Design, Synthesis, and Evaluation of Ultrashort Self-Assembling Peptide-Conducting Polymer Hybrid Hydrogels for Tissue Engineering

Abstract

Hydrogels are soft, viscoelastic, and semi-solid materials that are of great interest in biomedical applications for tissue engineering. This is due to their ability to mimic the natural environments of cells, arising from their porosity, high water content, and biocompatible mechanical stiffness. Hydrogels act as a scaffold for cell growth and proliferation and is used in wound healing and tissue regeneration applications. One possible use for this can be for the treatment of peripheral nerve neuropathy. Current research in non-surgical therapies for this disease is focusing on using implantable guidance conduits, since injured peripheral nerves are known to heal on their own, provided there are physical support structures in place for which hydrogels would be an ideal material. One way to prepare such hydrogel is by using self-assembling peptides. Peptides have been widely studied in various therapeutic applications, known for their adaptability and modifiability. For the treatment of peripheral nerve tissues specifically, electrical stimulation has been shown to promote cell growth and axonal extension. Conductive materials which show promise for such therapeutic uses are conducting polymers (CPs). CPs have the flexibility and versatility of plastics with conductivity ranges comparable to that of metals. The research in this thesis aims to explore the integrated preparation of peptide hydrogels in the presence of CP as a hybrid scaffold for therapeutic applications in peripheral nerve tissue regeneration. The first part of this thesis describes the design, synthesis, and characterisation of self-assembling peptides based on the existing ultrashort gelation sequence HG2.81. The following analogues were designed to complement the CP by both covalent and non-covalent conjugation. In the covalent method, peptides PS 1Nal, PS 2Nal and PS Trp were synthesised and coupled to thiophenes as a linker to co-polymerise into the CP backbone. For the non-covalent method, analogues 2.81COOH and PS Nap were synthesised with introduced negative charges, to form electrostatic interaction with the positively charged CP and act as its counterions for conductivity. All peptides were synthesised using solid phase peptide synthesis on solid support resin and purified using RP-HPLC to >95% purity. Methods of preparing the peptide-CP hydrogels and mechanisms of in situ polymerisation were extensively explored in Chapter 2. Results from covalent and non-covalent methods of conjugation showed that only 2.81COOH was able to form a cohesive and homogenous stable gel in the presence of the CP. This was attributed to its excellent self-assembly kinetics within minutes and C-terminal negative charge which complements the CP. In situ polymerisation of CP was confirmed by visual colour change observations from colourless to dark blue/black and by UV-Visible spectroscopy, where polaron peaks (700 - 900 nm) and bipolarons peaks (>1200 nm) correspond to the conductive state of the CP. The secondary structure of the peptide in the 2.81COOH-CP gel was characterised using circular dichroism (CD), however, signals show that the aromatic side groups of the peptide overlaps with the β-sheet in the aromatic region. Fourier transform infrared spectroscopy (FTIR) characterisation showed a peak at around 1630 cm-1 in the amide I region which corresponds the β-sheet conformation and confirmed the peptide's secondary structure. Transmission electron microscopy (TEM) was used to characterise the peptide fibril network nanostructure. Scanning electron microscopy (SEM) was used to image the lyophilised gels to reveal its porosity and scaffold morphology ideal for allowing diffusion of factors important for cell growth and repair, such as oxygen, nutrients, and the removal of metabolic waste. The second section of this thesis focuses on the evaluation of the properties of 2.81COOH-CP gels at varied concentrations of peptide and CP, based on the ideal characteristics of peripheral nerve cell culture scaffolds (Chapter 3). Hemolysis and cell viability assays performed on 2.81COOH at concentrations ranging from 0.96 and 250 μM, and for gel samples at 0.2% - 1% w/v concentrations, which indicated that they are non-toxic and biocompatible. Cyclic voltammetry and conductivity measurements were performed to analyse the gels' electrochemistry, which showed quasi-reversible reversible redox processes and conductivity within ranges of 10-1 to 10-3 S cm-1. Oscillatory rheology frequency sweeps demonstrated that the gels were viscoelastic and mechanically stable, with stiffness within the 100 - 1000 Pa range ideal for peripheral nerve cell cultures. Rheology strain sweeps as well as the self-healing and injectability of the gels were also tested to demonstrate its ability to withstand strain forces and shear stress. Data showed that 2.81COOH-CP gels were readily able to self-heal after liquefying and was injectable on smooth surfaces and in solution, which is promising for therapeutic applications and routes of administration of the gel. Results in this thesis have shown that ultrashort self-assembling peptides can combine with CP as a 2.81COOH-CP hybrid gel with potential to be further developed for tissue regeneration for peripheral nerve cells.