Modulating the mechanical properties of self-assembled peptide hydrogels via native chemical ligation

Abstract

Hydrogels produced from self-assembling peptides and peptide derivatives are being investigated as synthetic extracellular matrices for defined cell culture substrates and scaffolds for regenerative medicine. In many cases, however, they are less stiff than the tissues and extracellular matrices they are intended to mimic, and they are prone to cohesive failure. We employed native chemical ligation to produce peptide bonds between the termini of fibrillized beta-sheet peptides to increase gel stiffness in a chemically specific manner while maintaining the morphology of the self-assembled fibrils. Polymerization, fibril structure, and mechanical properties were measured by SDS-PAGE, mass spectrometry, TEM, circular dichroism, and oscillating rheometry; and cellular responses to matrix stiffening were investigated in cultures of human umbilical vein endothelial cells (HUVECs). Ligation led to a fivefold increase in storage modulus and a significant enhancement of HUVEC proliferation and expression of CD31 on the surface of the gels. The approach was also orthogonal to the inclusion of unprotected RGD-functionalized self-assembling peptides, which further increased proliferation. This strategy broadens the utility of self-assembled peptide materials for applications that require enhancement or modulation of matrix mechanical properties by providing a chemoselective means for doing so without significantly disrupting the gels' fibrillar structure.

Redox modulated hydrogelation of a self-assembling short peptide amphiphile

“…Environmental stimuli including pH [16][17][18][19], ionic strength and/or metal ions [18,[20][21][22], temperature [23], light [24] and enzyme-triggers [25], provide powerful approaches for modulating hydrogelation. Furthermore, introduction of microenvironment-sensitive amino acid residues into peptide sequences is also an important strategy for controlling peptide self-assembly and hydrogelation [9,[26][27][28].…”

Self-Assembled Peptides: Characterisation and In Vivo Response

“…Once the matrix has been formed based upon the supramolecular properties of the fibrils, efforts have been made to improve the stiffness post assembly. In one example, chemical crosslinking via native chemical ligation (NCL) has been used to introduce covalent bonds between fibrils [53]. Importantly for biological applications, this is a mild, aqueous reaction that allows the linkage of the N terminal Cys residue with a C terminal thioester on 11 residue peptides, yielding a fivefold increase in storage modulus without disrupting the fibril.…”

Modular self-assembling biomaterials for directing cellular responses

“…Encouraging successes have already been reported using ligand-presenting self-assembling biomaterials to influence a variety of cell behaviors. Representative examples include the differentiation of neural progenitor cells, 16 the adhesion and spreading of fibroblasts, 21 the proliferation and protein expression of endothelial cells, 26,28 the proliferation, differentiation, and migration of osteoblasts, 24 and the intracellular uptake of the material itself. 29 However, the modularity of these materials has not yet been fully exploited, as many simultaneously co-assembled factors have yet to be systematically tuned and optimized.…”

“…We employed native chemical ligation32 as a chemoselective way of accomplishing this by designing short peptide thioesters capable of self-assembling and then oligomerizing into covalently stabilized networks (Figure 2). 28 Other laboratories have also recently utilized chemical ligation in combination with peptide selfassembly to produce long collagen-mimetic 33 or α-helical fibrils. 34 In the case of β-sheet fibrillar gels studied in our laboratory, native chemical ligation led to as much as a six-fold stiffening of the gels without significantly altering the morphology of the self-assembled fibrils.…”

“…34 In the case of β-sheet fibrillar gels studied in our laboratory, native chemical ligation led to as much as a six-fold stiffening of the gels without significantly altering the morphology of the self-assembled fibrils. 28 Ligation could be performed in a range of peptide concentrations, providing a way of decoupling matrix stiffness from peptide concentration, and therefore presumably from porosity. The chemistry was also cytocompatible in cultures of primary human endothelial cells, and matrix stiffening by ligation led to a significant increase in cell proliferation over non-ligated gels (Figure 2c).…”