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Unlike naturally derived peptides, computationally designed sequences offer programmed self-assembly and charge display. Herein, new tetrameric, coiled coil-forming peptides were computationally designed ranging from 8 to 29 amino acids in length. Experimental investigations revealed that only the sequences having three or more heptads (i.e., 21 or more amino acids) exhibited coiled coil behavior. The shortest stable coiled coil sequence had a melting temperature ( T m ) of approximately 58 ± 1 °C, making it ideal for thermoreversible assembly over moderate temperatures. Effects of pH and monovalent salt were examined, revealing structural stability over a pH range of 4 to 11 and an enhancement in T m with the addition of salt. The incorporation of the coiled coil as a hydrogel cross-linker results in a thermally and mechanically reversible hydrogel. A subsequent demonstration of the hydrogel printed through a syringe illustrated one of many potential uses from 3D printing to injectable hydrogel drug delivery.

Peptide-based materials are diverse candidates for self-assembly into modularly designed and stimuli-responsive nanostructures with precisely tunable compositions. Here, we genetically fused computationally designed coiled coil-forming peptides to the N- and C-termini of compositionally distinct multistimuli-responsive resilin-like polypeptides (RLPs) of various lengths. The successful expression of these hybrid polypeptides in bacterial hosts was confirmed through techniques such as gel electrophoresis, mass spectrometry, and amino acid analysis. Circular dichroism spectroscopy and ultraviolet-visible turbidimetry demonstrated that despite the fusion of disparate structural and responsive units, the coiled coils remained stable in the hybrid polypeptides, and the sequence-encoded differences in thermoresponsive phase separation of the RLPs were preserved. Cryogenic transmission electron microscopy and coarse-grained modeling showed that after thermal annealing in solution, the hybrid polypeptides adopted a closed loop conformation and assembled into nanofibrils capable of further hierarchically organizing into cluster structures and ribbon-like structures mediated by the self-association tendency of the RLPs.

Protein hydrogels represent an important and growing biomaterial for a multitude of applications, including diagnostics and drug delivery. We have previously explored the ability to engineer the thermoresponsive supramolecular assembly of coiled-coil proteins into hydrogels with varying gelation properties, where we have defined important parameters in the coiled-coil hydrogel design. Using Rosetta energy scores and Poisson-Boltzmann electrostatic energies, we iterate a computational design strategy to predict the gelation of coiled-coil proteins while simultaneously exploring five new coiled-coil protein hydrogel sequences. Provided this library, we explore the impact of in silico energies on structure and gelation kinetics, where we also reveal a range of blue autofluorescence that enables hydrogel disassembly and recovery. As a result of this library, we identify the new coiled-coil hydrogel sequence, Q5, capable of gelation within 24 h at 4 °C, a more than 2-fold increase over that of our previous iteration Q2. The fast gelation time of Q5 enables the assessment of structural transition in real time using small-angle X-ray scattering (SAXS) that is correlated to coarse-grained and atomistic molecular dynamics simulations revealing the supramolecular assembling behavior of coiled-coils toward nanofiber assembly and gelation. This work represents the first system of hydrogels with predictable self-assembly, autofluorescent capability, and a molecular model of coiled-coil fiber formation.

With the ability to design their sequences and structures, peptides can be engineered to realize a wide variety of functionalities and structures. Herein, computational design was used to identify a set of 17 peptides having a wide range of putative charge states but the same tetrameric coiled-coil bundle structure. Calculations were performed to identify suitable locations for ionizable residues (D, E, K, and R) at the bundle's exterior sites, while interior hydrophobic interactions were retained. The designed bundle structures spanned putative charge states of -32 to +32 in units of electron charge. The peptides were experimentally investigated using spectroscopic and scattering techniques. Thermal stabilities of the bundles were investigated using circular dichroism. Molecular dynamics simulations assessed structural fluctuations within the bundles. The cylindrical peptide bundles, 4 nm long by 2 nm in diameter, were covalently linked to form rigid, micron-scale polymers and characterized using transmission electron microscopy. The designed suite of sequences provides a set of readily realized nanometer-scale structures of tunable charge that can also be polymerized to yield rigid-rod polyelectrolytes.

Computationally designed peptides form desired antiparallel, tetrameric coiled-coil bundles that hierarchically assemble into a variety of well-controlled nanostructures depending on aqueous solution conditions. The bundles selectively self-assemble into different structures: nanotubes, platelets, or needle-like structures at solution pH values of 4.5, 7, and 10, respectively. The self-assembly produces hollow tubes or elongated needle-like structures at pH conditions associated with charged bundles (pH 4.5 or 10); at neutral pH, near the pI of the bundle, a plate-like self-assembled structure forms. Transmission electron microscopy and small-angle X-ray scattering show the nanotubes to be uniform with a tube diameter of ∼13 nm and lengths of up to several μm, yielding aspect ratios >1000. Combining the measured nanostructure geometry with the apparent charged states of the constituent amino acids, a tilted-bundle packing model is proposed for the formation of the homogeneous nanotubes. This work demonstrates the successful use of assembly pathway control for the construction of nanostructures with diverse, well-structured morphologies associated with the folding and self-association of a single type of molecule.

There is intense interest in developing novel methods for the sustained delivery of low levels of clinical therapeutics. MAX8 is a peptide-based beta-hairpin hydrogel that has unique shear thinning properties that allow for immediate rehealing after the removal of shear forces, making MAX8 an excellent candidate for injectable drug delivery at a localized injury site. The current studies examined the feasibility of using MAX8 as a delivery system for nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), two neurotrophic growth factors currently used in experimental treatments of spinal cord injuries. Experiments determined that encapsulation of NGF and BDNF within MAX8 did not negatively impact gel formation or rehealing and that shear thinning did not result in immediate growth factor release. ELISA, microscopy, rheology, and Western blotting experiments collectively demonstrate the functional capabilities of the therapeutic-loaded hydrogels to (i) maintain a protective environment against in vitro degradation of encapsulated therapeutics for at least 28 days; and (ii) allow for sustained release of NGF and BDGF capable of initiating neurite-like extensions of PC12 cells, most likely due to NGF/BDGF signaling pathways. Importantly, while the 21 day release profiles could be tuned by adjusting the MAX8 hydrogel concentration, the initial shear thinning of the hydrogel (e.g., during injection) does not induce significant premature loss of the encapsulated therapeutic, most likely due to effective trapping of growth factors within structurally robust domains that are maintained during the application of shear forces. Together, our data suggests that MAX8 allows for greater dosage control and sustained therapeutic growth factor delivery, potentially alleviating side effects and improving the efficacy of current therapies.

The MAX1 β-hairpin peptide (VKVKVKVK-V(D)PPT-KVKVKVKV-NH2) has been shown to form nanofibrils having a cross-section of two folded peptides forming a hydrophobic, valine-rich core, and the polymerized fibril exhibits primarily β-sheet hydrogen bonding.1-7 These nanofibrils form hydrogel networks through fibril entanglements as well as fibril branching.8 Fibrillar branching in MAX1 hydrogel networks provide the ability to flow under applied shear stress and immediately reform a hydrogel solid on cessation of shear. New β-hairpins were designed to limit branching during nanofibril growth because of steric specificity in the assembled fibril hydrophobic core. The nonturn valines of MAX1 were substituted by 2-naphthylalanine (Nal) and alanine (A) residues, with much larger and smaller side chain volumes, respectively, to obtain LNK1 (Nal)K(Nal)KAKAK-V(D)PPT-KAKAK(Nal)K(Nal)-NH2. LNK1 was targeted to self-associate with a specific "lock and key" complementary packing in the hydrophobic core in order to accommodate the Nal and Ala residue side chains. The experimentally observable manifestation of reduced fibrillar branching in the LNK1 peptide is the lack of solid hydrogel formation after shear in stark contrast to the MAX1 branched fibril system. Molecular dynamics simulations provide a molecular picture of interpeptide interactions within the assembly that is consistent with the branching propensity of MAX1 vs LNK1 and in agreement with experimental observations.

Synthetic hydrogels containing covalently integrated soft and deformable drug depots capable of releasing therapeutic molecules in response to mechanical forces are attractive candidates for the treatment of degenerated tissues that are normally load bearing. Herein, radically cross-linkable block copolymer micelles (xBCM) assembled from an amphiphilic block copolymer consisting of hydrophilic poly(acrylic acid) (PAA) partially modified with 2-hydroxyethyl acrylate, and hydrophobic poly(n-butyl acryclate) (PnBA) were employed as the drug depots and the microscopic cross-linkers for the preparation of hyaluronic acid (HA)-based, hydrogels. HA hydrogels containing covalently integrated micelles (HAxBCM) were prepared by radical polymerization of glycidyl methacrylate (GMA)-modified HA (HAGMA) in the presence of xBCMs. When micelles prepared from the parent PAA-b-PnBA without any polymerizable double bonds were used, hydrogels containing physically entrapped micelles (HApBCM) were obtained. The addition of xBCMs to a HAGMA precursor solution accelerated the gelation kinetics and altered the hydrogel mechanical properties. The resultant HAxBCM gels exhibit an elastic modulus of 847 ± 43 Pa and a compressive modulus of 9.2 ± 0.7 kPa. Diffusion analysis of Nile Red (NR)-labeled xBCMs employing fluorescence correlation spectroscopy confirmed the covalent immobilization of xBCMs in HA networks. Covalent integration of dexamethasone (DEX)-loaded xBCMs in HA gels significantly reduced the initial burst release and provided sustained release over a prolonged period. Importantly, DEX release from HAxBCM gels was accelerated by intermittently applied external compression in a strain-dependent manner. Culturing macrophages in the presence of DEX-releasing HAxBCM gels significantly reduced cellular production of inflammatory cytokines. Incorporating mechano-responsive modules in synthetic matrices offers a novel strategy to harvest mechanical stress present in the healing wounds to initiate tissue repair.

A design strategy to control the thermally triggered folding, self-assembly, and subsequent hydrogelation of amphiphilic beta-hairpin peptides in a pH-dependent manner is presented. Point substitutions of the lysine residues of the self-assembling peptide MAX1 were made to alter the net charge of the peptide. In turn, the electrostatic nature of the peptide directly influences the solution pH at which thermally triggered hydrogelation is permitted. CD spectroscopy and oscillatory rheology show that peptides of lower net positive charge are capable of folding and assembling into hydrogel material at lower values of pH at a given temperature. The pH sensitive folding and assembling behavior is not only dependent on the net peptide charge, but also on the exact position of substitution within the peptide sequence. TEM shows that these peptides self-assemble into hydrogels that are composed of well-defined fibrils with nonlaminated morphologies. TEM also indicates that fibril morphology is not influenced by making these sequence changes on the hydrophilic face of the hairpins. Rheology shows that the ultimate mechanical rigidity of these peptide hydrogels is dependent on the rate of folding and self-assembly. Peptides that fold and assemble faster afford more rigid gels. Ultimately, this design strategy yielded a peptide MAX1(K15E) that is capable of undergoing thermally triggered hydrogelation at physiological buffer conditions (pH 7.4, 150 NaCl, 37 degrees C).

We present the first neutron spin echo (NSE) measurements of self-assembling peptide hydrogel networks to study the fibril dynamics on the nanometer and nanosecond length and time scales. MAX1 and MAX8 are synthetic beta-hairpin peptides that undergo triggered self-assembly at the nanoscale to form a physically cross-linked network of fibrils with a defined cross-section. When subjected to physiological pH and ionic strength (pH 7.4, 150 mM NaCl), the soluble peptides fold into a beta-hairpin and, subsequently, self-assemble to form a structurally rigid hydrogel stabilized by noncovalent cross-links. The sequence of MAX8 is identical to MAX1 with the exception of one single amino acid substitution that reduces the net charge on the peptide. As a result, faster folding and self-assembly kinetics are observed for MAX8 at the same peptide concentration and identical buffer conditions, and gels with a larger storage modulus are formed. NSE measurements of the peptide hydrogels demonstrate that the self-assembled peptide fibrils can be described as semiflexible chains on nanolength and time scales. Alteration of the peptide sequence affected the nanoscale dynamics of the hydrogels but not to an extent comparable to the large difference observed in the bulk viscoelasticity. Small angle neutron scattering (SANS) of the hydrogels reveals increased scattering for MAX8 at low wavevectors, an indication of a heterogeneous network with a tighter mesh size. Therefore, we conjecture that the difference in elastic modulus arises from differences in assembly kinetics that result in increased fibrillar branching and physical cross-links rather than a change in the fibril nanostructure or persistence length.

A de novo designed three-stranded beta-sheet (TSS1) has been prepared that undergoes temperature-induced folding and self-assembly to afford a network of beta-sheet rich fibrils that constitutes a mechanically rigid hydrogel. Circular dichroism and infrared spectroscopies show that TSS1 folds and self-assembles into a beta-sheet secondary structure in response to temperature. Rheological measurements show that the resulting hydrogels are mechanically rigid [at pH 9, G' = 1750-9000 Pa, and at pH 7.4, G' = 8500 Pa] and that the storage modulus can be modulated by temperature and peptide concentration. Nanoscale structure analysis by transmission electron microscopy and small angle neutron scattering indicate that the hydrogel network is comprised of fibrils that are about 3 nm in width, consistent with the width of TSS1 in the folded state. A unique property of the TSS1 hydrogel is its ability to shear-thin into a low viscosity gel upon application of shear stress and immediately recover its mechanical rigidity upon termination of stress. This attribute allows the hydrogel to be delivered via syringe to a target site with spatial and temporal resolution. Finally, experiments employing C3H10t1/2 mesenchymal stem cells seeded onto the hydrogel and incubated for 24 h indicate that the TSS1 hydrogel surface is noncytotoxic, supports cell adhesion, and allows cell migration.

The synthesis of poly(methyl methacrylate-co-methacryloxysuccinimide-graft-poly(ethylene glycol)) (PMMA-co-PMASI-g-PEG) via living free radical polymerization provides a convenient route to well-defined amphiphilic graft copolymers having a controllable number of reactive functional groups, variable length PEG grafts, and low polydispersity. These copolymers were shown to form PMMA-core/PEG-shell nanoparticles upon hydrophobic collapse in water, with the hydrodynamic size being defined by the molecular weight of the backbone and the PEG grafts. Functionalization of these polymeric nanoparticles with a 1,4,7,10-tetraazacyclododecanetetraacetic acid (DOTA) ligand capable of chelating radioactive 64Cu nuclei enabled the biodistribution and in vivo positron emission tomography of these materials to be studied and directly correlated to the initial structure. Results indicate that nanoparticles with increasing PEG chain lengths show increased blood circulation and low accumulation in excretory organs, suggesting the possible use of these materials as stealth carriers for medical imaging and systemic administration.