned with bioactive functional groups to promote chronic wound healing and bone regeneration. These scaffolds address the existing knowledge gaps in the field and provide a novel approach to promote tissue regeneration. The thesis has been organized into five chapters. Chapter 1 introduces the self-assembled peptide scaffolds, with emphasis on their significance in wound healing and bone regeneration. An overview is provided on the peptide self-assembly, interaction involved, factors affecting the process of self-assembly, nanostructure formation along with their biomedical applications in wound healing and bone regeneration. The role of extracellular matrix (ECM) and the need for biomaterials mimicking native extracellular matrices is also explained. The chapter also presents comprehensive literature survey, knowledge gap/problem definition, thesis objectives and scope, and organization of thesis. Chronic wounds are a major healthcare burden worldwide, seriously affecting the life quality of patients. The pathophysiological mechanisms of chronic wounds are complex and, therefore, multipronged approaches that address several different biological mechanisms are desirable. Conventional bandages, gauzes, and hydrogels merely provides a physical barrier and absorb the wound exudates keeping the moist environment. However, they overlook the underlying complex cellular mechanisms in chronic wounds. Therefore, in chapter 2, we have developed a multifunctional, nanofibrous lauric acid-peptide conjugate gel incorporating bioactive Y2O3 nanoparticles targeting various aspects of chronic wound. The gel exhibited potent ROS scavenging and bactericidal properties against E. coli and S. aureus, the prevalent strains at the wound site. The material was cytocompatible and provided a matrix for cell migration and proliferation, thus, resulting in efficient wound healing. The Y2O3-loaded gel also exhibited the angiogenic properties by activating hypoxia-induced cellular pathways. The peptide gel provides a drug-free, multifunctional approach for wound healing with proangiogenic, ROS scavenging, and antibacterial properties. Diabetes mellitus is a chronic disease characterized by hyperglycemia due to defects in insulin secretion or function. Elevated proteases and dysfunctional cellular pathways in diabetes compromises the angiogenesis. The strategies of exogenously delivering growth factors, angiogenic drugs, and gene therapies have major challenges of immunological reactions, degradation, and batch-to-batch variability in efficacy. Therefore, chapter 3 involves the development of growth factor-free proangiogenic cyclic hexapeptide (PWLSEKs) nanotubes. Nanotubes have heparin-mimicking functional groups to endogenously effect the angiogenic cellular pathways to promote diabetic wound healing. The nanotubes exhibited excellent cytocompatibility and induced no immunogenic response. The proangiogenic studies on hyperglycemic human umbilical vein endothelial cells (HG-HUVECs) showed an upregulated expression of proangiogenic marker proteins and genes. The nanotubes elevated the endothelial tube formation with a significant increase in tube length, number of nodes, junctions, and master segments. The in vitro wound healing studies on HG-HUVECs showed an increase in 2D and 3D-cell migration and invasion, thus, resulting in efficient wound healing in diabetic conditions. Proangiogenic cyclic peptide nanotubes, therefore, offer a promising approach for accelerating diabetic wound healing without the need for exogenous growth factors, drugs, and glycosaminoglycans, like heparin. The Bone Health and Osteoporosis Foundation estimates the total cost of care associated with osteoporotic and non-union fractures will reach $95 billion in 2040. The use of gold standard, allografts, are impeded by potential infection, limited availability, a high non-union rate, and risk of consequent surgeries. Thus, there is requirement of bio-interactive materials inducing the osteogenesis and bone mineralization. The use of natural enzymes in regenerative scaffolds are hampered by their vulnerability to denaturation, time, cost, and effort required for their purification and processing. Therefore, the development of synthetic biomaterials mimicking enzymes is critical for tissue regeneration. In chapter 4, we focused on developing alkaline phosphatase (ALP)-mimicking cyclic peptide nanotubes to induce osteogenesis and bone mineralization. The nanotubes consist of histidine residues with imidazole rings in close proximation, which is a critical group in the functional domain of ALP. Nanotubes demonstrated compatibility with murine pre-osteoblast MC3T3-E1 cells along with a notable ROS scavenging and anti-inflammatory properties. The enhanced phosphatase activity and formation of bone-like nodules showed osteogenic differentiation and bone mineralization. Subsequently, the biomaterial was found to upregulate the expression of genes marking osteogenic differentiation, namely osteopontin, osteocalcin, alkaline phosphatase, and runt-related transcription factor-2, following incubation periods of 7 and 14 days. Furthermore, the nanotubes were shown to inhibit osteoclastogenesis by reducing the expression of critical cytokines involved in this process, RANKL and TRAP. The developed biomaterial promoted the differentiation of preosteoblast cells into osteoblasts, which is a significant challenge for the currently available bone grafts. These biocompatible enzyme-like, peptide scaffolds can be exploited to develop novel multifunctional biomaterials for bone regeneration. Chapter 5 presents the major conclusions of this thesis, contributions made to the field of peptide-based scaffolds for promoting wound healing and bone regeneration, and future perspectives, which includes future research directions and potential clinical applications.