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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.

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 ofmuscle tissue regeneration, and markedly enhanced the engraftment of muscle stem cells in injured and noninjured muscles in mice.

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.

Soft materials in nature are formed through programmed self-assembly of biomolecules to create complex architectures and optimized physical properties. It is therefore a key challenge in biomaterials science and engineering to understand the principles that govern the structure and properties of such materials, and the interactions between their different components. This work explores the self-assembly of a class of biomimetic molecules, their supramolecular interactions within multi-component systems, and their physical properties across length scales. The materials studied here are Peptide Amphiphiles (PAs), which mimic the self-assembly of peptides and lipids in biological systems to create ordered nanostructures that resemble natural biopolymers. PAs can assemble into various shapes such as twisted ribbons, flat sheets, long cylinders, and micelles, based on the sequence of peptides in the molecule and the charge on the headgroup. High aspect ratio PA nanofibers form networks that can be used as cell scaffolds for regenerative medicine applications. In the first section of this dissertation, nano structures formed by a series of charged hydrophobic PAs were investigated using high resolution atomic force microscopy (AFM). These PAs self-assembled into twisted ribbons of different widths and periodicity. A correlation was observed between the width and pitch lengths of these nanostructures, and a self-assembly model was developed to explain the morphology of the nanostructures based on a balance between the energetically favorable packing of molecules in the center of the nanostructures, the unfavorable packing at the edges, and the deformations due to packing of twisted β-sheets. The morphological polydispersity of PA nanostructures was determined by peptides sequences, and the model indicated that this phenomenon is related to the twisting of their internal β-sheets. There was a change in the supramolecular chirality of the nanostructures as the peptide sequence was systematically modified, even though only amino acids with L configuration were used. The nanostructures switched from left-handed to right-handed twist as alanines in the sequence were replaced by larger and more hydrophobic valines. In addition, increasing the electrostatic charge on the assemblies changed their morphology from ribbons to cylinders, and then to micelles. Interestingly, a subpopulation of the nanostructures formed a DNA-like double helix structure with alternating major and minor grooves. A physical model was developed by combining known theories of chiral amphiphilic membranes and peptide twist, which established the key role of assembly stiffness in the formation of the double helix. As the stiffness of the assemblies increased, the ribbons become more asymmetric, and shifted towards forming double helices instead of twisted ribbons. When used as cellular scaffolds, the stiffness of these nanostructures also controlled the remodeling capacity of the material. Coatings of flexible fibers primarily deformed during cell spreading and migration, while coatings of rigid fibers primarily degraded. This effect was associated with the presence of more entanglements in flexible fiber networks, which stabilize the coatings. In the second section, multi-component systems of different PAs were investigated using combined fluorescence microscopy and AFM, and both uniformly mixed and self-sorted network architectures were found. The supramolecular organization of the individual components was found to control the final state of the mixtures, where the self-sorting was governed by the complementarity of hydrogen bond orientations in the molecules. This approach was used to study the co-assembly of protein-binding PAs, whose functional activities are known to be modified by the nanostructure. A new approach to create interpenetrating supramolecular networks with sequestered binding epitopes was developed using a combination of self-sorting and uniformly mixing bioactive components. In the third section, the physical properties of PAs were studied from the length scale of molecular organization up to the microscale dynamics of nanofiber networks. Modeling of diffraction patterns showed that the molecules within nanostructures are arranged in a rectangular lattice, and are tilted in the direction parallel to the hydrogen bonding. The persistence length of PA fibers, a measure of bending stiffness, was comparable to those of natural structural biopolymers like actin and collagen. PA fiber networks also exhibited glassy dynamics at the micron scale, like most fibrous components of the ECM, which supports their use as biomimetic scaffolds. The dynamics of the networks were sensitive to the stiffness of nanofibers only at high concentrations, where the collisions between fibers dominate the properties of the networks. Together, these studies provide a deeper understanding of the nanoarchitectures and physical properties of materials formed by self-assembling peptide-based biomolecules.

Accurate measurements of kinetic rate constants for interacting biomolecules is crucial for understanding the mechanisms underlying intracellular signalling pathways. The magnitude of binding rates plays a very important molecular regulatory role which can lead to very different cellular physiological responses under different conditions. Here, we extend the k-space image correlation spectroscopy (kICS) technique to study the kinetic binding rates of systems wherein: (a) fluorescently labelled, free ligands in solution interact with unlabelled, diffusing receptors in the plasma membrane and (b) systems where labelled, diffusing receptors are allowed to bind/unbind and interconvert between two different diffusing states on the plasma membrane. We develop the necessary mathematical framework for the kICS analysis and demonstrate how to extract the elevant kinetic binding parameters of the underlying molecular system from fluorescence video-microscopy image time-series. Finally, by examining real data for two model experimental systems, we demonstrate how kICS can be a powerful tool to measure molecular transport coefficients and binding kinetics.

The pigment hemozoin is a natural by-product of the metabolism of hemoglobin by the parasites which cause malaria. Previously, hemozoin was demonstrated to have a very high nonlinear optical response enabling third harmonic generation (THG) imaging. In this study, we present a complete characterization of the nonlinear THG response of natural hemozoin in malaria-infected red blood cells, as well as in pure isostructural synthesized hematin anhydride, in order to determine optimal imaging parameters for detection. Our study demonstrates the wavelength range for optimal pulsed femtosecond laser excitation of THG from hemozoin crystals. In addition, we show the hemozoin crystal detection as a function of crystal size, incident laser power, and the emission response of the hemozoin crystals to different incident laser polarization states. Our systematic measurements of the nonlinear optical response from hemozoin establish detection limits, which are essential for the optimal design of malaria detection technologies that exploit the THG response of hemozoin. Figure Combined overlay image of THG (bright crystals in blue, one scan per frame) and TP autofluorescence (oval cells in red, average of 15 sequential frame scans) of natural hemozoin crystals and red blood cells (infected with FCR-3 Plasmodium falciparum ), respectively, collected at the laser excitation wavelength of 1170 nm with 100 mW average incident power and pixel dwell time of 5 μs

Injuries in the adult Central Nervous System (CNS) lead to permanent and debilitating loss of function, as injured CNS neurons do not typically regenerate. However, mechanical forces can trigger rapid growth in adult CNS neurons, and may represent an alternative therapy for neural repair. Previous work in our lab has shown that synapses can be induced to form in adult neurons by contact with a functionalized bead, and mechanical force can be used to induce electrically functional connections between neuronal populations. In this thesis, we present the development of new technique to measure the mechanical properties of induced neurites. This tool, called a micropipette force probe, provides a force resolution of 0.2 nN and a temporal resolution of 50 ms. We find that the force required to initiate neurites is on the order of 10 nanonewtons, and observe structural changes that cause nanonewton fluctuations in neurite tension. Compared to alternative techniques like Atomic Force Microscopy, this technique enables higher throughput investigation of induced neurites and parallelized recruitment of synapses.