A supramolecular polymer-collagen microparticle slurry for bone regeneration with minimal growth factor.

Recombinant bone morphogenetic protein-2 (BMP-2) is a potent osteoinductive growth factor that can promote bone regeneration for challenging skeletal repair and even for ectopic bone formation in spinal fusion procedures. However, serious clinical side effects related to supraphysiological dosing highlight the need for advances in novel biomaterials that can significantly reduce the amount of this biologic. Novel biomaterials could not only reduce clinical side effects but also expand the indications for use of BMP-2, while at the same time lowering the cost of such procedures. To achieve this objective, we have developed a slurry containing a known supramolecular polymer that potentiates BMP-2 signaling and porous collagen microparticles. This slurry exhibits a paste-like consistency that stiffens into an elastic gel upon implantation making it ideal for minimally invasive procedures. We carried out in vivo evaluation of the novel biomaterial in the rabbit posterolateral spine fusion model, and discovered efficacy at unprecedented ultra-low BMP-2 doses (5 µg/implant). This dose reduces the growth factor requirement by more than 100-fold relative to current clinical products. This observation is significant given that spinal fusion involves ectopic bone formation and the rabbit model is known to be predictive of human efficacy. We expect the novel biomaterial can expand BMP-2 indications for difficult cases requiring large volumes of bone formation or involving patients with underlying conditions that compromise bone regeneration.

Optimization of peptide amphiphile-lipid raft interaction by changing peptide amphiphile lipophilicity.

Various peptide amphiphile (PA) molecules have been developed to promote bone regeneration. Previously we discovered that a peptide amphiphile with a palmitic acid tail (C16) attenuates the signaling threshold of leucine-rich amelogenin peptide (LRAP)-mediated Wnt activation by increasing membrane lipid raft mobility. In the current study, we found that treatment of murine ST2 cells with an inhibitor (Nystatin) or Caveolin-1-specific siRNA abolishes the effect of C16 PA, indicating that Caveolin-mediated endocytosis is required. To determine whether hydrophobicity of the PA tail plays a role in its signaling effect, we modified the length of the tail (C12, C16 and C22) or composition (cholesterol). While shortening the tail (C12) decreased the signaling effect, lengthening the tail (C22) had no prominent effect. On the other hand, the cholesterol PA displayed a similar function as the C16 PA at the same concentration of 0.001% w/v. Interestingly, a higher concentration of C16 PA (0.005%) is cytotoxic while cholesterol PA at the higher concentration (0.005%) is well-tolerated by cells. Use of the cholesterol PA at 0.005% enabled a further reduction of the signaling threshold of LRAP to 0.20 nM, compared to 0.25 nM at 0.001%. Caveolin-mediated endocytosis is also required for cholesterol PA, as evidenced by Caveolin-1 siRNA knockdown experiments. We further demonstrated that the noted effects of cholesterol PA are also observed in human bone marrow mesenchymal stem cells (BMMSCs). Taken together, these results indicate that the cholesterol PA modulates lipid raft/caveolar dynamics, thereby increasing receptor sensitivity for activation of canonical Wnt signaling. STATEMENT OF SIGNIFICANCE: Cell signaling involves not only the binding of growth factors (or other cytokines) and cognate receptors, but also their clustering on the cell membrane. However, little or no work has been directed thus far toward investigating how biomaterials can serve to enhance growth factor or peptide signaling by increasing diffusion of cell surface receptors within membrane lipid rafts. Therefore, a better understanding of the cellular and molecular mechanism(s) operating at the material-cell membrane interface during cell signaling has the potential to change the paradigm in designing future biomaterials and regenerative medicine therapeutics. In this study, we designed a peptide amphiphile (PA) with a cholesterol tail to enhance canonical Wnt signaling by modulating lipid raft/caveolar dynamics.

Heterocyclic Chromophore Amphiphiles and their Supramolecular Polymerization.

Supramolecular polymerization of π-conjugated amphiphiles in water is an attractive approach to create functional nanostructures. Here, we report on the synthesis, optoelectronic and electrochemical properties, aqueous supramolecular polymerization, and conductivity of polycyclic aromatic dicarboximide amphiphiles. The chemical structure of the model perylene monoimide amphiphile was modified with heterocycles, essentially substituting one fused benzene ring with thiophene, pyridine or pyrrole rings. All the heterocycle-containing monomers investigated underwent supramolecular polymerization in water. Large changes to the monomeric molecular dipole moments led to nanostructures with low electrical conductivity due to diminished interactions. Although the substitution of benzene with thiophene did not notably change the monomer dipole moment, it led to crystalline nanoribbons with 20-fold higher electrical conductivity, due to enhanced dispersion interactions as a result of the presence of sulfur atoms.

Sustained-release losartan from peptide nanofibers promotes chondrogenesis.

Introduction: The central pathologic feature of osteoarthritis (OA) is the progressive loss of articular cartilage, which has a limited regenerative capacity. The TGF-ß1 inhibitor, losartan, can improve cartilage repair by promoting hyaline rather that fibrous cartilage tissue regeneration. However, there are concerns about side effects associated with oral administration and short retention within the joint following intra-articular injections. To facilitate local and sustained intra-articular losartan delivery we have designed an injectable peptide amphiphile (PA) nanofiber that binds losartan. The aims of this study are to characterize the release kinetics of losartan from two different PA nanofiber compositions followed by testing pro-regenerative bioactivity on chondrocytes. Methods: We tested the impact of electrostatic interactions on nanostructure morphology and release kinetics of the negatively charged losartan molecule from either a positively or negatively charged PA nanofiber. Subsequently, cytotoxicity and bioactivity were evaluated in vitro in both normal and an IL-1ß-induced OA chondrocyte model using ATDC5. Results: Both nanofiber systems promoted cell proliferation but that the positively-charged nanofibers also significantly increased glycosaminoglycans production. Furthermore, gene expression analysis suggested that losartan-encapsulated nanofibers had significant anti-inflammatory, anti-degenerative, and cartilage regenerative effects by significantly blocking TGF-ß1 in this in vitro system. Discussion: The results of this study demonstrated that positively charged losartan sustained-release nanofibers may be a novel and useful treatment for cartilage regeneration and OA by blocking TGF-ß1.

Artificial extracellular matrix scaffolds of mobile molecules enhance maturation of human stem cell-derived neurons.

Human induced pluripotent stem cell (hiPSC) technologies offer a unique resource for modeling neurological diseases. However, iPSC models are fraught with technical limitations including abnormal aggregation and inefficient maturation of differentiated neurons. These problems are in part due to the absence of synergistic cues of the native extracellular matrix (ECM). We report on the use of three artificial ECMs based on peptide amphiphile (PA) supramolecular nanofibers. All nanofibers display the laminin-derived IKVAV signal on their surface but differ in the nature of their non-bioactive domains. We find that nanofibers with greater intensity of internal supramolecular motion have enhanced bioactivity toward hiPSC-derived motor and cortical neurons. Proteomic, biochemical, and functional assays reveal that highly mobile PA scaffolds caused enhanced ß1-integrin pathway activation, reduced aggregation, increased arborization, and matured electrophysiological activity of neurons. Our work highlights the importance of designing biomimetic ECMs to study the development, function, and dysfunction of human neurons.

Modeling Interactions within and between Peptide Amphiphile Supramolecular Filaments.

Many peptides are able to self-assemble into one-dimensional (1D) nanostructures, such as cylindrical fibers or ribbons of variable widths, but the relationship between the morphology of 1D objects and their molecular structure is not well understood. Here, we use coarse-grained molecular dynamics (CG-MD) simulations to study the nanostructures formed by self-assembly of different peptide amphiphiles (PAs). The results show that ribbons are hierarchical superstructures formed by laterally assembled cylindrical fibers. Simulations starting from bilayer structures demonstrate the formation of filaments, whereas other simulations starting from filaments indicate varying degrees of interaction among them depending on chemical structure. These interactions are verified by observations using atomic force microscopy of the various systems. The interfilament interactions are predicted to be strongest in supramolecular assemblies that display hydrophilic groups on their surfaces, while those with hydrophobic ones are predicted to interact more weakly as confirmed by viscosity measurements. The simulations also suggest that peptide amphiphiles with hydrophobic termini bend to reduce their interfacial energy with water, which may explain why these systems do not collapse into superstructures of bundled filaments. The simulations suggest that future experiments will need to address mechanistic questions about the self-assembly of these systems into hierarchical structures, namely, the preformation of interactive filaments vs equilibration of large assemblies into superstructures.

3D Printing of Supramolecular Polymer Hydrogels with Hierarchical Structure.

Liquid crystalline hydrogels are an attractive class of soft materials to direct charge transport, mechanical actuation, and cell migration. When such systems contain supramolecular polymers, it is possible in principle to easily shear align nanoscale structures and create bulk anisotropic properties. However, reproducibly fabricating and patterning aligned supramolecular domains in 3D hydrogels remains a challenge using conventional fabrication techniques. Here, a method is reported for 3D printing of ionically crosslinked liquid crystalline hydrogels from aqueous supramolecular polymer inks. Using a combination of experimental techniques and molecular dynamics simulations, it is found that pH and salt concentration govern intermolecular interactions among the self-assembled structures where lower charge densities on the supramolecular polymers and higher charge screening from the electrolyte result in higher viscosity inks. Enhanced hierarchical interactions among assemblies in high viscosity inks increase the printability and ultimately lead to greater nanoscale alignment in extruded macroscopic filaments when using small nozzle diameters and fast print speeds. The use of this approach is demonstrated to create materials with anisotropic ionic and electronic charge transport as well as scaffolds that trigger the macroscopic alignment of cells due to the synergy of supramolecular self-assembly and additive manufacturing.

Fast and programmable locomotion of hydrogel-metal hybrids under light and magnetic fields.

The design of soft matter in which internal fuels or an external energy input can generate locomotion and shape transformations observed in living organisms is a key challenge. Such materials could assist in productive functions that may range from robotics to smart management of chemical reactions and communication with cells. In this context, hydrated matter that can function in aqueous media would be of great interest. Here, we report the design of hydrogels containing a scaffold of high-aspect ratio ferromagnetic nanowires with nematic order dispersed in a polymer network that change shape in response to light and experience torques in rotating magnetic fields. The synergistic response enables fast walking motion of macroscopic objects in water on either flat or inclined surfaces and also guides delivery of cargo through rolling motion and light-driven shape changes. The theoretical description of the response to the external energy input allowed us to program specific trajectories of hydrogel objects that were verified experimentally.

Supramolecular-covalent hybrid polymers for light-activated mechanical actuation.

The development of synthetic structures that mimic mechanical actuation in living matter such as autonomous translation and shape changes remains a grand challenge for materials science. In living systems the integration of supramolecular structures and covalent polymers contributes to the responsive behaviour of membranes, muscles and tendons, among others. Here we describe hybrid light-responsive soft materials composed of peptide amphiphile supramolecular polymers chemically bonded to spiropyran-based networks that expel water in response to visible light. The supramolecular polymers form a reversibly deformable and water-draining skeleton that mechanically reinforces the hybrid and can also be aligned by printing methods. The noncovalent skeleton embedded in the network thus enables faster bending and flattening actuation of objects, as well as longer steps during the light-driven crawling motion of macroscopic films. Our work suggests that hybrid bonding polymers, which integrate supramolecular assemblies and covalent networks, offer strategies for the bottom-up design of soft matter that mimics living organisms.

Enhanced Out-of-Plane Conductivity and Photovoltaic Performance in n = 1 Layered Perovskites through Organic Cation Design.

Layered perovskites with the formula (R-NH3)2PbI4 have excellent environmental stability but poor photovoltaic function due to the preferential orientation of the semiconducting layer parallel to the substrate and the typically insulating nature of the R-NH3+ cation. Here, we report a series of these n = 1 layered perovskites with the form (aromatic- O-linker-NH3)2PbI4 where the aromatic moiety is naphthalene, pyrene, or perylene and the linker is ethyl, propyl, or butyl. These materials achieve enhanced conductivity perpendicular to the inorganic layers due to better energy level matching between the inorganic layers and organic galleries. The enhanced conductivity and visible absorption of these materials led to a champion power conversion efficiency of 1.38%, which is the highest value reported for any n = 1 layered perovskite, and it is an order of magnitude higher efficiency than any other n = 1 layered perovskite oriented with layers parallel to the substrate. These findings demonstrate the importance of leveraging the electronic character of the organic cation to improve optoelectronic properties and thus the photovoltaic performance of these chemically stable low n layered perovskites.

Covalent-supramolecular hybrid polymers as muscle-inspired anisotropic actuators.

Skeletal muscle provides inspiration on how to achieve reversible, macroscopic, anisotropic motion in soft materials. Here we report on the bottom-up design of macroscopic tubes that exhibit anisotropic actuation driven by a thermal stimulus. The tube is built from a hydrogel in which extremely long supramolecular nanofibers are aligned using weak shear forces, followed by radial growth of thermoresponsive polymers from their surfaces. The hierarchically ordered tube exhibits reversible anisotropic actuation with changes in temperature, with much greater contraction perpendicular to the direction of nanofiber alignment. We identify two critical factors for the anisotropic actuation, macroscopic alignment of the supramolecular scaffold and its covalent bonding to polymer chains. Using finite element analysis and molecular calculations, we conclude polymer chain confinement and mechanical reinforcement by rigid supramolecular nanofibers are responsible for the anisotropic actuation. The work reported suggests strategies to create soft active matter with molecularly encoded capacity to perform complex tasks.