Self-Assembly of Fibronectin Mimetic Peptide-Amphiphile Nanofibers

Abstract

Single-tailed peptide-amphiphiles have been shown to form nanofibers in solution and gel after screening of their electrostatic charges, and those containing cell-binding motifs are promising as tissue engineering scaffolds. A fibronectin-mimetic peptide sequence was developed, containing both the primary binding domain RGD and the synergy binding domain PHSRN, which has shown superior cell adhesion properties over simple RGD sequences and fibronectin in 2D culture. In order to test this sequence in a 3D environment in the future, we have designed a C(16) single-tailed peptide-amphiphile, PR_g (with a peptide headgroup of GGGSSPHSRN(SG)(5)RGDSP), that forms nanofibers and a gel in solution without any screening of its positive charge. In this study, we characterized the self-assembly properties of the PR_g peptide-amphiphile via critical micelle concentration (CMC) measurements, circular dichroism (CD) spectroscopy, cryo-transmission electron microscopy (cryo-TEM), small angle neutron scattering (SANS), and rheology measurements. The CMC of the PR_g amphiphile was determined to be 38 microM. CD measurements showed that even though the peptide formed an unordered secondary structure, the peptide-amphiphile's spectrum after aging resembled more the spectrum of an alpha+beta protein. Cryo-TEM images of a 100 microM peptide-amphiphile solution showed individual nanofibers with a diameter of approximately 10 nm and lengths on the order of several micrometers. Images taken at higher concentrations (1 mM) show a high degree of bundling among the nanofibers, and at even higher concentrations (3 and 4 mM) SANS measurements also indicated that the peptide-amphiphile formed rod-shaped structures in solution. The peptide-amphiphile gel was monitored by parallel-plate rheometry, and the elastic modulus (G') was greater than the viscous modulus (G''), which indicates that PR_g forms a gel. The shear modulus for a 2 day old gel was measured to be approximately 500 Pa, which is within the modulus range for living tissue; thus, the PR_g gel shows potential as a possible scaffold for tissue engineering.