Self-Assembly for the Synthesis of Functional Biomaterials

Abstract

The use of self-assembly for the construction of functional biomaterials is a highly promising and exciting area of research, with great potential for the treatment of injury or disease. By using multiple noncovalent interactions, coded into the molecular design of the constituent components, self-assembly allows for the construction of complex, adaptable, and highly tunable materials with potent biological effects. This review describes some of the seminal advances in the use of self-assembly to make novel systems for regenerative medicine and biology. Materials based on peptides, proteins, DNA, or hybrids thereof have found application in the treatment of a wide range of injuries and diseases, and this review outlines the design principles and practical applications of these systems. Most of the examples covered focus on the synthesis of hydrogels for the scaffolding or transplantation of cells, with an emphasis on the biological, mechanical, and structural properties of the resulting materials. In addition, we will discuss the distinct advantages conferred by self-assembly (compared with traditional covalent materials), and present some of the challenges and opportunities for the next generation of self-assembled biomaterials.

Figure 1. Self-assembling peptides based on the β-sheet

(A) Schematic of the ionic self-complementary peptides developed by Zhang and coworkers. The peptides pack into sheets and fibers based on hydrophobic interactions on one face of the molecule, and complementary ionic interaction on the other. These fibers then form a highly porous hydrogel. (B) Peptides with alternating hydrophilic and hydrophobic residues assemble into β-sheet structures (left) that form twisted ribbons (right) and bundle into larger fibers. (C) The Q11 peptide (blue) developed by the Collier lab assembles into nanofibers due to β-sheet formation. Appending a peptide epitope (red) results in a high density of the signal on the fibril surface. (D) Amphiphilic tri-block peptides developed by the Hartgerink group. The central hydrophobic block forces self-assembly via hydrophobic between molecules and hydrogen bonding along the fiber axis. Charged lysine residues provide solubility, and balance the hydrophobic forces. (E) The β-hairpin molecule MAX1, which can fold under appropriate conditions and form intramolecular hydrogen bonds. (F) Upon charge screening in cell culture media, the β-hairpin molecule can fold and then subsequently assemble into long nanofibers due to intermolecular interactions. [(A): ref. ; (B): ref. ; (C): ref. ; (D): ref. ; (E): ref. ; (F): ref. 38]

Figure 2. Peptide materials using the coiled-coil motif

(A) Top-down view of two α-helices interacting via coiled-coil interaction. The a and d residues in the heptad repeat associate due to hydrophobic interactions, whereas ionic interactions like salt bridges between the e and g residues further stabilize the structure. (B) Two side-on views of the same coiled-coil structure, further illustrating the intermolecular associations that hold it together. (C) Redesigning the a-helix motif to create “sticky ends” based on charge-charge interactions allowed the assembly of peptides into nanofibers. (D) The coiled-coil interactions originally designed by the Woolfson group in Ref. , where ionic interactions between the b and c positions resulted in the formation of fibers, but not hydrogels. (E) Using weaker interactions (e.g. hydrophobic, H-bonding) between residues b, c, and f resulted in hydrogels. [(A, B): ref. ; (C): ref. ; (D, E): ref. 48]

Figure 3. Hydrogels constructed from collagen-mimetic peptides

(A) The structure of a peptide synthesized by the Chmielewski group, with metal-binding groups at the ends and middle of a peptide bearing collagen-mimetic repeats. The peptides self-assemble into triple helices, and addition of metal ions crosslinks them into a hydrogel. (B) Three peptides with collagen-like repeats, developed by the Hartgerink laboratory, self-assemble into staggered, heterotrimeric helices. Interactions between the ends and middle of these helices result in the formation of bundled nanofibers that form a hydrogel. (C) Schematic of the sticky-end interactions that result in formation of the nanofibers observed. Arrows indicate charged residues that promote additional inter-helical bundling. [(A): ref. ; (B, C): ref. 54]

Figure 4. Peptide amphiphiles and their applications

(A) Chemical structure of a representative peptide amphiphile (PA), and its assembly into nanofibers, with the four primary regions highlighted. Region I: unbranched alkyl (usually C₁₆) tail; region II: a β-sheet forming segment to promote H-bonding along the fiber axis; region III: charged amino acids for solubility; region IV: a peptide epitope to imbue the material with biological signaling. (B) TEM image demonstrating the self-assembly of PAs into high aspect ratio nanofibers, often several microns in length. (C) Injection of a PA solution containing the IKVAV epitope into the damaged spinal cord of a mouse resulted in regeneration of both ascending and descending axons (ii), whereas a control saline injection did not (i). (D) A VEGF-mimetic PA was able to stimulate angiogenesis in a mouse hind-limb ischemia model, restoring more blood flow to the injured limb after 28 days (top image) compared with the peptide alone, or a PA with a non-bioactive sequence. (E) Schematic of self-assembled sac formation upon injection of a polyelectrolyte solution into an oppositely charged PA solution. (F) SEM demonstrating the hierarchical structure of the sac membrane, including the dense arrangement of fibers perpendicular to the membrane (region 3). (G) SEM images of a PA-based monodomain gel, with fibers aligned in the direction of shear (left); a PA solution that was not heat-annealed shows randomly oriented fibers (right). [(A, B): ref. ; (C): ref. ; (D): ref. ; (E, F): ref. ; (G): ref. 70]

Figure 5. Self-assembling short aromatic peptides

(A) Self-assembly of the Fmoc-FF peptide, as determined by computational modeling. The peptide forms antiparallel β-sheet hydrogen bonds (i); different β-sheets are brought together by π-π stacking between Fmoc groups (orange), with interdigitated phenyl side groups (purple) from the phenylalanine residues (ii); the twist of the β-sheets results in a cylindrical arrangement, shown in top (iii) and side (iv) views. (B) Structure of the Nap-FFGEY peptide, showing the site of phosphorylation that drives the structural transition. The peptide forms a β-sheet structure (i, ii), resulting in further association into nanotube-like structures (iii, iv). [(A): ref. ; (B): ref. 87]

Figure 6. Protein-based self-assembled materials

(A) Structure of the telechelic, coiled-coil based polypeptide designed by the Tirrell group. (B) Association of the end helices into coiled-coils results in hydrogel formation. (C) Chemical structure of the amphiphilic K_xL_y block co-polypeptide designed by the Deming laboratory. (D) The leucine α-helices self-assemble due to the hydrophobic effect, forming extended tapes and fibers. (E) Naturally-derived WW domains form a noncovalent complex with PPxY domains. The strength of the interaction between the two can be tuned by changing the specific motif; in this example, the Nedd4.3 variant of the WW domain has a weaker interaction with PPxY than the CC43 variant. (F) Incorporating repeating WW and PPxY domains into separate protein polymers allows for the construction of a hydrogel through protein association interactions. [(A): ref. ; (B): ref. ; (C, D): ref. ; (E,F): ref. 107]

Figure 7. Hydrogels constructed from DNA

(A) DNA structures in the shape of X, Y, or T can form extended networks due to complementary sticky ends; addition of a ligase enzyme covalently fixes these links into a hydrogel. (B) By incorporating plasmid DNA into this cross-linked gel, the resulting “P-gel” pads can be used in a cell-free translation system to produce protein more efficiently than if the plasmid was free in solution. (C) Y-shaped DNA monomers can form a hydrogel by association of cytosine-rich i-motifs; the magnification shows one such i-motif at the junction of two DNA arms. (D) A Y-shaped DNA scaffold can be brought together by the addition of a dsDNA linker to form a hydrogel without the need for enzymatic linking. Heating the material can disrupt the sticky-end interactions and melt the gel; alternatively, an enzyme that cleaves a sequence in the middle of the crosslinking strands can be used. [(A): ref. ; (B): ref. ; (C): ref. ; (D): ref. 115]