Tunable Macroscopic Alignment of Self-Assembling Peptide Nanofibers.

Progress in the design and synthesis of nanostructured self-assembling systems has facilitated the realization of numerous nanoscale geometries, including fibers, ribbons, and sheets. A key challenge has been achieving control across multiple length scales and creating macroscopic structures with nanoscale organization. Here, we present a facile extrusion-based fabrication method to produce anisotropic, nanofibrous hydrogels using self-assembling peptides. The application of shear force coinciding with ion-triggered gelation is used to kinetically trap supramolecular nanofibers into aligned, hierarchical macrostructures. Further, we demonstrate the ability to tune the nanostructure of macroscopic hydrogels through modulating phosphate buffer concentration during peptide self-assembly. In addition, increases in the nanostructural anisotropy of fabricated hydrogels are found to enhance their strength and stiffness under hydrated conditions. To demonstrate their utility as an extracellular matrix-mimetic biomaterial, aligned nanofibrous hydrogels are used to guide directional spreading of multiple cell types, but strikingly, increased matrix alignment is not always correlated with increased cellular alignment. Nanoscale observations reveal differences in cell-matrix interactions between variably aligned scaffolds and implicate the need for mechanical coupling for cells to understand nanofibrous alignment cues. In total, innovations in the supramolecular engineering of self-assembling peptides allow us to decouple nanostructure from macrostructure and generate a gradient of anisotropic nanofibrous hydrogels. We anticipate that control of architecture at multiple length scales will be critical for a variety of applications, including the bottom-up tissue engineering explored here.

Tunable Macroscopic Alignment of Self-Assembling Peptide Nanofibers.

Fibrous proteins that comprise the extracellular matrix (ECM) guide cellular growth and tissue organization. A lack of synthetic strategies able to generate aligned, ECM-mimetic biomaterials has hampered bottom-up tissue engineering of anisotropic tissues and led to a limited understanding of cell-matrix interactions. Here, we present a facile extrusion-based fabrication method to produce anisotropic, nanofibrous hydrogels using self-assembling peptides. The application of shear force coinciding with ion-triggered gelation is used to kinetically trap supramolecular nanofibers into aligned, hierarchical structures. We establish how modest changes in phosphate buffer concentration during peptide self-assembly can be used to tune their alignment and packing. In addition, increases in the nanostructural anisotropy of fabricated hydrogels are found to enhance their strength and stiffness under hydrated conditions. To demonstrate their utility as an ECM-mimetic biomaterial, aligned nanofibrous hydrogels are used to guide directional spreading of multiple cell types, but strikingly, increased matrix alignment is not always correlated with increased cellular alignment. Nanoscale observations reveal differences in cell-matrix interactions between variably aligned scaffolds and implicate the need for mechanical coupling for cells to understand nanofibrous alignment cues. In total, innovations in the supramolecular engineering of self-assembling peptides allow us to generate a gradient of anisotropic nanofibrous hydrogels, which are used to better understand directed cell growth.

Stabilization of Synthetic Collagen Triple Helices: Charge Pairs and Covalent Capture.

Collagen mimetic peptides are composed of triple helices. Triple helical formation frequently utilizes charge pair interactions to direct protein assembly. The design of synthetic triple helices is challenging due to the large number of competing species and the overall fragile nature of collagen mimetics. A successfully designed triple helix incorporates both positive and negative criteria to achieve maximum specificity of the supramolecular assembly. Intrahelical charge pair interactions, particularly those involved in lysine-aspartate and lysine-glutamate pairs, have been especially successful both in driving helix specificity and for subsequent stabilization by covalent capture. Despite this progress, the important sequential and geometric relationships of charged residues in a triple helical context have not been fully explored for either supramolecular assembly or covalent capture stabilization. In this study, we compare the eight canonical axial and lateral charge pairs of lysine and arginine with glutamate and aspartate to their noncanonical, reversed charge pairs. These findings are put into the context of collagen triple helical design and synthesis.

Dynamic Imine Bonding Facilitates Mannan Release from a Nanofibrous Peptide Hydrogel.

Recently, there has been increased interest in using mannan as an immunomodulatory bioconjugate. Despite notable immunological and functional differences between the reduced (R-Man) and oxidized (O-Man) forms of mannan, little is known about the impact of mannan oxidation state on its in vivo persistence or its potential controlled release from biomaterials that may improve immunotherapeutic or prophylactic efficacy. Here, we investigate the impact of oxidation state on the in vitro and in vivo release of mannan from a biocompatible and immunostimulatory multidomain peptide hydrogel, K2(SL)6K2 (abbreviated as K2), that has been previously used for the controlled release of protein and small molecule payloads. We observed that O-Man released more slowly from K2 hydrogels in vitro than R-Man. In vivo, the clearance of O-Man from K2 hydrogels was slower than O-Man alone. We attributed the slower release rate to the formation of dynamic imine bonds between reactive aldehyde groups on O-Man and the lysine residues on K2. This imine interaction was also observed to improve K2 + O-Man hydrogel strength and shear recovery without significantly influencing secondary structure or peptide nanofiber formation. There were no observed differences in the in vivo release rates of O-Man loaded in K2, R-Man loaded in K2, and R-Man alone. These data suggest that, after subcutaneous injection, R-Man naturally persists longer in vivo than O-Man and minimally interacts with the peptide hydrogel. These results highlight a potentially critical, but previously unreported, difference in the in vivo behavior of O-Man and R-Man and demonstrate that K2 can be used to normalize the release of O-Man to that of R-Man. Further, since K2 itself is an adjuvant, a combination of O-Man and K2 could be used to enhance the immunostimulatory effects of O-Man for applications such as infectious disease vaccines and cancer immunotherapy.

Cation-π Interactions and Their Role in Assembling Collagen Triple Helices.

Cation-π interactions play a significant role in the stabilization of globular proteins. However, their role in collagen triple helices is less well understood and they have rarely been used in de novo designed collagen mimetic systems. In this study, we analyze the stabilizing and destabilizing effects in pairwise amino acid interactions between cationic and aromatic residues in both axial and lateral sequential relationships. Thermal unfolding experiments demonstrated that only axial pairs are stabilizing, while the lateral pairs are uniformly destabilizing. Molecular dynamics simulations show that pairs with an axial relationship can achieve a near-ideal interaction distance, but pairs in a lateral relationship do not. Arginine-π systems were found to be more stabilizing than lysine-π and histidine-π. Arginine-π interactions were then studied in more chemically diverse ABC-type heterotrimeric helices, where arginine-tyrosine pairs were found to form the best helix. This work helps elucidate the role of cation-π interactions in triple helices and illustrates their utility in designing collagen mimetic peptides.

Single-atom replacement as a general approach towards visible-light/near-infrared heavy-atom-free photosensitizers for photodynamic therapy.

Photodynamic therapy has become an emerging strategy for the treatment of cancer. This technology relies on the development of photosensitizers (PSs) that convert molecular oxygen to cytotoxic reactive oxygen species upon exposure to light. In this study, we have developed a facile and general strategy for obtaining visible light/near-infrared-absorbing PSs by performing a simple sulfur-for-oxygen replacement within existing fluorophores. Thionation of carbonyl groups within existing fluorophore cores leads to an improvement of the singlet oxygen quantum yield and molar absorption coefficient at longer wavelengths (deep to 600-800 nm). Additionally, these thio-based PSs lack dark cytotoxicity but exhibit significant phototoxicity against monolayer cancer cells and 3D multicellular tumor spheroids with IC50 in the micromolar range. To achieve tumor-specific delivery, we have conjugated these thio-based PSs to an antibody and demonstrated their tumor-specific therapeutic activity.

Critical material content in modern conventional U.S. vehicle electronics.

Critical materials (CMs) are vital to modern technology. Components of modern vehicles can be recycled to recover and reuse the CMs to help ensure a supply of these materials. Electronic components from a 2015 GMC Sierra truck (21 components) and 2016 Toyota Camry sedan (10 components) were analyzed for CMs. The components were processed via size reduction, aqua regia leaching and dissolution, and final solutions were analyzed for metal content. It was found that most electronic components of both vehicles contain CMs. The most concentrated CMs in the components were Sn, Nb, and Tb. Nd and Co were found in several of the magnetic components. CM economic value was found to be low compared to the overall value of the components, and the CM content would not allow for a viable pathway for recycling. Remanufacturing of components may be a more economic option of reuse in the future.