Self-assembling multidomain peptide hydrogels: designed susceptibility to enzymatic cleavage allows enhanced cell migration and spreading

Abstract

Multidomain peptides are a class of amphiphilic self-assembling peptides with a modular ABA block motif in which the amphiphilic B block drives self-assembly while the flanking A blocks, which are electrostatically charged, control the conditions under which assembly takes place. Previously we have shown that careful selection of the amino acids in the A and B blocks allow one to control the self-assembled fiber length and viscoelastic properties of formed hydrogels. Here we demonstrate how the modular nature of this peptide assembler can be designed for biological applications. With control over fiber length and diameter, gelation conditions, and viscoelastic properties, we can develop suitable materials for biological applications. Going beyond a simple carrier for cell delivery, a biofunctional scaffold will interact with the cells it carries, promoting advantageous cell-matrix interactions. We demonstrate the design of a multidomain peptide into a bioactive variant by incorporation of a matrix metalloprotease 2 (MMP-2) specific cleavage site and cell adhesion motif. Gel formation and rheological properties were assessed and compared to related peptide hydrogels. Proteolytic degradation by collagenase IV was observed in a gel weight loss study and confirmed by specific MMP-2 degradation monitored by mass spectrometry and cryo-transmission electron microscopy (cryo-TEM). Combination of this cleavage site with the cell adhesion motif RGD resulted in increased cell viability and cell spreading and encouraged cell migration into the hydrogel matrix. Collectively the structural, mechanical, and bioactive properties of this multidomain peptide hydrogel make it suitable as an injectable material for a variety of tissue engineering applications.

Figure 1

Storage (black) and loss moduli (grey) of MDPs 1-5 containing variants with and without cleavage sequence, cell adhesion motif and variable length flanking region.

Figure 2

Hydrogels from MDP4 (blue, open) and MDP1 (black, filled) as a control were incubated with trypsin (squares) or collagenase IV (diamonds) and compared to a negative control in PBS. The weight of the hydrogels were determined each day. Data points show averages of three samples. Weight loss is depicted as a percentage where the weight at the beginning of the experiment is 100%.

Figure 3

MALDI-TOF mass spectrometry of MDP1 and 4 before and 48 hours after incubation with MMP-2. Whereas the control peptide shows a single peptide before and after digestion, the cleavage peptide is degraded and multiple fragments can be observed. The expected fragments after cleavage KSLSLS and LRGSLSLSLK are both present. Additional fragments could be identified as alternative cleavage products, whereas some of the peaks remained unidentified.

Figure 4

CryoTEM images before (a) and after (b) incubation with MMP-2, the nanofibers disintegrate leaving small aggregated structures which do not crosslink.

Figure 5

Cell viability of in MDP1, 2, 4 and 5 as determined by MTT assay after 3, 7 and 14 days in culture. Symbols and bars represent mean values and standard deviations (n=10). Viability is significantly lower in the unmodified peptide, presence of the cell adhesion motif and the cleavage site increase cellular activity, the combination of both in MDP5 results in highest viability and indicates that this is the most favorable environment for cell proliferation among these four peptide hydrogels.

Figure 6

Fluorescently labeled cells visualized by confocal microscopy (magnification 63×) barely spread in the parent peptide MDP1 (A), but they show enlarged cell bodies when either the cell attachment motif (B) or the cleavage site is present (C). Combination of both bioactive peptide motifs results in enhanced cell spreading (D).

Figure 7

Green-fluorescent cells were seeded on top of MDP2 without cleavage site (A, B) and MDP5 where the cleavage motif is present (C, D). Images show cells after 1 day (A, C) and after 5 days (B, D) in culture. Whereas cells remain as a monolayer on top of MDP2 hydrogels, they migrate into MDP5.