Mark T. McClendon

Self-assembling hydrogel scaffolds for photocatalytic hydrogen production

Integration into a soft material of all the molecular components necessary to generate storable fuels is an interesting target in supramolecular chemistry. The concept is inspired by the internal structure of photosynthetic organelles, such as plant chloroplasts, which colocalize molecules involved in light absorption, charge transport and catalysis to create chemical bonds using light energy. We report here on the light-driven production of hydrogen inside a hydrogel scaffold built by the supramolecular self-assembly of a perylene monoimide amphiphile. The charged ribbons formed can electrostatically attract a nickel-based catalyst, and electrolyte screening promotes gelation. We found the emergent phenomenon that screening by the catalyst or the electrolytes led to two-dimensional crystallization of the chromophore assemblies and enhanced the electronic coupling among the molecules. Photocatalytic production of hydrogen is observed in the three-dimensional environment of the hydrogel scaffold and the material is easily placed on surfaces or in the pores of solid supports.

Tubular hydrogels of circumferentially aligned nanofibers to encapsulate and orient vascular cells.

There is a great clinical need for tissue engineered blood vessels that could be used to replace or bypass damaged arteries. The success of such grafts will depend strongly on their ability to mimic the cellular and matrix organization found in native arteries, but currently available cell scaffolds such as electrospun fibers or hydrogels lack the ability to simultaneously encapsulate and align cells. Our laboratory has recently developed liquid crystalline solutions of peptide amphiphile nanofibers that form aligned domains at exceedingly low concentrations (<1 wt%), and can be trapped as gels with macroscopic alignment using low shear rates and ionic crosslinking. We describe here the use of these systems to fabricate tubes with macroscopic circumferential alignment and demonstrate their potential as arterial cell scaffolds. The nanofibers in these tubes were circumferentially aligned by applying small amounts of shear in a custom built flow chamber prior to gelation. Small angle X-ray scattering confirmed that the direction of nanofiber alignment was the same as the direction of shear flow. We also show the encapsulation of smooth muscle cells during the fabrication process without compromising cell viability. After two days in culture the encapsulated cells oriented their long axis in the direction of nanofiber alignment thus mimicking the circumferential alignment seen in native arteries. Cell density roughly doubled after 12 days demonstrating the scaffolds ability to facilitate necessary graft maturation. Since these nanofiber gels are composed of >99% water by weight, the cells have abundant room for proliferation and remodeling. In contrast to previously reported arterial cell scaffolds, this new material can encapsulate cells and direct cellular organization without the requirement of external stimuli or gel compaction.

Gd(III)-labeled peptide nanofibers for reporting on biomaterial localization in vivo.

Bioactive supramolecular nanostructures are of great importance in regenerative medicine and the development of novel targeted therapies. In order to use supramolecular chemistry to design such nanostructures, it is extremely important to track their fate in vivo through the use of molecular imaging strategies. Peptide amphiphiles (PAs) are known to generate a wide array of supramolecular nanostructures, and there is extensive literature on their use in areas such as tissue regeneration and therapies for disease. We report here on a series of PA molecules based on the well-established β-sheet amino acid sequence V3A3 conjugated to macrocyclic Gd(III) labels for magnetic resonance imaging (MRI). These conjugates were shown to form cylindrical supramolecular assemblies using cryogenic transmission electron microscopy and small-angle X-ray scattering. Using nuclear magnetic relaxation dispersion analysis, we observed that thermal annealing of the nanostructures led to a decrease in water exchange lifetime (τm) of hundreds of nanoseconds only for molecules that self-assemble into nanofibers of high aspect ratio. We interpret this decrease to indicate more solvent exposure to the paramagnetic moiety on annealing, resulting in faster water exchange within angstroms of the macrocycle. We hypothesize that faster water exchange in the nanofiber-forming PAs arises from the dehydration and increase in packing density on annealing. Two of the self-assembling conjugates were selected for imaging PAs after intramuscular injections of the PA C16V3A3E3-NH2 in the tibialis anterior muscle of a murine model. Needle tracts were clearly discernible with MRI at 4 days postinjection. This work establishes Gd(III) macrocycle-conjugated peptide amphiphiles as effective tracking agents for peptide amphiphile materials in vivo over the timescale of days.

Electrospinning Bioactive Supramolecular Polymers from Water

Electrospinning is a high-throughput, low-cost technique for manufacturing long fibers from solution. Conventionally, this technique is used with covalent polymers with large molecular weights. We report here the electrospinning of functional peptide-based supramolecular polymers from water at very low concentrations (<4 wt %). Molecules with low molecular weights (<1 kDa) could be electrospun because they self-assembled into one-dimensional supramolecular polymers upon solvation and the critical parameters of viscosity, solution conductivity, and surface tension were optimized for this technique. The supramolecular structure of the electrospun fibers could ensure that certain residues, like bioepitopes, are displayed on the surface even after processing. This system provides an opportunity to electrospin bioactive supramolecular materials from water for biomedical applications.

Sulfated glycopeptide nanostructures for multipotent protein activation

Biological systems have evolved to utilize numerous proteins with capacity to bind polysaccharides for the purpose of optimizing their function. A well-known subset of these proteins with binding domains for the highly diverse sulfated polysaccharides are important growth factors involved in biological development and tissue repair. We report here on supramolecular sulfated glycopeptide nanostructures, which display a trisulfated monosaccharide on their surfaces and bind five critical proteins with very different polysaccharide binding domains. Binding does not disrupt the filamentous shape of the nanostructures or their internal β-sheet backbone, but must involve accessible adaptive configurations to interact with such different proteins. The glycopeptide nanostructures amplified signaling of bone morphogenetic protein 2 significantly more than the natural sulfated polysaccharide heparin, and promoted regeneration of bone in the spine with a protein dose that is 100-fold lower than expected. These super-bioactive nanostructures may enable many therapies in the horizon involving proteins.

Injectable biomimetic liquid crystalline scaffolds enhance muscle stem cell transplantation

Significance Most research aiming to achieve muscle regeneration focuses on the biology of “muscle stem cells,” but delivery methods that enhance transplantation efficiency of these cells are at early stages. We report on a liquid crystalline scaffold that encapsulates the cells and gels upon injection in vivo without requiring an external stimulus. As a unique structural feature, the scaffold contains nanofibers that align preferentially with surrounding natural muscle fibers. The biomimetic scaffold can have a stiffness that matches that of muscle, has great ability to retain growth factors, and has a biodegradation rate that is compatible with regeneration time scales. Most importantly, the scaffold enhances engraftment efficiency of the cells in injured muscle, and without injury when combined with growth factors. Muscle stem cells are a potent cell population dedicated to efficacious skeletal muscle regeneration, but their therapeutic utility is currently limited by mode of delivery. We developed a cell delivery strategy based on a supramolecular liquid crystal formed by peptide amphiphiles (PAs) that encapsulates cells and growth factors within a muscle-like unidirectionally ordered environment of nanofibers. The stiffness of the PA scaffolds, dependent on amino acid sequence, was found to determine the macroscopic degree of cell alignment templated by the nanofibers in vitro. Furthermore, these PA scaffolds support myogenic progenitor cell survival and proliferation and they can be optimized to induce cell differentiation and maturation. We engineered an in vivo delivery system to assemble scaffolds by injection of a PA solution that enabled coalignment of scaffold nanofibers with endogenous myofibers. These scaffolds locally retained growth factors, displayed degradation rates matching the time course of muscle tissue regeneration, and markedly enhanced the engraftment of muscle stem cells in injured and noninjured muscles in mice.

Reversible self-assembly of superstructured networks

Chemically reversible hydrogels The dynamic reorganization of some cellular biopolymers in response to signals has inspired efforts to create artificial materials with similar properties. Freeman et al. created hydrogels based on peptide amphiphiles that can bear DNA strands that assemble into superstructures and that disassemble in response to chemical triggers. The addition of DNA conjugates induced transitions from micelles to fibers and bundles of fibers. The resulting hydrogels were used as an extracellular matrix mimic for cultured cells. Switching the hydrogel between states also switched astrocytes between their reactive and naïve phenotypes. Science, this issue p. 808 Large-scale redistribution of molecules in a supramolecular material generates chemically reversible superstructures. Soft structures in nature, such as protein assemblies, can organize reversibly into functional and often hierarchical architectures through noncovalent interactions. Molecularly encoding this dynamic capability in synthetic materials has remained an elusive goal. We report on hydrogels of peptide-DNA conjugates and peptides that organize into superstructures of intertwined filaments that disassemble upon the addition of molecules or changes in charge density. Experiments and simulations demonstrate that this response requires large-scale spatial redistribution of molecules directed by strong noncovalent interactions among them. Simulations also suggest that the chemically reversible structures can only occur within a limited range of supramolecular cohesive energies. Storage moduli of the hydrogels change reversibly as superstructures form and disappear, as does the phenotype of neural cells in contact with these materials.

Electrophysiological assessment of a peptide amphiphile nanofiber nerve graft for facial nerve repair

Facial nerve injury can cause severe long‐term physical and psychological morbidity. There are limited repair options for an acutely transected facial nerve not amenable to primary neurorrhaphy. We hypothesize that a peptide amphiphile nanofiber neurograft may provide the nanostructure necessary to guide organized neural regeneration. Five experimental groups were compared, animals with (1) an intact nerve, (2) following resection of a nerve segment, and following resection and immediate repair with either a (3) autograft (using the resected nerve segment), (4) neurograft, or (5) empty conduit. The buccal branch of the rat facial nerve was directly stimulated with charge balanced biphasic electrical current pulses at different current amplitudes whereas nerve compound action potentials (nCAPs) and electromygraphic responses were recorded. After 8 weeks, the proximal buccal branch was surgically reexposed and electrically evoked nCAPs were recorded for groups 1–5. As expected, the intact nerves required significantly lower current amplitudes to evoke an nCAP than those repaired with the neurograft and autograft nerves. For other electrophysiologic parameters such as latency and maximum nCAP, there was no significant difference between the intact, autograft, and neurograft groups. The resected group had variable responses to electrical stimulation, and the empty tube group was electrically silent. Immunohistochemical analysis and transmission electron microscopy confirmed myelinated neural regeneration. This study demonstrates that the neuroregenerative capability of peptide amphiphile nanofiber neurografts is similar to the current clinical gold standard method of repair and holds potential as an off‐the‐shelf solution for facial reanimation and potentially peripheral nerve repair.

Abstract 3175: Sulfated glycopeptide nanostructures activate and extend bone morphogenetic protein 4 ability to increase pediatric malignant glioma chemotherapeutic efficacy

Pediatric gliomas, particularly high-grade gliomas, are among the most formidable and devastating cancers in children. These tumors have remained incurable, regardless of the many treatment approaches attempted. We recently identified a small population of glioma cells with stem-like features in pediatric gliomas (glioma stem cells: GSCs), that may be responsible, for therapeutic resistance in pediatric gliomas. Signaling by bone morphogenetic protein 4 (BMP4), an essential molecule for central nervous system (CNS) development, increases GSC therapeutic sensitivity, and whose activation is a promising adjuvant for glioma treatment. Mechanisms through which BMP4 increases therapeutic sensitivity need to be elucidated as this can lead to the identification of additional targets for treating malignant gliomas in children. Delivery systems for administering BMP4 in a clinical setting also need to be developed. Here, we show that BMP4 increases chemosensitivity by decreasing H3 lysine 4 trimethylation (H3K4me3) at the promoter of multidrug resistant gene 1 (MDR1) that, in turn, results in increased BMP4 expression. This appears to be the result of BMP4 decreasing levels of hSETD1A, a critical methyltransferase for H3K4me3. Our work also demonstrates the first use of a novel sulfated glycopeptide (glycol-PA) nanostructure as a vector for delivery of BMP4. Glycol-PA markedly extended and enhanced BMP4 function, and increased chemotherapeutic anti-tumor activity against pediatric malignant glioma cells in culture. Overall, our results expand understanding of how BMP4 brings about glioma therapeutic sensitization through epigenetic mechanisms, and show that highly bioactive glyco-PA nanostructures have potential as a novel delivery mechanism for treating pediatric malignant glioma, as well as other tumors. Citation Format: Guifa Xi, Benjamin Best, Sonali Nayak, Mark McClendon, Barbara Mania-Farnell, John Kessler, Charles David James, Samuel Stupp, Tadanori Tomita. Sulfated glycopeptide nanostructures activate and extend bone morphogenetic protein 4 ability to increase pediatric malignant glioma chemotherapeutic efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3175.

Correction to Gd(III)-Labeled Peptide Nanofibers for Reporting on Biomaterial Localization in Vivo

Page 7328. In Figure ​Figure2B2B, the leftmost panel is a cryogenic TEM image of PA2 rather than PA1. We include here a corrected panel for PA1. No conclusions or analysis in the work are affected by this correction. Figure 2B Image of PA1 nanofibers after thermal annealing. Scale bar is 200 nm.