Directed intermixing in multicomponent self-assembling biomaterials

Abstract

The noncovalent coassembly of multiple different peptides can be a useful route for producing multifunctional biomaterials. However, to date, such materials have almost exclusively been investigated as homogeneous self-assemblies, having functional components uniformly distributed throughout their supramolecular structures. Here we illustrate control over the intermixing of multiple different self-assembling peptides, in turn providing a simple but powerful means for modulating these materials' mechanical and biological properties. In β-sheet fibrillizing hydrogels, significant increases in stiffening could be achieved using heterobifunctional cross-linkers by sequestering peptides bearing different reactive groups into distinct populations of fibrils, thus favoring interfibril cross-linking. Further, by specifying the intermixing of RGD-bearing peptides in 2-D and 3-D self-assemblies, the growth of HUVECs and NIH 3T3 cells could be significantly modulated. This approach may be immediately applicable toward a wide variety of self-assembling systems that form stable supramolecular structures.

Figure 1

Schematic for controlling intermixing within multi-peptide co-assemblies. (A) Table of all of peptides used (functional domain in red, fibrillizing domain in blue). In the intermixed case (B), peptides were combined in the dry state and assembled into protofibrils in water, pH 2.8. Gels were then produced by overlaying these intermixed solutions with PBS. In the separately assembled case (C), different peptides were directed to form protofibrils in water, in separate containers. These protofibrils were then mixed together and overlaid with PBS to form gels composed of distinct, unmixed fibril populations.

Figure 2

Peptides formed protofibrils in water, pH 2.8 (A, C) and mature fibrils in PBS (B). Peptide solutions were prepared in water (27 mM Q11/3 mM total ligand-bearing peptides), then diluted to 0.25 mM in either water (A) or PBS (B) and spotted onto TEM grids. Fibrils appeared wider and stained more darkly with uranyl acetate when formed in PBS (B). (C) Oscillating rheometry at 0.2% strain indicated that the storage and loss moduli of solutions of fibrils (27 mM Q11/3 mM ligand-bearing peptides as indicated) were significantly diminished compared to fully gelled Q11 peptides, which exhibit storage moduli in the range of 10–30 kPa. (G′ solid symbols, G″ open symbols). Circular dichroism with the same formulations of separately assembled and intermixed peptides in water revealed beta-sheet signatures (D).

Figure 3

Quantification of peptide intermixing using immunoelectron microscopy. Q11 was co-assembled with biotin-RGD-Q11 (A, D), myc-Q11 (B, E), or both biotin-RGD-Q11 and myc-Q11 (C, F, G), using either intermixing or separately assembling protocols. All samples were exposed to both 5 nm gold particles specific for the biotin tags and 15 nm gold particles specific for the myc tags. Bar = 100 nm (A–C). In D–G, each data point represents a single fibril, plotted on coordinates that illustrate the number of each particle type bound per 100 nm of fibril length. Fibrils binding only biotin-specific NPs are green, fibrils binding only myc-specific NPs are red, and fibrils binding both are blue. Fibrils containing only biotin-RGD-Q11 bound predominantly biotin-specific NPs (A, D), with the opposite being true for fibrils containing only myc-Q11 (B, E). In single-label controls, the non-cognate particles bound with a background level of 9–17% (percentages shown in H, right 2 columns). Separately assembled fibrils were evenly divided into two populations binding only one of each particle type, with a similar background level (F, H, left column), whereas intermixed fibrils predominantly stained bound both particle types (C, G, H, left center column). *Fisher’s exact test, p= 2.8 × 10⁻¹⁹.

Figure 4

Gel viscoelasticity was dependent on the time periods allowed for protofibrillization in water and gelation in PBS (A). The degree of intermixing did not significantly influence gel storage moduli for the formulations used within cell culture experiments (B, 27 mM Q11/3 mM RGD-Q11).

Figure 5

Directed intermixing can be used to control the stiffening of hydrogel assemblies. Storage and loss moduli shown for gels prior to the addition of cross-linker (A) and after (B); legend is below the data. Uncross-linked hydrogels containing separately assembled or intermixed Lys-Q11 and Cys-Q11 peptides exhibited storage moduli similar to gels made exclusively of Q11 (A). Upon application of SM(PEG)₁₂, hydrogels containing both separately assembled Lys-Q11 and Cys-Q11 peptides stiffened to a much greater degree than all other hydrogels (B, black squares). All gels were composed of 30 mM total peptide including either 1.5 mM Cys-Q11, 1.5 mM Lys-Q11, or both. HPLC (C) and MALDI mass spectrometry (D) were used to separate PEGylated species and identify the appropriate cross-links between Cys-Q11 and Lys-Q11, respectively.

Figure 6

Cell growth was significantly influenced by the intermixing of RGD ligands (27mM Q11/3mM RGD-Q11 gels, A–F). HUVEC growth (A) measured at 3 days by MTS (p < 0.01, n = 3). Calcein-AM stained HUVECs at day 3 on gels of Q11 (B), intermixed RDG fibrils (C), separately assembled RGD-Q11/Q11 (D), and intermixed RGD-Q11/Q11 (E). Growth of NIH 3T3 fibroblasts encapsulated within gels (p < 0.05, n = 3).