Bradley L. Nilsson

Self-assembled amino acids and dipeptides as noncovalent hydrogels for tissue engineering

Noncovalent hydrogels derived from the self-assembly of peptides and proteins have demonstrated advantages over covalent hydrogels for three-dimensional cell scaffolding applications. There is growing interest in exploiting minimal, self-assembling dipeptides and amino acids as hydrogel networks that support cell culture applications, but significant questions persist concerning the mechanism of self-assembly and the relationship between the molecular structure of the assembled materials and their emergent viscoelastic and biochemical properties. This review will critically assess current progress in the use of minimal self-assembling peptides and functionalized amino acids to create hydrogels, with a focus on the challenges of understanding the structure and function of these materials and on the outlook for the use of these modular and dynamic materials as robust networks for tissue engineering.

A Reductive Trigger for Peptide Self-Assembly and Hydrogelation

Stimulus-responsive peptide self-assembly provides a powerful method for controlling self-assembly as a function of environment. The development of a reductive trigger for peptide self-assembly and subsequent hydrogelation is described herein. A self-assembling peptide sequence, Ac-C(FKFE)(2)CG-NH(2), was cyclized via disulfide bonding of the flanking cysteine residues. The macrocyclic form of this peptide enforces a conformational restraint that prevents adoption of the beta-sheet conformation that is required for self-assembly. Upon reduction of this disulfide bond, the peptide relaxes into the preferred beta-sheet conformation, and immediate self-assembly into fibrillar superstructures occurs. At sufficient peptide concentration, self-assembly is accompanied by the formation of rigid, viscoelastic hydrogels.

Review self‐assembly of amphipathic β‐sheet peptides: Insights and applications

Amphipathic peptides composed of alternating polar and nonpolar residues have a strong tendency to self‐assemble into one‐dimensional, amyloid‐like fibril structures. Fibrils derived from peptides of general (XZXZ)n sequence in which X is hydrophobic and Z is hydrophilic adopt a putative β‐sheet bilayer. The bilayer configuration allows burial of the hydrophobic X side chain groups in the core of the fibril and leaves the polar Z side chains exposed to solvent. This architectural arrangement provides fibrils that maintain high solubility in water and has facilitated the recent exploitation of self‐assembled amphipathic peptide fibrils as functional biomaterials. This article is a critical review of the development and application of self‐assembling amphipathic peptides with a focus on the fundamental insight these types of peptides provide into peptide self‐assembly phenomena.

Self-assembly and hydrogelation promoted by F5-phenylalanine

Phenylalanine (Phe)-derived molecules have been exploited as low molecular weight hydrogelators. Perturbing the hydrophobic and π–π interactions that promote self-assembly and hydrogelation of these derivatives will facilitate improved understanding of hydrogelation phenomena and the design of small molecule hydrogelators with novel properties. The efficient self-assembly and hydrogelation of Fmoc-protected pentafluorophenylalanine (Fmoc-F5-Phe) are reported herein. Suspensions of Fmoc-F5-Phe in water undergo rapid self-assembly to entangled fibrillar structures within minutes, giving rise to rigid supramolecular gels. Self-assembly occurs at concentrations as low as 2 mM (0.1 wt%). Variation of the fluorinated aromatic side chain or N-terminal functionalization perturbs hydrogelation, implicating fluorous and π–π interactions as the primary determinants for molecular recognition and self-assembly. The hydrophobic and electronic properties of F5-Phe provide remarkable potential for functional self-assembly in a minimal amino acid scaffold.

Effect of C-Terminal Modification on the Self-Assembly and Hydrogelation of Fluorinated Fmoc-Phe Derivatives

The development of hydrogels resulting from the self-assembly of low molecular weight (LMW) hydrogelators is a rapidly expanding area of study. Fluorenylmethoxycarbonyl (Fmoc) protected aromatic amino acids derived from phenylalanine (Phe) have been shown to be highly effective LMW hydrogelators. It has been found that side chain functionalization of Fmoc-Phe exerts a significant effect on the self-assembly and hydrogelation behavior of these molecules; fluorinated derivatives, including pentafluorophenylalanine (F(5)-Phe) and 3-F-phenylalanine (3-F-Phe), spontaneously self-assemble into fibrils that form a hydrogel network upon dissolution into water. In this study, Fmoc-F(5)-Phe-OH and Fmoc-3-F-Phe-OH were used to characterize the role of the C-terminal carboxylic acid on the self-assembly and hydrogelation of these derivatives. The C-terminal carboxylic acid moieties of Fmoc-F(5)-Phe-OH and Fmoc-3-F-Phe-OH were converted to C-terminal amide and methyl ester groups in order to perturb the hydrophobicity and hydrogen bond capacity of the C-terminus. Self-assembly and hydrogelation of these derivatives was investigated in comparison to the parent carboxylic acid compounds at neutral and acidic pH. It was found that hydrogelation of the C-terminal acids was highly sensitive to solvent pH, which influences the charge state of the terminal group. Rigid hydrogels form at pH 3.5, but at pH 7 hydrogel rigidity is dramatically weakened. C-terminal esters self-assembled into fibrils only slowly and failed to form hydrogels due to the higher hydrophobicity of these derivatives. C-terminal amide derivatives assembled much more rapidly than the parent carboxylic acids at both acidic and neutral pH, but the resultant hydrogels were unstable to shear stress as a function of the lower water solubility of the amide functionality. Co-assembly of acid and amide functionalized monomers was also explored in order to characterize the properties of hybrid hydrogels; these gels were rigid in unbuffered water but significantly weaker in phosphate buffered saline. These results highlight the complex nature of monomer/solvent interactions and their ultimate influence on self-assembly and hydrogelation, and provide insight that will facilitate the development of optimal amino acid LMW hydrogelators for gelation of complex buffered media.

The influence of side-chain halogenation on the self-assembly and hydrogelation of Fmoc-phenylalanine derivatives

Fmoc-protected aromatic amino acids, including Fmoc-phenylalanine (Fmoc-Phe), Fmoc-tyrosine (Fmoc-Tyr), and Fmoc-pentafluorophenylalanine (Fmoc-F5-Phe), have been shown to undergo efficient self-assembly and to promote hydrogelation in aqueous solvents. In order to probe the electronic and steric role of the benzyl side-chain in hydrophobic and π–π interactions during self-assembly, the hydrogelation behavior of monohalogenated (F, Cl, Br) Fmoc-Phe side-chain derivatives was assessed. Incorporation of single halogen substituents on the aromatic side-chain of Fmoc-Phe dramatically enhances the efficient self-assembly of these amino acid derivatives (relative to Fmoc-Phe) into amyloid-like fibrils that promote hydrogelation in aqueous solvents. The position of halogen substitution (ortho, meta, para) and the halogen itself (F, Cl, Br) exert a strong influence on the self-assembly rate and on the bulk rheological properties of the resultant hydrogel. These results demonstrate that minimal atomic substitutions can be used to tune self-assembly and gelation of small molecule hydrogelators.

Tuning β-Sheet Peptide Self-Assembly and Hydrogelation Behavior by Modification of Sequence Hydrophobicity and Aromaticity

Peptide self-assembly leading to cross-β amyloid structures is a widely studied phenomenon because of its role in amyloid pathology and the exploitation of amyloid as a functional biomaterial. The self-assembly process is governed by hydrogen bonding, hydrophobic, aromatic π-π, and electrostatic Coulombic interactions. A role for aromatic π-π interactions in peptide self-assembly leading to amyloid has been proposed, but the relative contributions of π-π versus general hydrophobic interactions in these processes are poorly understood. The Ac-(XKXK)(2)-NH(2) peptide was used to study the contributions of aromatic and hydrophobic interactions to peptide self-assembly. Position X was globally replaced by valine (Val), isoleucine (Ile), phenylalanine (Phe), pentafluorophenylalanine (F(5)-Phe), and cyclohexylalanine (Cha). At low pH, these peptides remain monomeric because of repulsion of charged lysine (Lys) residues. Increasing the solvent ionic strength to shield repulsive charge-charge interactions between protonated Lys residues facilitated cross-β fibril formation. It was generally found that as peptide hydrophobicity increased, the required ionic strength to induce self-assembly decreased. At [NaCl] ranging from 0 to 1000 mM, the Val sequence failed to assemble. Assembly of the Phe sequence commenced at 700 mM NaCl and at 300 mM NaCl for the less hydrophobic Ile variant, even though it displayed a mixture of random coil and β-sheet secondary structures over all NaCl concentrations. β-Sheet formation for F(5)-Phe and Cha sequences was observed at only 20 and 60 mM NaCl, respectively. Whereas self-assembly propensity generally correlated to peptide hydrophobicity and not aromatic character the presence of aromatic amino acids imparted unique properties to fibrils derived from these peptides. Nonaromatic peptides formed fibrils of 3-15 nm in diameter, whereas aromatic peptides formed nanotape or nanoribbon architectures of 3-7 nm widths. In addition, all peptides formed fibrillar hydrogels at sufficient peptide concentrations, but nonaromatic peptides formed weak gels, whereas aromatic peptides formed rigid gels. These findings clarify the influence of aromatic amino acids on peptide self-assembly processes and illuminate design principles for the inclusion of aromatic amino acids in amyloid-derived biomaterials.

Complementary π–π Interactions Induce Multicomponent Coassembly into Functional Fibrils

Noncovalent self-assembled materials inspired by amyloid architectures are useful for biomedical applications ranging from regenerative medicine to drug delivery. The selective coassembly of complementary monomeric units to provide ordered multicomponent fibrils is a possible strategy for enhancing the sophistication of these noncovalent materials. Herein we report that complementary π-π interactions can be exploited to promote the coassembly of phenylalanine (Phe) derivatives that possess complementary aromatic side-chain functionality. Specifically, equimolar mixtures of Fmoc-Phe and Fmoc-F(5)-Phe, which possess side-chain groups with complementary quadrupole electronics, readily coassemble to form two-component fibrils and hydrogels under conditions where Fmoc-Phe alone fails to self-assemble. In addition, it was found that equimolar mixtures of Fmoc-Phe with monohalogenated (F, Cl, and Br) Fmoc-Phe derivatives also coassembled into two-component fibrils. These results collectively indicate that face-to-face quadrupole stacking between benzyl side-chain groups does not account for the molecular recognition between Phe and halogenated Phe derivatives that promote cofibrillization but that coassembly is mediated by more subtle π-π effects arising from the halogenation of the benzyl side chain. The use of complementary π-π interactions to promote the coassembly of two distinct monomeric units into ordered two-component fibrils dramatically expands the repertoire of noncovalent interactions that can be used in the development of sophisticated noncovalent materials.

Effects of Varied Sequence Pattern on the Self-Assembly of Amphipathic Peptides

Amphipathic peptides have an increased propensity to self-assemble into amyloid-like β-sheet fibrils when their primary sequence pattern consists of alternating hydrophobic and hydrophilic amino acids. These fibrils adopt a bilayer architecture composed of two β-sheets laminated to bury the hydrophobic side chains of the β-sheet in the bilayer interior, leaving the hydrophilic side chains exposed at the bilayer surface. In this study, the effects of altering the sequence pattern of amphipathic peptides from strictly alternating hydrophobic/hydrophilic repeats to more complex patterning of hydrophobic and hydrophilic residues on self-assembly of the resulting sequences is reported. Self-assembly of the Ac-(FKFE)2-NH2 peptide was compared to that of four related sequences with varied amino acid sequence patterning: Ac-(FK)2(FE)2-NH2, Ac-KEFFFFKE-NH2, Ac-(KFFE)2-NH2, and Ac-FFKEKEFF-NH2. The Ac-(FKFE)2-NH2 and Ac-(FK)2(FE)2-NH2 peptides effectively self-assembled at high (1.0 mM) and low (0.2 mM) concentrations (pH 3-4) into β-sheet nanoribbons that were 8 and 4 nm wide, respectively. The Ac-KEFFFFKE-NH2 peptide failed to self-assemble at low concentration (pH 3-4), but self-assembled into distinct nanotapes that were ~20 nm in width at high concentration. Ac-(KFFE)2-NH2 and Ac-FFKEKEFF-NH2 failed to self-assemble into fibril/tape-like materials at either high or low concentration at pH 3-4, although Ac-FFKEKEFF-NH2 formed micelle-like aggregates at higher concentrations. At neutral pH, similar self-assembly behavior was observed for each peptide as was observed at acidic pH. An exception was the Ac-FFKEKEFF-NH2 peptide, which formed ~20 nm nanotapes at neutral pH. These results indicate that amino acid sequence patterns exert a profound influence on self-assembly propensity and morphology of the resulting materials even when the overall hydrophobicity or charge of the related peptides are identical. Sequence pattern variation can thus be exploited as a variable in the creation of novel materials composed of self-assembled peptides.

Amyloid-binding small molecules efficiently block SEVI (semen-derived enhancer of virus infection)- and semen-mediated enhancement of HIV-1 infection.

Semen was recently shown to contain amyloid fibrils formed from a self-assembling peptide fragment of the protein prostatic acid phosphatase. These amyloid fibrils, termed semen-derived enhancer of virus infection, or SEVI, have been shown to strongly enhance HIV infectivity and may play an important role in sexual transmission of HIV, making them a potential microbicide target. One novel approach to target these fibrils is the use of small molecules known to intercalate into the structure of amyloid fibrils, such as derivatives of thioflavin-T. Here, we show that the amyloid-binding small molecule BTA-EG6 (the hexa(ethylene glycol) derivative of benzothiazole aniline) is able to bind SEVI fibrils and effectively inhibit both SEVI-mediated and semen-mediated enhancement of HIV infection. BTA-EG6 also blocks the interactions of SEVI with HIV-1 virions and HIV-1 target cells but does not cause any inflammation or toxicity to cervical epithelial cells. These results suggest that an amyloid-binding small molecule may have utility as a microbicide, or microbicidal supplement, for HIV-1.