Review

S. I. Stupp et al.

DOI: 10.1002/ijch.201300046

Supramolecular Nanofibers of Peptide Amphiphiles for Medicine Matthew J. Webber,[a, b] Eric J. Berns,[a, b] and Samuel I. Stupp*[b, c, d, e]

Abstract: Peptide nanostructures are an exciting class of supramolecular systems that can be designed for novel therapies with great potential in advanced medicine. This paper reviews progress on nanostructures based on peptide amphiphiles capable of forming one-dimensional assemblies that emulate in structure the nanofibers present in extracellular matrices. These systems are highly tunable using supramolecular chemistry, and can be designed to signal cells directly with bioactive peptides. Peptide amphiphile nanofibers can also be used to multiplex functions through

co-assembly and designed to deliver proteins, nucleic acids, drugs, or cells. We illustrate here the functionality of these systems, describing their use in regenerative medicine of bone, cartilage, the nervous system, the cardiovascular system, and other tissues. In addition, we highlight recent work on the use of peptide amphiphile assemblies to create hierarchical biomimetic structures with order beyond the nanoscale, and also discuss the future prospects of these supramolecular systems.

Keywords: amphiphiles · nanostructures · peptides · self-assembly · supramolecular chemistry

1. Introduction Since the recognition of pioneering work by Lehn, Pederson, and Cram with the 1987 Nobel Prize in Chemistry, supramolecular chemistry has gained considerable momentum and captured broad interest in many areas of science. “Chemistry beyond the molecule” is the pathway to design highly organized systems across multiple length scales without covalent bonds.[1] It is now clear that supramolecular chemistry is an integral part of the exponentially growing field of self-assembly, particularly in the context of materials.[2] Supramolecular self-assembly is a powerful strategy to craft functions in organic materials that require nanoscale structure with molecules interacting in highly specific ways. This could involve guiding structure with networks of hydrogen bonds, electrostatic or electronic interactions, hydrophobic-hydrophilic separation of molecular segments, and repulsive forces in assemblies of molecules, among many other complex interactions. The opportunity in supramolecular materials is to recreate the highly structured but dynamic environment of biological systems using the collective energies and reversibility of many non-covalent bonds. There are many highly functional self-assembling supramolecular structures in biological systems that provide great inspiration for materials design. The two-dimensional assembly of lipids creates cell membranes, which form a highly selective barrier between the intracellular milieu and the extracellular space while also providing a dynamic platform for cell signaling. Even though there is growing awareness that cell membranes are more 530

complex in their organization,[3] the self-assembly of phospholipids to form bilayers is fundamental to membrane form.[4] Another group of interesting structures are those that enclose and deliver cargo, as is the case for viral capsids formed by protein self-assembly to encapsulate genetic code.[5] Fibrous, one-dimensional objects of self-assembled components are also common in the biological world. These filament-like structures provide me[a] M. J. Webber, E. J. Berns Department of Biomedical Engineering Northwestern University, Evanston, Illinois, 60208 (USA) [b] M. J. Webber, E. J. Berns, S. I. Stupp Institute for BioNanotechnology in Medicine Northwestern University, Chicago, Illinois, 60611 (USA) [c] S. I. Stupp Department of Materials Science and Engineering Northwestern University, Evanston, Illinois, 60208 (USA) Cook Hall, Room 1127 2220 Campus Drive Evanston, IL 60208 (USA) phone: (847) 491 3002 fax: (847) 491 3010 e-mail: [email protected] [d] S. I. Stupp Department of Chemistry Northwestern University, Evanston, Illinois, 60208 (USA) [e] S. I. Stupp Department of Medicine Northwestern University, Chicago, Illinois, 60611 (USA)

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chanical support to cells and tissues and also harbor signaling cues for cells, as is the case for extracellular matrix components such as fibronectin and collagen; meanwhile, self-assembled filamentous components of the cytoskeleton (microtubules, microfilaments, and intermediate filaments) are critical for cell motility, division, and intracellular transport.[6] The direct involvement of one-dimensional supramolecular structures in cellular processes important during the development of tissues and organs provides biomimetic inspiration for the design of materials for regenerative medicine. These processes include cell migration, proliferation, and stem cell differentiation and are instructed largely by signals orchestrated in the fibrous bed of the extracellular matrix. Furthermore, materials Matthew J. Webber completed his undergraduate studies in chemical engineering at the University of Notre Dame (2006), and obtained both a Master’s degree in 2009 and a Ph.D. in 2011 in biomedical engineering from Northwestern University. His doctoral research focused on the development of peptide amphiphiles for cardiovascular applications. He is currently a postdoctoral fellow at the Massachusetts Institute of Technology. Eric Berns completed his undergraduate studies in biology and animal science at the University of Massachusetts in Amherst and obtained a Master’s degree in biomedical engineering from Boston University (USA) in 2007. He completed his Ph.D. in biomedical engineering in 2013 at Northwestern University, in the laboratory of Prof. Samuel I. Stupp. His doctoral research focused on the development of peptide amphiphiles and aligned scaffolds for neural regeneration.

2. Self-Assembly of Peptide Amphiphiles 2.1 Design of Peptide Amphiphiles

Samuel I. Stupp is Board of Trustees Professor of Chemistry, Materials Science, and Medicine at Northwestern University. He also serves as Director of the Institute for BioNanotechnology in Medicine and of the Louis A. Simpson and Kimberly K. Querrey Center for Regenerative Nanomedicine at Northwestern. He has degrees in chemistry and materials science from the University of California at Los Angeles and Northwestern University. His research is focused on self-assembly and supramolecular chemistry of materials, with special interest in the areas of energy and medicine.

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designed to promote regeneration should ideally use biologically derived components such as lipids, amino acids, nucleotides, and saccharides in order to maximize biocompatibility and also to ensure that they can be fully biodegradable after fulfilling their functions. Peptides, specifically, are a promising platform for the design of self-assembled materials with control over structural features at the nanoscale. The chemical design versatility derived from a diverse amino acid library and precise control over amino acid sequence affords a variety of possible secondary, tertiary, and quaternary structures through folding, hydrogen bonding, and hydrophobic interactions. Over the years, the Stupp laboratory has synthesized several classes of molecules that are rationally designed to assemble into supramolecular nanostructures as components of materials.[2b,7] This review describes the peptide amphiphiles developed by the group, which self-assemble in aqueous media into bioactive supramolecular nanoscale filaments. These “supramolecular polymers” emulate the architecture of fibrous components in the extracellular matrix of cells and can form soluble assemblies, gels, and coatings on solid substrates, as well as membranes and microcapsules in combination with covalent polymers.[7g,8] These systems, inspired by the fibrillar self-assembled proteins found in nature, have broad potential within a number of targets in regenerative medicine. Here, we highlight the progress made thus far in the development of these systems, beginning with molecular design and characterization of structures, and then transitioning into a number of in vivo studies aimed at their translation into clinical therapies. In addition, we will highlight recent work aimed at the preparation of assemblies with hierarchical order across several length scales and project what future these systems may have in designing novel therapies.

The typical peptide amphiphile (PA) incorporates a short hydrophobic segment on one end of a more hydrophilic oligopeptide sequence.[7g,8d,9] Figure 1 illustrates a representative chemical structure of a PA molecule, which is generally composed of four key structural regions.[7g,10] The first segment, the hydrophobic domain, typically consists of a long, traditionally saturated, alkyl tail. The second segment, immediately adjacent to the tail, consists of a short peptide sequence that promotes hydrogen bonding by formation of intermolecular b-sheets. A third segment typically contains acidic or basic amino acids to provide charge and enhance solubility in water and also to trigger structural changes such as gelation through pH changes or addition of salts. Finally, the fourth segment, at the terminus opposite the hydrophobic tail, is used to integrate within the molecule a bioactive signal, which

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Figure 1. A) Molecular structure of a representative peptide amphiphile with four rationally designed chemical entities. B) Molecular graphics illustration of the peptide amphiphile molecule and its self-assembly into nanofibers as well as an illustration of the cross section of these fibers, highlighting the extensive hydration of the peptide shell. C) TEM micrograph of IKVAV nanofibers. D) SEM micrograph of IKVAV nanofiber gel network. Reprinted with permission from Niece et al.[15h] (Part C) and Silva et al.[10] (Part D).

may consist of an epitope to interact with cell receptors, a segment that binds proteins or biomolecules, or a pharmacological agent. The first generation of this class of PAs containing these four structural regions were designed to self-assemble into cylindrical nanostructures of high aspect ratio that display on their surfaces a high concentration of bioactive signals. The hydrophobic tail is primarily responsible for the strongly amphiphilic nature of the molecule, and is a crucial molecular element in these self-assembling supramolecular systems. The amphiphilic character of the molecule results in hydrophobic collapse of the alkyl moiety into the core of the nanostructure. This allows the attached peptides to be specifically displayed on the nanostructure surface, where they are accessible to cells, proteins, or other biological targets. This hydrophobic tail can be tuned through the use of alkyl chains with varying lengths, or alternatively can be replaced with other hydrophobic moieties.[8d] The alkyl block is most often incorporated through the addition of palmitic acid, or a similar alkyl acid, to the N-terminal amine of the peptide using 532

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solid-phase synthetic methods and standard peptide coupling reagents. The short peptide domain adjacent to the alkyl segment is typically composed of amino acids with a strong propensity to form b-sheet hydrogen bonds. The principal axes of these b-sheets are primarily parallel to the long axis of the assembled nanofiber. This unique feature is largely what governs the one-dimensional assemblies formed by PA molecules developed by the Stupp laboratory.[11] The b-sheet driving force for self-assembly into high aspect ratio nanostructures is not limited to PAs, as other self-assembling systems have demonstrated the use of b-sheetforming peptide domains to prepare nanostructures and entangled nanofiber hydrogels.[12] The third element in the design strategy of PAs is to include a number of charged amino acids following the hydrophobic peptide sequence. This number should be enough to ensure adequate solubility in aqueous conditions and to assist with purification. In solutions of low ionic strength (i.e., long Debye length), electrostatic repulsion between like-charged residues can inhibit supra-

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Supramolecular Nanofibers of Peptide Amphiphiles for Medicine

molecular nanofiber formation or limit their dimensions. Thus, nanofiber growth can be triggered by changing pH or, alternatively, by increasing the concentration of screening electrolytes in the solution and thus shortening the Debye length. This salt-responsive design element is a critical feature in the development of therapies based on PAs. PA molecules can be combined with bioactive entities, such as proteins, growth factors, DNA, or cells, to form a liquid cocktail with very low viscosity that can be injected using a syringe. Upon injection into the tissue, however, electrolytes in the physiological environment can immediately promote supramolecular self-assembly of PAs into nanofibers and additionally promote the formation of entangled nanofiber networks at high concentrations. The signatures of this process of salt-mediated changes can be the formation of nanofiber gels that encapsulate a bioactive payload or cells, highly viscous solutions of PA nanofibers, or simply longer and more stable nanofibers. The final element in the design of PAs developed by the Stupp laboratory, which makes them highly versatile and applicable for a number of therapeutic targets, is the incorporation and presentation of peptide epitopes on the end of the molecule opposite the hydrophobic domain. This enables bioactive signals to be displayed at controllable densities on the surface of the supramolecular nanofiber. For example, when interfacing PAs with cells, it is often desirable to display an epitope that promotes cell adhesion. Cells adhere to native extracellular matrix (ECM) through the formation of focal adhesions between cell membrane integrins and various ECM proteins.[13] Thus, the incorporation of cell-adhesion sequences displayed on the surface of PA nanofibers enables these nanostructures to both structurally and functionally mimic native ECM. A commonly used short peptide epitope that promotes such cell adhesion is RGDS. This epitope is present in many ECM proteins, notably fibronectin, and is specifically responsible for cell adhesion to these proteins.[14] Thus, this epitope has been frequently incorporated into PAs as a functional epitope for biological interaction with cells.[8g,15] Another common epitope is the laminin-derived IKVAV, a pentapeptide sequence crucial for neuron cell attachment, migration, and neurite outgrowth.[16] The IKVAV epitope has been specifically incorporated into PAs to provide bioactivity for neural applications.[10,17] Recent work has demonstrated the possibility of using light in order to control peptide epitope presentation on the PA nanofiber surface.[18] By incorporating a photocleavable nitrobenzyl ester group between the b-sheet region and the bioactive epitope, light could be used to remove a cell-adhesion epitope. This could be a useful strategy to dynamically modify the bioactivity of PA nanofibers in situ in order to alter the time course of bioactivity or to pattern gelled substrates with region-specific control of bioactivity.

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2.2 PA Self-Assembly Mechanisms

The driving forces that govern PA self-assembly in aqueous environments arise from three primary energy contributions: hydrophobic collapse of the alkyl tails accompanied by various enthalpic and entropic effects, hydrogen bonding and side chain interactions among the central peptide segments, and electrostatic repulsion among the charged amino acids. The final nanostructure in an aqueous environment, including its size, shape and interfacial curvature, should result from the complex interplay of all of these interactions. Extensive characterization of PA assemblies made from a large diversity of chemical architectures using transmission electron microscopy (TEM), cryogenic-TEM,[19] atomic force microscopy (AFM), and small-angle X-ray scattering (SAXS) confirm their strong tendency to self-assemble into high aspect ratio one-dimensional assemblies that are cylindrical in shape. Interestingly, a single hydrated PA molecule is predicted to have a tapered shape as a result of the hydrophobicity of the alkyl tail and the hydration of the polar or charged amino acids in the head group, and calculations predict that tapered molecules should self-assemble into objects with spherical geometry to allow molecules to pack most effectively.[20] Thus, the nature of molecular shape alone (i.e., tapered vs. linear) does seem to predict well the nature of the assembly and PA assemblies. More likely, the rigid and extended shape of b-sheets formed by amino acids adjacent to the hydrophobic tail is the subunit that encodes assembly of PAs into one-dimensional nanostructures. We have performed molecular simulations to understand the self-assembly behavior of PAs in water.[21] The model used in this work takes into account only hydrophobic interaction and intermolecular hydrogen bonding, neglecting repulsive forces due to charged head groups. In these simulations, if strong b-sheet hydrogen bonding is eliminated from the assembly, the hydrophobic interactions of these molecules predict spherical micelles of finite size. In this case, this micellization adheres to a closed association pathway with a nucleation mechanism above a critical micellization temperature and/or concentration. In contrast, if hydrophobic interactions are neglected, hydrogen bonding alone results in an open association pathway characterized by assemblies of one-dimensional b-sheets with a broad size distribution that result from step-by-step aggregation of molecules via hydrogen bonding. For a system in which both hydrophobic interactions and hydrogen bonding coexist, the kinetics and resultant nanostructure geometry differ depending on the strength of intermolecular hydrogen bonding. With relatively weak hydrogen bonding, spherical micelles may form with random b-sheets interspersed throughout their corona. In this case, the ordering of bsheets is compromised to allow the assembly to adopt a spherical geometry. Increasing the hydrogen-bonding energy, however, disrupts this spherical geometry, result-

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ing in long one-dimensional cylindrical fibers where bsheets grow along the long axis of the nanofiber. Therefore, it is presumed to be this combined effect of intermolecular hydrogen bonding among the peptide segments and the hydrophobic collapse of alkyl tails that leads to the formation of cylindrical nanofibers in dilute aqueous solutions, as opposed to assemblies of spherical geometry as the shape of the solvated individual molecule would predict. In experimental studies, the first four amino acids closest to the hydrophobic core, in the so-called b-sheet region, were found to be a critical determinant in the formation of cylindrical micelles.[22] When the hydrogen bonding between these first four amino acids was disrupted, spherical micelles formed. 2.3 Internal Structure of PA Nanofibers

The molecular packing of PA molecules within nanofibers governs not only the morphological and structural features of the nanofibers, but also their functional properties including the organization and spacing of epitopes presented on the surface. As a result, this internal packing within PA nanofibers should greatly affect molecular dynamics and biological signaling of these nanostructures and therefore their performance as biomaterials and therapies. While PAs commonly have a linear peptide segment, the molecular architecture can be modified to include branched structures via orthogonal chain growth from the e-amine of a lysine residue or cyclic peptide segments on the end opposite the hydrophobic domain. These changes in molecular architecture are predicted to affect molecular packing and peptide density on the nanofiber surface. Studies have shown that the binding of avidin to a biotin-functionalized nanofiber was highly dependent on the molecular architecture and, therefore, the molecular packing of PA molecules within the nanostructures.[15f] Additional work has evaluated the effect of molecular structure on the assembly and density of presented epitopes, using linear, branched, and cyclic peptide architectures.[15d,k] In this work, it was found that branched structures resulted in enhanced cell signaling, thought to be due to enhanced epitope accessibility as a result of increased molecular spacing. We have also studied the molecular packing and internal structure of nanofibers using a number of spectroscopic techniques including circular dichroism (CD), nuclear magnetic resonance (NMR), transmission infrared spectroscopy (IR), polarizationmodulation infrared reflection-absorption spectroscopy (PM-IRRAS), and UV-Vis absorption and photoluminescence measurements. These studies have revealed the presence of b-sheets oriented parallel to the long axis of the fibers, contributing to the nanofiber morphology.[23] Assessing hydration of PA nanofibers using Stern Volmer quenching with PA molecules containing pendant tryptophan or pyrene fluorophores demonstrated that the peptide shell of PA nanofibers remains well solvated even 534

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in the assembled or gelled conditions.[24] Even fluorophores buried deeper within the hydrophobic core of the nanostructure demonstrated some quenching from the aqueous quencher, though less in comparison to those located closer to the nanofiber surface. These studies suggest a high degree of hydration in the peptide shell of PA assemblies. The molecular architecture of the PA leads to various degrees of internal order for the hydrophobic tails, detected using infrared techniques. This suggests that order of the hydrophobic segment correlates, to some extent, with order in the peptidic domain.[23] PAs with a branched peptide domain yielded nanofibers with the lowest degree of internal order, likely due to more disordered molecular packing in the peptide corona. Studies using PAs derivatized with diacetylene-containing hydrophobic tails suggest that a higher degree of internal order exists in the nanofibers formed by the linear PA.[25] In this work, diacetylene groups incorporated within the hydrophobic tail were polymerized to create polydiacetylene backbones. Because this is known to be a topotactic reaction, that polymerization occurred within the assembled nanofiber demonstrates a high degree of internal order. Moreover, this polymerization yielded more highly conjugated backbones when the diacetylene tail was conjugated to a linear PA than when it was conjugated to a PA with a branched peptide architecture, likely indicating increased order in molecular packing for the linear PA architecture.[25] The finding that PA assemblies are highly hydrated yet highly ordered peptides, and the ability to vary this degree of organization through design of the peptide domain, provide a very rich opportunity to rationally design PA molecules with control over epitope display and dynamics for control of bioactivity in these nanostructures. Co-assembly of multiple PAs into a single nanostructure offers another method to change the composition, internal structure, and possibly the dynamics of the assemblies. This adds an additional functionality to the PA nanofiber, allowing for bioactive epitopes to be multiplexed in a synergistic way to regulate cell activity. Towards this goal, we have demonstrated co-assembly of two oppositely charged PAs, each presenting a different bioactive sequence, in aqueous solution at physiological pH.[15h] In this case, electrostatic attraction between molecules contributes to the formation of mixed nanofibers that simultaneously present both biological signals on their surface. The degree of mixing was further studied by CD and FRET, which suggested strong co-assembly of different PAs, with the possibility of microdomains of the co-assembled molecules along the length of the nanofiber.[26] Mixing of PAs can also be a useful strategy to increase the spacing of epitopes for optimal recognition by proteins or receptors using methods where an epitopepresenting PA is mixed with a shorter, non-bioactive diluent molecule which would allow the epitope to be displayed protruding from the fiber surface. Such a strategy

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has been used to optimize RGDS epitope spacing and maximize cell adhesion to PA nanofibers.[15k,l] Along the same lines, we have demonstrated the possibility of co-assembling PAs with opposite peptide polarities, the N!C direction relative to the attachment point of the hydrophobic tail. Traditional PAs synthesized in our laboratory have either a free acid or a free amide group presented on the C-terminus, and the N-terminus is amidated with an alkyl chain. However, it is possible that some epitopes may require a free N-terminus in order to be bioactive. Therefore, in this work a new class of peptide amphiphiles was designed and synthesized in order to reverse the peptide polarity by attachment of an alkyl tail at the C-terminus, allowing for the peptide synthesis to result in presentation of the N-terminal domain on the surface of the nanostructure.[27] Further, co-assembly of PAs with opposite peptide polarities could enable multiplexing C-terminal and N-terminal bioactivities on the same nanofiber. 2.4 Control of Nanostructure Morphology

One method to control the shape of assembled nanostructures is through modifications in the design of the b-sheet peptide segment. Though most evidence has shown that self-assembly of PAs into cylindrical nanostructures is remarkably tolerant to changes within this central peptide region, in recent work we have found that certain modifications in this region can greatly alter the geometry of the assembled nanostructure. One example has shown that introducing sequences with alternating hydrophobic and hydrophilic amino acids causes the PA to assemble in such a way as to eliminate all interfacial curvature and generate completely flat nanobelts.[28] Peptides with such an alternating sequence of hydrophobic and hydrophilic amino acids are known to have a strong propensity to form b-sheet structures. The structural motif used in this work to produce nanobelts, VEVE, orients the hydrophilic and hydrophobic side chains on opposite sides of the peptide backbone. In aqueous conditions, the valine surfaces have a tendency to associate in order to minimize solvent exposure, leading to dimerization of two PA molecules and creating, effectively, a molecule with similar geometry to that of a lipid with two hydrophobic alkyl tails. When assembling molecules with this shape, they tend to favor a flat (bilayer) packing geometry over a cylindrical one (Figure 2A). Increasing the number of VE dimeric repeats results in flat nanobelts with reduced width and increased b-sheet twisting.[29] If this alternating sequence is disrupted, exchanging the VEVE peptide segment with VVEE, the resulting nanostructures recapture their interfacial curvature and form traditional cylindrical nanofibers.[28] This work suggests that cylindrical geometries in PA assemblies may require weak intermolecular side chain interactions in the peptide sequence. At the same time, one expects assemblies with curvature to be Isr. J. Chem. 2013, 53, 530 – 554

Figure 2. A) Peptide amphiphiles with alternating hydrophobic and hydrophilic amino acid side chains can form flat, wide nanobelts. B) UV irradiation can be used to transform assemblies from quadruple helical fiber morphology to single fibers by photochemical cleavage of a 2-nitrobenzyl group. C) Electrostatic repulsion by amino acids in the charged region of the peptide amphiphile can enable transformation from spherical micelles to cylindrical nanofibers upon charge screening with divalent calcium ions. D) A phenylalanine-containing PA demonstrating a temporal transformation of nanostructure from twisted ribbons to helical ribbons. Reprinted with permission from: Cui et al.[28] (Part A), Muraoka et al.[30a] (Part B), Goldberger et al.[17d] (Part C), and Pashuck et al.[31] (Part D).

highly hydrated relative to flat dense assemblies. Hydration in curved assemblies may be critical for effective biological signaling through epitopes, both because of dy-

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namics as well as an entropic driving force upon epitope binding to receptors, other proteins, or biopolymers in the extracellular matrix. Another modification that can be made to the peptide region near the core of the assembly in order to alter nanostructure morphology is to incorporate bulky groups with the intent of altering the molecular packing. We have designed PA molecules that incorporate a photocleavable 2-nitrobenzyl group in the area between the palmitoyl tail and the peptide domain.[30] One PA designed with this photocleavable group self-assembled into quadruple helical nanostructures with a uniform helical pitch of ~ 92 nm when the 2-nitrobenzyl group was present.[30a] The quadruple helical structures were determined to be made from bundles of four individual nanofibers of 11 nm diameter. After irradiation to cleave the 2-nitrobenzyl group from the PA molecules, the helical structures transform into the typical cylindrical nanofibers (Figure 2B). When the peptide sequence was altered, in part, to present an RGDS motif for interaction with cells, the 2-nitrobenzyl modification resulted in the formation of spherical nanostructures.[30b] Again in this work, the common cylindrical shape was recovered following lightinduced cleavage of the 2-nitrobenzyl group, and at sufficiently high concentrations the solution underwent a solgel transformation upon exposure to light and cleavage of the 2-nitrobenzyl group. This molecule also allowed us to evaluate the dependence of cell signaling and bioactivity on nanostructure geometry by assessing the bioactivity of the RGDS epitope in both the spherical and cylindrical conformations. Data on cell-adhesion markers in this work point to an enhanced signaling capacity of epitopes presented on cylindrical nanofibers relative to the same epitope presented on spherical nanostructures. While incorporating a peptide epitope can impart PA nanofibers with biological function, its presence can negatively impact the assembly into desired nanofiber morphologies. For example, a recent study found that display of the IKVAV sequence – a sequence rich in hydrophobic amino acids – can promote aggregation of PAs into fused nanofiber bundles through interdigitation of the hydrophobic sequences on adjacent fibers.[17d] The propensity to aggregate was reduced by incorporating additional charged amino acids into the charged region of the molecules, increasing electrostatic repulsion in this region of the assembly. The addition of a sufficient number of charged residues disrupted b-sheet formation and induced micelle assembly rather than cylindrical assemblies in monovalent salt solutions. Screening the charges with divalent cations by addition of CaCl2 induced a transition from micelle to cylindrical nanofibers that were not aggregated as was the case for IKVAV PA versions with fewer charged amino acids (Figure 2C). Moreover, disruption of the bundling via electrostatic control significantly enhanced the bioactivity of IKVAV PA molecules, as measured by neurite growth. 536

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The b-sheet peptide region can be modified with bulky aromatic residues in order to alter the molecular packing and geometry of the assembled nanostructure. Recent work from our group (Figure 2D) has shown that the use of phenylalanine residues in this region leads initially to the formation of twisted ribbon structures which, over time, transition into helical ribbons.[31] This molecule allows both b-sheet formation and aromatic stacking as the intermolecular forces governing its assembly, likely leading to the planar structures. The observed transition in nanostructure twist occurs in the absence of any external stimuli, and rather is a transition over time. Thus, it is hypothesized that such a temporal change in nanostructure geometry is a result of a transition toward more stable molecular packing within the assembly. The control of length in PA nanofibers remains a challenge. As expected, designs that lead to poor internal packing tend to yield short fibers, and those with short linear peptides with high tendency toward b-sheet formation tend to generate long fibers.[32] We designed and synthesized a dumbbell-shaped template molecule with an oligo(p-phenylene ethynylene) core to control nanostructure formation and length.[33] In this templating approach, the hydrophobic backbone of the molecule was designed to instruct the self-assembly of PAs into nanofibers of a precise length through association with the hydrophobic portion of the polymer. AFM studies revealed that the length of the PA aggregate structures were consistent with that of the template when the two were mixed at a molar ratio of 200 : 1 (PA:template). However, this approach is only practical for cylindrical assemblies of very low aspect ratio, which no longer could be described as one-dimensional nanostructures. 2.5 Control of Nanofiber Gel Properties

Self-assembled PA nanostructures, at concentrations on the order of 1 % by weight (or even lower) in water, can entangle into gel networks under charge-screening conditions. Work has been done to control the gelation kinetics and rheological properties of these resulting gel networks through molecular design and counterion selection. The gelation kinetics of peptide amphiphile nanofiber networks can be tuned through modifications to the internal peptide sequence (b-sheet region) while keeping the bioactive domain constant, which allows for the creation of PA nanofiber gels with gelation times ranging from minutes to nearly an hour.[34] When the b-sheet region was modified to include more bulky and hydrophilic residues, replacing AAAAGGG with SLSLGGG, the time for gelation was found to increase significantly. This control in molecular design gives these systems flexibility as an injectable biomaterial by allowing the gelation time to be tuned to the desired application. A terminal IKVAV epitope, presented on these nanofibers with drastically different gelation times, showed no significant change in bio-

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activity and promoted the viability and neurogenesis of encapsulated neural progenitor cells. Another recent study by our group systematically assessed how changes in the internal peptide sequence can result in differences in rheological properties of the nanofiber gel.[35] These PAs were designed by varying the number and sequence of valine and alanine residues in the b-sheet domain. A strong correlation was found between gel stiffness and the degree to which the b-sheet axis twisted with respect to the long axis of the nanofiber. Molecules that assembled with the least amount of twisting and disorder in their b-sheets, as determined by CD and IR studies, were also found to produce gels with the highest mechanical stiffness. The ability to control the mechanical properties of a bulk gel through molecular design of the b-sheet-forming sequence on individual PA molecules establishes the importance of molecular packing on the overall organization and dynamics of these systems and provides the ability to finely control the bulk mechanical properties of these assemblies. PA gel properties can also be affected by the pH of the gelation solution or the presence of counterions, as both factors contribute to electrostatic screening of charged residues and enable the fiber entanglements and bundling necessary to form a gel network. It is known, for example, that screening by divalent and trivalent counterions results in more mechanically robust nanofiber gels, likely because such ions enable intermolecular and even interfiber ionic cross-linking whereas monovalent ions and changes in pH do not.[36] Moreover, even among ions with the same valency, d-block divalent ions such as Fe2 + , Zn2 + , and Cu2 + produce mechanically stiffer gels than sblock divalent ions such as Mg2 + , Ca2 + , and Ba2 + . Another means to control PA self-assembly and gelation is through pH. One interesting strategy in which we have used pH to influence the self-assembly and gelation of PA molecules demonstrated the ability to use light to trigger self-assembly and gelation of a liquid PA solution within liposomes.[37] Liquid solutions of a PA that was designed to self-assemble in acidic conditions were used to hydrate a dried phospholipid film, resulting in the PA solution being encapsulated within liposomes. Subsequently, a photoacid generator was used to trigger the assembly of these PAs by generating acid upon exposure to light. The encapsulation and light-triggered PA assembly were supported through the use of confocal fluorescent microscopy, SEM, FTIR and CD. Such a technique could allow the preparation of particles where PAs displaying bioactive epitopes could be delivered as densely packed nanofibers within the interior of liposomes for use in delivery of a bioactive PA payload to specific tissues or tumors. 2.6 Internal Covalent Capture of PA Assemblies

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chemical properties of these supramolecular nanofibers. The first work using PAs in our group incorporated cysteine residues within the peptide sequence to harness their ability to make disulfide bonds under oxidizing conditions.[7g,8d] In this work, PAs containing several cysteines were shown to form intermolecular hydrogen bonding upon nanofiber formation. Upon oxidation, intermolecular disulfide bonds formed that effectively cross-linked the nanofiber via covalent capture into a high molecular weight fibrous polymer. Due to the reversible nature of the disulfide bond, this cross-linking could be reversed by reducing agents. Another method that has been used to polymerize PA molecules and trap their supramolecular assemblies is through the incorporation of diacetylene groups in the hydrophobic segment.[25,38] Using UV irradiation, we have successfully polymerized PA molecules within nanofibers through the creation of a polydiacetylene backbone while retaining their cylindrical shape. This topotactic polymerization strategy in filamentous PA nanostructures does not interfere with the nanostructure shape control of the b-sheet, as the distance between flanking substituents is not changed as a result of the diacetylene polymerization. 2.7 Encapsulation of Small Molecules and Carbon Nanotubes

The hydrophobic core of PA nanofibers could make possible the encapsulation of payloads of hydrophobic drugs or small molecules, which could then be specifically delivered via targeting by epitopes on the nanofiber surface. Pyrene, a small hydrophobic molecule, was selected to establish this potential to encapsulate hydrophobic smallmolecule drugs within the PA nanofiber.[39] Pyrene encapsulation and aggregation within the core of PA nanofibers was inferred from the formation of a pyrene excimer. PA nanofibers in these studies were prepared from molecules with either cholesterol or palmitic acid hydrophobic domains. This excimer formation was also observed using peptide amphiphiles in which the pyrene is covalently linked as part of the hydrophobic tail. In another study, a monomeric precursor to a conducting polymer, poly(3,4-ethylenedioxythiophene) (PEDOT), was also successfully encapsulated within the hydrophobic core of PA nanofibers.[40] Following oxidative polymerization, PEDOT was formed and confined within the core of the insulating peptide amphiphile nanofiber. Such a redoxactive supramolecular nanostructure, formed by self-assembly of PAs presenting epitopes and surrounding a conducting core polymer, could be useful to prepare matrices that are both biologically and electronically active for biological signaling. PAs have also been used to encapsulate carbon nanotubes (CNTs) and assist in dispersing these in water (Figure 3A).[41] The encapsulation of nanotubes was confirmed using TEM and optical absorbance spectroscopy. This non-covalent functionalization of CNTs using PA

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Figure 3. A) PAs can be used to encapsulate multiwalled carbon nanotubes, simultaneously providing a method for non-covalently functionalizing the surface and enabling dispersion of the carbon nanotubes in an aqueous medium. B) TEM and electron diffraction showing the crystallographic alignment of hydroxyapatite nanocrystals that mineralize on PA nanofibers. C) Demonstration of a method for delivering and controlling the release rate of the cancer drug doxorubicin by enzymatically switching PA assemblies from a cylindrical nanofiber morphology to a disassembled state. Reprinted with permission from: Arnold et al.[41] (Part A), Spoerke et al.[43] (Part B) and Webber et al.[46] (Part C).

occurs without compromising their structural, electronic, or optical properties. The assembly of PA molecules on the surfaces of carbon nanotubes has the potential for adding a biofunctional shell coating these one-dimensional conductors, and similar to the PEDOT case, could be used to integrate bioactivity that combines biological and electronic signals. 2.8 Templating Mineral on PA Nanofiber Surfaces

The surface of the PA nanofibers can be customized at the molecular level to template mineralization. The first PAs designed by our laboratory were designed to template in a biomimetic fashion hydroxyapatite crystals, the primary mineral in bone and dentin.[7g] In these studies, 538

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PAs displayed phosphorylated serine residues in order to template mineralization. Phosphorylated serine, a nonstandard amino acid, is highly abundant in proteins that naturally template calcium phosphate in mineralized tissues.[42] Using the PA to template mineral produced hydroxyapatite mineral with the crystallographic c-axis aligned parallel to the long axis of the PA nanofiber. This is similar to the crystallographic orientation of hydroxyapatite crystals in bone with respect to the long axis of collagen fibers. The significance of a crucial enzyme, alkaline phosphatase, was subsequently determined to play a crucial role in the biomineralization of PA networks in three dimensions (Figure 3B).[43] The temporal control provided by the enzymatically mediated harvesting of phosphate ions enables PA nanofibers that present phosphorylated serine to nucleate hydroxyapatite on their surface, allowing for spatially controlled biomimetic mineralization of a three-dimensional PA scaffold. The ability of PAs to nucleate mineral could have implications in the development of therapies that promote regeneration in defects of bone and teeth. The stability of PA nanofibers allows mineralization under different, and even extreme, conditions. PAs have been used as templates for the nucleation and growth of cadmium sulfide (CdS) nanocrystals.[44] In these studies, nanofiber gels prepared from a PA with a phosphorylated serine residue were first broken up through a combination of mechanical agitation and sonication to form a dilute aqueous suspension. It was reasoned that, similar to nucleation of hydroxyapatite, the phosphorylated serine and acidic residues on the PA could provide the coordination necessary to adequately sequester Cd2 + . Cadmium nitrate tetrahydrate (Cd(NO3)2·4H2O) was then added as a source of Cd2 + and the mixture was subsequently exposed to hydrogen sulfide (H2S) gas, initiating the templated growth of the cadmium sulfide nanocrystals. Nanostructures with different morphologies were formed depending on the various molar ratios of Cd2 + :PA. In systems containing cadmium concentrations in slight excess to the PA (Cd2 + : PA = 2.4 : 1), individual CdS nanocrystals of 3–5 nm in diameter could be clearly seen to decorate the PA fibers. 2.9 Attachment of MRI Contrast Agents

Magnetic resonance imaging (MRI) is one of the most powerful diagnostic techniques in medicine, as it is able to provide three-dimensional structural information of living tissue at very high resolutions. One limitation to the technology, however, is the circulation time of the small-molecule contrast agents used for imaging. Previously, we covalently attached a molecule (DOTA) to the PA that chelates gadolinium in order to increase the relaxivity of the MRI agent.[15b,c] Even with MRI agent conjugation, the PA molecules retained their ability to selfassemble into cylindrical nanofibers or spheres in aque-

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ous conditions. The rotational correlation time of the MRI agent was increased upon self-assembly of the conjugated PA into nanostructures, suggesting enhanced relaxivity of the self-assembly. It was also found that the molecules relaxivity and imaging properties can be influenced by the position at which the PA molecule is conjugated with the DOTA derivative, showing an enhancement in relaxivity when the DOTA/Gd(III) complex was placed closer to the hydrophobic region. It was possible to image nanofiber gel networks of these MRI agent conjugated PAs using MRI.[15c] Future work can use this technology to examine PA constructs in vivo in order to track the fate of an implanted PA nanofiber gel and also to evaluate the efficacy of IV-administered PAs as a prolonged blood-pool contrast agent. 2.10 Drug Delivery Using PAs

The PA hydrophobic domain has demonstrated an ability to solubilize and deliver hydrophobic small molecules for applications in drug delivery. Chemotherapeutics with poor aqueous solubility, such as camptothecin, can be encapsulated in this core in order to improve their aqueous solubility over 50-fold.[45] This method of delivering the drug resulted in an anti-tumor effect in a breast cancer model. Enzyme-responsive nanofibers were also designed and used to prepare an enzyme trigger for drug delivery from PAs.[46] In this work, a PA was prepared that contained in its peptide domain a consensus substrate sequence for protein kinase A (PKA). In the presence of PKA, the serine residue of this consensus sequence becomes phosphorylated. The design of the PA featured a weak b-sheet domain, which enabled the molecule to switch from a nanofiber assembly to an unassembled state as a result of the serine phosphorylation (Figure 3C). This process could be reversed with the use of alkaline phosphatase, which cleaved the phosphate from the serine and restored the supramolecular nanofiber assembly. Using this specific enzyme, which is a known cancer biomarker, we solubilized the chemotherapeutic doxorubicin in the core of the nanofiber. The drug release rate was increased in the presence of PKA, and the nanofiber-delivered drug demonstrated improved toxicity in a cancer cell line that specifically produces this enzyme. The PA also provides an ideal platform for covalent chemistry that can enable drug release over time through hydrolysis. This was achieved by synthesizing a novel protected hydrazide-modified Fmoc-lysine residue that could be used in solid-phase synthesis of PAs or other peptides.[47] This residue could be incorporated in various positions during synthesis of the peptide segment of the molecule, and then following purification could be coupled to ketone- or aldehyde-containing drugs to form a hydrazone linkage. This affords long-term release of the compound over a period of months, but does not interfere with the ability of the PA to assemble into supramolecIsr. J. Chem. 2013, 53, 530 – 554

ular nanofibers or entangle into hydrogel networks. We recently reported on the preparation of a PA capable of releasing an anti-inflammatory drug, dexamethasone (Dex), by conjugation using this hydrazone linkage strategy.[48] Nanofiber gels of this PA demonstrated sustained release of soluble Dex for over one month in physiological media, while maintaining the anti-inflammatory activity of the drug in vitro. The ability of this gel to mitigate the inflammatory response in cell transplantation strategies was evaluated using cell-surrogate polystyrene microparticles suspended in the nanofiber gel that were then subcutaneously injected into a mouse. Live animal luminescence imaging using the chemiluminescent reporter molecule luminol showed a significant reduction in inflammation at the site where particles were injected with Dex-PA compared to the site of injection of particles within a control PA in the same animal. Histological evidence suggested a marked reduction in the number of infiltrating inflammatory cells when particles were delivered within Dex-PA nanofiber gels and very little inflammation was observed at either three days or 21 days postimplantation. The use of Dex-PA could facilitate localized anti-inflammatory activity as a component of biomaterials designed for various applications in regenerative medicine and could specifically be a useful module for PAbased therapies. Another interesting PA-based drug delivery strategy involved the therapeutic delivery of carbon monoxide from PA nanofiber gels.[49] Ruthenium-containing carbonyl compounds have been demonstrated to spontaneously release CO, a known anti-inflammatory signaling gas, in aqueous conditions. We recently prepared the first example of a material for therapeutic CO delivery by attaching a pendant ruthenium glycinate to a PA. This PA was capable of nanofiber gel formation, and spontaneously released CO in aqueous conditions. In vitro studies demonstrated that treatment of cardiomyocytes with this PA protected these cells from hydrogen peroxideinduced oxidative stress.

3. PA Supramolecular Assemblies for Regenerative Medicine 3.1 Regenerative Medicine

The desire for high quality of life and longevity demands continuous development of new therapeutic strategies aimed at regeneration of tissues and organs when function becomes compromised by injury, disease, congenital defects, or age. The field of regenerative medicine seeks to develop such therapies, with a focus on restoring form and function through the replacement or restoration of lost, damaged, aged, or dysfunctional cells and tissue. Promising targets include diseases and injuries of the nervous system such as paralysis, Parkinsons, Alzheimers, and multiple sclerosis, all of which are limited by an

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innate inability of the nervous system to regenerate. Heart disease and heart failure, which are among the leading causes of mortality worldwide, would benefit from new therapies to regenerate myocardium, another tissue with limited natural regenerative capacity. Regeneration of insulin-producing pancreatic b-cells could improve the lives of the many children inflicted with diabetes who suffer through regular insulin injections and blood glucose monitoring. The regeneration of adult teeth would be an improvement over dentures and dental implants. Damage to bones, tendons, ligaments, and cartilage are all an enormous source of pain and compromise active lifestyles. Surely, this is not an inclusive list of ailments that could benefit from new regenerative strategies. These conditions present complex problems, and therapies designed to combat them could benefit from an interdisciplinary perspective using knowledge from fields of stem cell science, developmental biology, molecular biology, genetics, materials science, chemistry, bioengineering, and tissue engineering.[50] In this section, we will review the recent progress our group has made in translating PA supramolecular assemblies as candidate therapies in pre-clinical work for diseases that affect a number of tissue sites, including therapies aimed at regeneration of the nervous system, cardiovascular system, pancreatic islets, bones, cartilage, and tooth enamel. While the basic element of a PA-based therapy is consistent throughout the work, the individual PA molecule can be varied and specifically tuned for a particular therapeutic target. The part of PA molecular structure that is most frequently modified for targets in regenerative medicine is the bioactive epitope region. This region can be tuned to maximize function for the regenerative processes required in a specific tissue. This would take into account the targeted cells and the specific signaling pathways that are important in achieving regenerative efficacy. Therefore, the bioactive sequences selected are chosen based on their ability to promote regeneration by favoring adhesion or proliferation of a specific cell type, facilitate the binding or delivery of a specific growth factor or signaling protein, or activate endogenous repair mechanisms within a diseased tissue. Other parameters, such as the biodegradation rate required for a specific target as well as mechanical properties of the artificial extracellular matrix required for a suitable cell response, may also lead to modifications of sequence in the PA structure. 3.2 PAs for Neural Regeneration

As human life expectancy increases, the demand for new therapies to alleviate the growing incidence of neurological disorders like Alzheimers and Parkinsons disease will, likewise, increase. Central to many neurological diseases and disorders is the loss of neuron connectivity or neuron death. Therefore, strategies that promote the gen540

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eration of new neurons to replace those lost from disease or that aim to restore connectivity in remaining neural tissue are being widely pursued. As the central nervous system has little capacity to regenerate itself through neurogenesis, stem cell therapy is one direction of scientific exploration, with the goal of differentiating stem or progenitor cells into neurons that integrate into the neural circuitry and restore function.[51] In conditions of traumatic injury to the nervous system, such as spinal cord injury, it is imperative to promote regeneration of axons that are damaged from the initial injury as well as those compromised by secondary damage.[52] Strategies to counteract the inhibitory effects on axonal regeneration that arise from the formation of a glial scar at the site of injury is a promising therapeutic approach to reversing paralysis from spinal cord injury. Over the past several years, a primary focus of our group has been to develop a PA-based therapy to promote axonal regeneration in the damaged spinal cord. PA nanofibers that present the laminin-derived IKVAV epitope sequence were developed to provide neural cells with a signal that is known to play a significant role in neurite outgrowth, cell attachment, migration, and differentiation. To evaluate the presentation of this epitope on the differentiation of neural cells, neural progenitor cells (NPCs) from the subventricular zone were cultured in nanofiber gels of IKVAV PA.[10] NPCs cultured within these gels underwent rapid and selective differentiation into neurons, evident by expression of the neuronal marker b-tubulin III (Figure 4). Meanwhile astrocytic differentiation, measured by glial fibrillary acidic protein (GFAP) expression, was suppressed compared to controls. NPCs cultured in the IKVAV gels also demonstrated significantly more neurite outgrowth than on control substrates PDL or laminin-coated coverslips, as well as in PA gels without a bioactive sequence. The promotion of selective differentiation into neurons when cultured within the PA was dependent on the concentration of the IKVAV PA, as gels formed with low concentrations of the IKVAV PA mixed with the control PA did not show the same ability to promote neuronal differentiation. Control PA gels in which the IKVAV peptide was added also did not promote neuronal differentiation. This suggests that the ability to display the epitope on the nanofibers at high concentrations plays an important role in the activity of the material. Additionally, changing nanofiber stiffness through modifying the b-sheet region and the peptide polarity has also been demonstrated to play an important role in the growth and maturation of neurons on PA nanofiber substrates.[53] The results from studies with NPCs demonstrating that the IKVAV PA promoted selective differentiation into neurons and increased neurite outgrowth, while simultaneously suppressing astrocytic differentiation, suggested that this PA might be useful as a therapy for spinal cord injury. In addition to its bioactivity, the PA is advanta-

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Figure 4. A) Immunohistochemistry of a neural progenitor cell (NPC) neurosphere encapsulated in a laminin-mimetic IKVAV PA gel for seven days, resulting in extensive neurite outgrowth. b-tubulin labeled green, GFAP labeled red. B) A higher percentage of NPCs differentiated into neurons (b-tubulin +) in IKVAV PA gels, compared to laminin and poly-d-lysine controls. C) A smaller percentage of NPCs differentiated into astrocytes (GFAP +) in IKVAV PA gels at seven days in vitro, compared to laminin and poly-d-lysine controls. D) At eleven weeks following spinal cord injury (SCI), descending motor fibers of mice show regeneration across the lesion in animals receiving IKVAV PA injection 24 hours after injury, but not in control animals. E) Mean mouse BBB locomotor scores show that IKVAV PA promotes functional recovery from SCI in mice, compared to control animals. Parts A–C adapted with permission from Silva et al.[10]  AAAS. Parts D and E reprinted with permission from Tysseling-Mattiace et al.[17c]

geous in that it can be injected as a liquid solution into damaged tissue, at which time the natural ionic conditions of the extracellular environment initiate its self-assembly into nanofibers in situ. Through a minimally invasive injection, the PA can be localized to the injury site without causing significantly more trauma to the already damaged spinal cord. To test whether the PA could promote recovery after such an injury, a spinal cord injury mouse model was employed.[17c] Material was injected into the damaged spinal cord 24 hours after injury, where it assembled and remained localized for more than two weeks. The presence of the nanofiber in the injury site resulted in a number of favorable effects. Immunohistochemistry revealed that astrogliosis was significantly reduced in spinal cords injected with IKVAV PA compared to controls. Apoptosis was reduced in and around the injury site. In addition, both motor and sensory axons were found to Isr. J. Chem. 2013, 53, 530 – 554

have regenerated across the lesion, whereas in control animals many axons abruptly ended at the boundary of the lesion, and those that did enter failed to cross the lesion (Figure 4). Functionally, mice receiving the IKVAV PA injections scored significantly higher on a scoring scale that measured motor recovery than did mice treated with IKVAV peptide, glucose, non-bioactive PA, and sham control groups. A follow-up study was carried out to determine if this therapy was useful in both compression and contusion injury models in both mice and rats, and also to gain better understanding of the mechanism behind the improved functional recovery in animals receiving the IKVAV PA therapy.[17b] In two different species, and in two different spinal cord injury models (compression and contusion), injection of IKVAV PA improved behavioral recovery. Careful analysis of the distribution and classification of neurons after the IKVAV PA

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treatment revealed that there were a greater number of serotonergic axons in the spinal cord caudal, but not rostral, to the lesion, relative to controls. This observation may explain part of the functional recovery, since serotonin is known to play a role in regulation of central pattern generators (CPG) and in locomotion. There were also higher trends in the total number of neurons near the lesion and long propriospinal tract connections between the lumbar and thoracic regions of the cord. To expand the range of potential PA systems and to study their effects on specific neuronal populations, a hybrid system composed of IKVAV PA and collagen (type 1) was developed and neurons from the cerebellar cortex were cultured on the gel surfaces.[17a] The cell responses of two subtypes of neurons – Purkinje cells and granule cells – were observed on gels composed of a range of PA concentrations. Granule cell density tripled on gels composed of  0.25 mg/mL IKVAV PA. On the other hand, Purkinje neurons were most dense at a concentration of 0.25 mg/mL IKVAV PA, and decreased at greater concentrations. The IKVAV PA concentration also had specific effects on dendrite and axon growth of these cells, showing that substrate composition could be used to control important aspects of the responses of specific cell populations. In the peripheral nervous system, the self-assembling PA platform has recently been shown to be useful in countering disorders resulting from cavernous nerve (CN) damage, which can lead to erectile dysfunction (ED).[54] Following damage to the CN, smooth muscles in the penis undergo apoptosis, leading to ED. In one study, the PA was used to trap and release sonic hedgehog (SHH) protein to the smooth muscle cells surrounding the corpora cavernosa in the penis by injecting the PA solution mixed with SHH directly into the corpora cavernosa, where the PA rapidly self-assembled into a network of nanofibers, trapping the protein in the process.[54b] This allowed SHH to be delivered to the cells over several days, resulting in significantly less smooth muscle apoptosis, compared to injections of PA with the control protein bovine serum albumin. The PA injections also showed no sign of immune response, investigated by CD3 protein immunohistochemical analysis. A second study investigated whether a related PA/SHH therapy could work directly towards regenerating the cavernous nerve, rather than simply mitigating the effects of its damage.[54a] In this investigation, a preformed gel composed of aligned PA nanofibers (the mechanism of alignment will be discussed in a later section) was applied bilaterally to crushed CNs of rats and was again used to deliver SHH. TEM analysis of the CN four weeks after injury revealed signs of regeneration in the PA-treated rats, showing intact myelinated and unmyelinated fibers as well as new axon sprouts, whereas control CNs contained degenerating unmyelinated fibers and demonstrated a breakdown of myelin around the myelinated axons. By six weeks, erectile function, as mea542

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sured by intracavernosal pressure, was improved 58 % for treatment with the PA scaffold in comparison to controls, indicating significant reinnervation in the penis. In another example of a PA gel designed to locally release a signaling protein, an IKVAV PA gel containing brain-derived neurotrophic factor (BDNF) was used to assist in an effort to regenerate the auditory nerve.[55] Following transplantation of stem cells into the auditory nerve, the PA gel was placed around the injury site, with the goal of reducing glial scar formation while releasing BDNF to the nearby implanted cells and surrounding tissue. PA gels with BDNF significantly increased survival and differentiation of the implanted stem cells, further demonstrating the ability of PAs to satisfy these critical requirements for an effective cell replacement therapy. 3.3 PAs for Cardiovascular Diseases

The treatment of ischemic diseases of the heart, peripheral vasculature, and chronic wounds could benefit from the promotion of angiogenesis, the process by which new blood vessels form from existing vasculature.[56] One promising PA material designed for the promotion of angiogenesis is termed a heparin-binding peptide amphiphile (HBPA). This molecule was designed to contain a CardinWeintraub heparin-binding sequence within its bioactive domain in order that it could specifically bind and present heparan sulfatelike glycosaminoglycans. By design, this oppositely charged biopolymer screens positive charges on the HBPA molecules, triggering PA selfassembly into nanofibers that display heparin on their surface.[57] The interaction between the PA and heparin was determined to have an association constant of 1.1  107, measured using isothermal titration calorimetry. Many potent signaling proteins, including fibroblast growth factor 2 (FGF-2), bone morphogenetic protein 2 (BMP-2) and vascular endothelial growth factor (VEGF), contain heparin-binding domains. In native tissue, these proteins interact with heparan sulfate through these binding domains for appropriate presentation to cell receptors. The display of heparin on the surface of HBPA nanofibers enables the complex to capture these potent signaling proteins and present them to receptors on cells in a biomimetic fashion. This is significant, as heparan sulfate is known to play a role as a cofactor in angiogenesis, specifically in VEGF and FGF-2 molecular biology through factor binding, protein-receptor complex stabilization, and extending the signaling lifetime of these factors through protection against proteolysis.[58] Nanogram quantities of VEGF and FGF-2 combined with HBPA-heparin and implanted into a surgical pocket of a rat cornea resulted in significant neovascularization of the cornea compared to the growth factors alone, HBPA-heparin without growth factors, and similar materials with and without growth factors.[57b] This result is significant as the cornea is an avascular tissue site, meaning

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any new blood vessels formed are indicative of a strong angiogenic response. Scrambling of the heparin-binding sequence presented on the HBPA diminished the observed angiogenic effect in an endothelial cell tube formation assay.[57a] Since the scrambled version of the PA and HBPA had similar association constants with heparin, the difference in bioactivity was attributed to a slower off-rate for the interaction between heparin and HBPA, stabilizing the protein from enzymatic degradation and leading to more efficient growth factor signaling. HBPA-heparan sulfate nanofiber gels were implanted subcutaneously in a mouse model through percutaneous injection and demonstrated excellent tissue reaction as well as in vivo retention for at least 30 days.[59] One exciting finding from this study was that, as the material was biodegraded, it was transformed into a highly vascularized connective tissue. This occurred for the HBPA-heparan sulfate gel without the addition of any exogenous growth factors, suggesting that this material can also potentiate endogenous angiogenic factors secreted by inflammatory cells and fibroblasts. In this study, a dynamic analysis of the tissue reaction using the skinfold chamber model showed no adverse effects from implantation of HBPA on the underlying microcirculation, and also reiterated the biocompatibility and angiogenic effects found in the subcutaneous model. These studies point to the potential to use the HBPA system for the healing of chronic wounds or to the enhance efficacy of skin flaps and grafts, as both of these conditions could benefit from an extensive subcutaneous granulation tissue such as that observed in these studies. The ability to bind and present growth factors could also have implications in the treatment of ischemic conditions of the myocardium, which often result following an infarction. The HBPA-heparin nanofiber gels were evaluated in this capacity in a mouse ischemia-reperfusion infarction model.[60] In these studies, angiogenic growth factors were delivered with the PA nanofiber gel to the left ventricle wall of the post-infarct myocardium of mice. This treatment resulted in significantly improved hemodynamic function of the left ventricle at 30 days following treatment. This result of enhanced ventricular contractility and increased systolic blood pressure was a significant increase over animals receiving an injection of only growth factors or an injection of saline. Interestingly, unlike in the subcutaneous model, there was no effect for the HBPA nanofiber alone in this tissue site. In the same report, the HBPA nanofiber gel loaded with growth factors was used to treat a chronically ischemic rat hind limb.[60a] In these studies, injection of the HBPA nanofiber gel resulted in enhanced perfusion of the ischemic limb through the development of larger arteries. In this case, there was no significant difference between the groups receiving HBPA with or without growth factors. Thus, the response in this particular tissue site was similar to that seen in the subcutaneous studies where the nanofiber gel Isr. J. Chem. 2013, 53, 530 – 554

was able to potentiate the angiogenic factors already present in the tissue. As an alternative to expensive recombinant growth factors traditionally used with the HBPA system, we recently reported on the development of a completely synthetic cell-free therapy that was based on PA nanostructures designed to mimic the activity of VEGF.[61] These PA nanofibers display on their surfaces a VEGF-mimetic peptide at high density. The VEGF-mimetic nanofibers were found to induce phosphorylation of VEGF receptors and promote pro-angiogenic behavior in endothelial cells, indicated by an enhancement in proliferation, survival and migration in vitro. In a chicken embryo assay, these nanostructures elicited an angiogenic response in the host vasculature (Figure 5). When evaluated in a mouse hindlimb ischemia model, the nanofibers increased tissue perfusion, functional recovery, limb salvage, and treadmill endurance compared to controls, which included the VEGF-mimetic peptide alone. Immunohistological evidence also demonstrated an increase in the density of microcirculation in the ischemic hind limb, suggesting the mechanism of efficacy of this promising potential therapy is linked to the enhanced microcirculatory angiogenesis that results from treatment with these polyvalent VEGFmimetic nanofibers. Atherosclerosis is becoming a global problem, affecting nearly 80 million people in the US alone. Though a variety of interventional strategies exist, these procedures have limited efficacy due primarily to the secondary complications of neointimal hyperplasia, reclosing of the arteries as a result of cell proliferation and migration into the lumen. The significance of nitric oxide as a naturally released substance that limits the proliferation and migration of vascular smooth muscle cells has prompted this compound to be evaluated from a synthetic delivery standpoint.[62] In an attempt to control nitric oxide release, peptide amphiphile nanofiber gels were mixed with diazeniumdiolate nitric oxide donors to prepare nitric oxidereleasing nanofiber gels.[63] Combination with the nanofiber gel demonstrated a prolonged release of the nitric oxide for up to four days using diazeniumdiolate donors with usual half-lives ranging from seconds to hours. This prolonged release profile could be advantageous in delivering NO for the treatment of neointimal hyperplasia. This was supported by the ability of the PANO donor combination to decrease smooth muscle cell proliferation and promote smooth muscle cell death in vitro. A rat carotid artery balloon injury model was used to assess the therapeutic potential of the combination. In these studies, nitric oxidereleasing PA nanofiber gels applied to the outer surface of the artery resulted in a reduction of neointimal hyperplasia by up to 77 % compared to controls. In addition, treatment with this PA-NO donor combination also reduced inflammation in the injury site.

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Figure 5. Representative images from chicken chorioallantoic membranes stimulated with VEGF-mimetic PA compared to controls of VEGF peptide, non-bioactive PA (Mutant PA), and saline. Increased blood vessel density at the site of stimulation is indicative of pro-angiogenic signaling by the VEGF-mimetic PA. On the right are images obtained by laser Doppler perfusion imaging of ischemic mouse hind limbs at the time when ischemia was created (Day 0) and 28 days after administering treatment. Images indicate improved blood flow in the limb with ischemic injury for treatment with VEGF PA compared to controls. Reprinted from Webber et al.[61] Copyright  2011 National Academy of Sciences, USA.

3.4 PAs to Facilitate Islet Transplantation

Type I diabetes mellitus results from autoimmune destruction of b-cells, the cells responsible for glucose sensing and insulin secretion within pancreatic islet cell masses. One potential strategy to combat type I diabetes is through transplantation of donor islets, traditionally into the circulatory system of the liver. However, the efficacy of this strategy is limited by poor islet viability and engraftment following transplant.[64] To improve islet engraftment and survival, the angiogenic HBPA-heparin nanofiber system was used to enhance the vasculature in the transplant site of a diabetic mouse model.[65] Presently, donor islets are transplanted into the liver because of its high level of vasculature. HBPA, combined with angiogenic factors FGF-2 and VEGF, was found to significantly enhance vasculature in an abdominal fat mass, the omentum. Such a transplant site would, in principle, be a more desirable location for transplantation of islets as, compared to the liver, it is less invasive and does not compromise the function of a vital organ. Islets transplanted along with the HBPA-heparin nanofiber gel and growth factors into the omentum resulted in a significant increase in the cure percentage of diabetic mice, indicating a restoration of normal blood glucose levels. This was not the case for the administration of islets with HBPA-heparin alone or with growth factors alone, indicating that the 544

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HBPA combined with growth factors was necessary to sufficiently vascularize the tissue site for appropriate islet engraftment. In addition to its use to facilitate islet transplant by increasing vascularization at the transplant site, HBPA and angiogenic growth factors have been used to prepare islets during in vitro pre-culture prior to transplantation.[66] Culturing islets in the presence of HBPA and growth factors resulted in enhanced cell viability, enhanced glucose sensitivity and resultant insulin production, and improved health and function of intra-islet endothelial cells. Such a finding is significant as donor islets are traditionally processed in vitro prior to transplantation to improve cell purity and limit immunogenicity. However, this pre-culture time can inadvertently result in reduced islet function and impaired viability. Therefore, the HBPA-growth factor nanofiber could be useful during this transition step to promote islet health and function, which could result in improved outcomes following transplantation. 3.5 PAs for Bone Regeneration

The regeneration of bone in sufficiently large defects resulting from traumatic bone loss, tumor resections, or infection remains a major challenge in orthopedic medicine.[67] One potential approach is to replace the tissue

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with a permanent metal implant. While these materials have mechanical properties that allow for weight-bearing applications, their rigidity can lead to problems with stress shielding and they do not allow for tissue ingrowth, which can result in the implant loosening over time. Titanium and its alloys are traditionally used for applications in hard tissue replacement due to their reasonable mechanical properties and established biocompatibility. However, these metallic implants do not have a bioactive component, limiting their ability to prompt regeneration. PAs were explored as a means to afford these materials with an added bioactivity that could, for example, prompt tissue growth and regeneration around the implant to assist in long-term implant fixation. PA nanofibers displaying the RGDS epitope were used to functionalize a nickel-titanium (NiTi) alloy, like that commonly used in stents, bone plates, and artificial joints, through the use of standard silanization and cross-linking chemistry.[8g,68] Modification of the NiTi with PAs resulted in significantly enhanced adhesion of pre-osteoblastic cells in vitro, while cells did not attach to the non-functionalized NiTi. For applications in bone, porous titanium implants are frequently used to facilitate tissue ingrowth and assist with implant fixation. It has been demonstrated that RGDSpresenting PA nanofiber gels could be prepared within the pores of such scaffolds.[15i,j] In this case, the PA molecules were post-gelled within the interconnected pores of titanium foam by subsequent introduction of screening salts. Pre-osteoblastic cells that were seeded within these PA-titanium foam hybrids remained viable, proliferated, and underwent osteogenic differentiation. These results indicated potential for the use of PA-filled porous metal constructs to treat bone defects, as demonstrated in a study using a rat femoral defect model.[15i] Though metallic implants are sufficient with respect to restoring load-bearing capacity, they have little true regenerative function. For cases such as segmental bone defects, where a portion of bone is removed due to trauma, infection, or disease, leaving a defect beyond the critical size for adequate healing, promoting regeneration of bone to bridge this defect is a desirable alternative. Currently, the gold standard for such conditions is an autograft of bone removed from an alternate site, such as the iliac crest of the pelvis, which is then transplanted to the defect to promote regeneration of new bone. However, this strategy still has many drawbacks, including pain from a second procedure, risk of infection, and donor site tissue morbidity. Furthermore, some patients may not have adequate bone available for autograft. Therefore, a synthetic strategy that could promote bone regeneration would be desirable. With the goal of developing a resorbable, organic matrix that could promote mineralization while supporting cell adhesion and directing differentiation of mesenchymal stem cells, a PA gel was designed, composed of both phosphoserine-containing PA and RGDS-containing PA.[69] The resulting gels were shown Isr. J. Chem. 2013, 53, 530 – 554

to nucleate the formation of spheroidal nodules of crystalline carbonated hydroxyapatite along the PA nanofibers. The gels also promoted osteogenic differentiation of mesenchymal stem cells, suggesting that the presence of phosphoserine residues played a role in directing differentiation. Building on this work, a phosphoserine-containing PA gel designed to promote biomimetic hydroxyapatite mineralization in the absence of cells, growth factors, or bone grafts, was able to promote regeneration in a bone defect model.[70] The PA nanofiber gel, implanted into a criticalsize rat femoral defect, resulted in a significant increase in the formation of new bone within the defect. While the defect was not entirely bridged with any of the evaluated materials, there was significant reduction in the defect distance for treatment with the phosphoserine PA combined with an RGDS PA. To trigger greater bone growth, a multicomponent system formed by a porous collagen scaffold filled with a heparin-binding PA gel along with heparan sulfate and bone morphogenetic protein-2 (BMP-2) was tested in the rat critical-size femoral defect model.[71] This strategy emulated native BMP-2 signaling by retaining and presenting BMP-2, which binds via a heparin-binding domain to the heparan sulfate, which itself is bound to heparin-binding PA gel within the collagen scaffold pores. This biomimetic approach led to a large volume of bone regeneration in the defect using amounts of BMP-2 one order of magnitude lower than required with injection of BMP-2 alone. In more than half of the animals receiving the scaffolds, continuous bone formation between the proximal and distal femur was present, whereas only one animal with a collagen scaffold with BMP-2 and HBPA bridged the gap, and no bridging was observed in other control groups (Figure 6). This clinically relevant finding suggests that amplification of growth factor activity may be possible through mimicking components of the extracellular environment with biomaterials. 3.6 PAs for Enamel Regeneration

As humans lack the capacity to develop new teeth and are, furthermore, living longer, the regeneration of teeth is a formidable challenge. Enamel, the outermost coating of teeth, is the hardest tissue in the body. The cells responsible for the production of enamel during development, the ameloblasts, subsequently die once their role is complete, preventing enamel regeneration during adulthood. RGDS-presenting PA nanofibers have been evaluated as scaffolds for ameloblast-like cells as well as primary enamel organ epithelial (EOE) cells that initiate the process of enamel formation.[15g] Treatment with branched RGDS PA nanofibers in vitro resulted in an enhancement in proliferation of these cells and increased their expression of amelogenin and ameloblastin, two proteins secreted by ameloblasts during enamel formation. In an organ culture model, embryonic mouse inci-

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Figure 6. Analysis of the in vivo bone regeneration capacity of collagen scaffolds (Coll) containing heparin-binding peptide amphiphile (HBPA) nanofibers presenting heparan sulfate (HS) and BMP-2. A) Representative femur reconstructions from microcomputed tomography of the various treatment groups. B) The number of animals used per condition and the number of animals with a bridged femur after treatment. C) Quantitative analysis of new bone volume (mm3) within the defect, measured by microcomputed tomography. Data are presented as mean  standard error of the mean; ***P < 0.001. Reprinted with permission from Lee et al.[71]

sors were injected with RGDS PA and cultured, indicating EOE proliferation and ameloblastic differentiation, demonstrated by an increase in expression of enamel-specific proteins. An in vivo study, wherein an embryonic mouse incisor was implanted into the kidney capsule, revealed ectopic enamel synthesis following injection of the PA into the tissue adjacent to the incisor.[72] This phenomenon could be controlled at chosen sites based on where the PA was injected, and was specific to RGDS epitope presentation on the PA, as a scrambled epitope did not prompt the ectopic formation of enamel. Such an artificially triggered formation of highly organized enamel mineral could provide insight into promoting cell-fabricated mineral in order to treat dental caries, a condition that is essentially ubiquitous in humans. Hartgerink and colleagues have also used PAs in vitro as a matrix for dental stem cells.[73] When encapsulated within the PA, stem cells derived from human exfoliated deciduous teeth proliferate and secrete a soft collagen matrix. Additionally, another variety of cell, dental pulp stem cells, differentiated into an osteoblast-like phenotype and deposited mineral within the nanofiber gel. The PA system combined with dental stem cells has potential to enable regeneration of both soft and mineralized 546

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dental tissue and could be used as a treatment for dental caries. 3.7 PAs for Cartilage Regeneration

Cartilage regeneration remains a significant target within regenerative medicine, due in large part to the inability of adults to naturally replace damaged cartilage coupled with the tremendous pain and suffering that result from osteoarthritis. Recently, we reported on a specially designed PA molecule that presented a novel binding sequence to transforming growth factor b-1 (TGFb-1), as an injectable matrix for cartilage regeneration.[74] The PA was designed to bind and release TGFb-1 by presenting a binding sequence derived from phage display. In a rabbit full-thickness chondral defect model, the PA presenting the binding sequence promoted extensive regeneration in the chondral defect, whether or not exogenous TGFb-1 was combined with the material (Figure 7). The results suggest that the binding PA is able to bind and retain endogenous factors that are present in the defect as part of the microfracture procedure. The new cartilage that forms is histologically similar to healthy cartilage, and fully fills the defect site. These studies suggest prom-

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Figure 7. Representative histological sections collected twelve weeks after treatment and stained with safranin O for glycosaminoglycans (A–D) and type II collagen immunostaining (E–H) in articular cartilage defects treated with (A, E) 100 ng/mL TGF-b1, (B, F) filler PA + 100 ng/ mL TGF-b1, (C, G) TGF-b1binding PA + 100 ng/mL TGF-b1, and (D, H) TGF-b1binding PA alone. The binding PA with (C, G) and without (D, H) TGF-b1 demonstrates the formation of new cartilage that was histologically similar to existing cartilage. Arrows denote the boundaries of the cartilage defect. Reprinted from Shah et al.[74] Copyright  2010 National Academy of Sciences, USA.

ise for a PA-based therapy to promote cartilage regeneration, an unaddressed issue within the field of regenerative medicine.

4. PA Assemblies with Hierarchical Structure Beyond the Nanoscale Structures in biological systems often have hierarchical ordering across scales and their functions are closely linked to their supramolecular complexity. Good examples are the matrices of connective tissues such as bone, muscle, cartilage, and also structures in the brain and spinal cord. Recent work in our laboratory has explored PA assemblies that exhibit structural order over length scales that are much larger than the dimensions of 1D assemblies of PA molecules described so far in this manuscript. We describe below three different systems in which this type of higher order has been discovered and Isr. J. Chem. 2013, 53, 530 – 554

rationalized in the context of molecular interactions in synergy with external energies or mixing with other components such as covalent polymers. These structures were observed to evolve as a result of thermally induced transformations, crystallization of supramolecular assemblies, or interactions with macromolecules bearing opposite charge to PA assemblies. 4.1 Massively Aligned PA Nanofiber Bundles

In very recent work, it was discovered that when solutions of certain PAs underwent a thermal annealing process followed by a slow cooling, nanofibers became organized into large bundles of aligned fibers (Figure 8).[75] The PAs in thermally treated solutions form large plaque-like structures at elevated temperatures that break into bundles of nanofibers during the cooling phase. We postulated that this plaque structure formation at elevated temperatures was driven by the entropy of restricted water

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Figure 8. A) A thermally treated peptide amphiphile solution (blue) dragged through a thin layer of aqueous CaCl2 to form a flexible string-like gel. B) SEM of aligned nanofiber bundles in these string-like gels. C) Calcein-labeled human mesenchymal stem cells cultured in the aligned nanofibrous gels align in the direction of the fibers. D) SEM of plaques that were captured by adding CaCl2 to PA solutions at 80 8C and E) SEM of plaques breaking into nanofiber bundles. Schematic representation of a plaque at F) high temperature and G) its rupture into fused nanofiber bundles upon cooling of the solution. H) Top: calcium fluorescence image of HL-1 cardiomyocytes encapsulated in a noodle-like string. Below: successive spatial maps of calcium fluorescence intensity traveling at 80 ms intervals, showing the propagation of an electrical signal throughout the entire string and demonstrating a functional cardiac syncytium. Figure adapted with permission from Zhang et al.[75]  2010 Nature Publishing.

molecules surrounding the peptide amphiphiles. By transitioning from a fiber to plaque morphology, the interface with water was decreased, reducing the number of restricted water molecules per PA. As the solution is cooled, Raleigh instabilities and fluctuations in surface tension lead to rupture of the plaque into bundled nanofibers, producing an aqueous lyotrophic liquid crystal at unusually low concentrations, on the order of 1 % by weight in water. These liquid crystalline solutions contain microdomains of aligned bundles of fibers. When the solution is pipetted across a surface into a solution of divalent cations, it rapidly forms a flexible, string-like gel composed of a monodomain of nanofibers aligned in the direction of the gel. Cells can be seeded on top of these gels, or suspended in the heat-cycled solution following annealing but prior to gelation, resulting in the encapsulation of cells within the aligned matrix. Whether grown on or within the gel, cells align their axis and grow processes in the direction of nanofiber alignment. When autocon548

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tractile cardiomyocytes were cultured within the gel, the cells proliferated, filling the entire structure. Within ten days, the cells formed extensive cell-cell contacts, enabling the cells to generate waves of electrical signals that propagated along the length of the aligned scaffold. These aligned gels hold promise as a bioactive material that could be used to reestablish communication between nerve or muscle cells, to guide cells spatially for migration from one location to another, or as a protein delivery vehicle as discussed in the neural regeneration section.[54a] Aligned PA gels were also explored as a material capable of nucleating oriented mineralization along PA nanofibers.[76] It was found that cylindrical nanofibers, but not similar flatter one-dimensional nanofibers, promoted mineralization along the long axis of the nanofibers. Furthermore, alignment of the nanofibers into monodomain gels enabled the templating of oriented crystals over multiple length scales, which may be useful in the context of regeneration of mineralized tissues with aligned features.

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In an attempt to recapitulate the microarchitecture of the medial layer of arteries, in which smooth muscle cells are highly aligned circumferentially, a method was developed to organize PA nanofibers circumferentially into a tubular hydrogel.[77] By applying a low shear rate during gelation in a cylindrical chamber, it was possible to create a highly circumferentially aligned PA gel while simultaneously encapsulating smooth muscle cells without compromising cell viability. Cells cultured for two days in the tubes grew processes oriented in the nanofiber direction and cell density doubled after 12 days. This work demonstrated the potential of using aligned PA scaffolds to create tissue-engineered replacements for damaged blood vessels. 4.2 Long-Range PA Nanofiber Crystallization

Another method was recently discovered to induce crystallization of PAs in solution, using a balance of repulsive forces from native or X-rayinduced charges on PA molecules combined with mechanical forces resulting from the entrapment of the nanofibers within domains in a larger network.[78] This crystal-like organization, resulting from the combination of charge repulsion of nanofibers in balance with mechanical compression mediated by these networks, is reminiscent of a tensegrity network. By either increasing the concentration of PA solutions or introducing charge through X-ray irradiation, fibers packed into a hexagonal crystalline phase with unusually large fiber surface-to-surface spacing, as much as 218  apart in the case of X-ray induced crystallization. Another important finding was the spontaneous reversibility of the process. When X-ray irradiation was removed, the solution slowly returned to a disordered state over time. The addition of screening ions suppresses this long-range order. This work sheds light on electrostatic mechanisms of control over ordered systems and may inform studies examining cellular mechanisms of control over structural assembly of objects such as cytoskeletal filaments. 4.3 PA-Polymer Hybrid Hierarchically Ordered Membranes

While most of the research mentioned thus far has examined higher-ordered systems of PAs, work has also been done to prepare and control multicomponent hybrid hierarchically ordered materials that form between PAs and biopolymers.[8e,79] A recent report discusses the formation of a highly ordered architecture that develops at the interface between the negatively charged polysaccharide, hyaluronic acid (HA), and an oppositely charged PA.[8e] When these two solutions come in contact, a diffusion barrier rapidly forms at the liquid-liquid interface, controlling subsequent growth of the membrane as osmotic pressure differences drive the polymer through the interfacial layer (Figure 9). Bundles of PA interact with the reptating high molecular weight polymer, resulting in the Isr. J. Chem. 2013, 53, 530 – 554

formation of fibrous bundles perpendicular to the interfacial barrier. This results in the formation of a thick membrane structure. If this HA solution is completely submerged in a PA solution, the membrane structure completely encloses the HA solution, rapidly forming a robust sealed sac-like structure that can be easily manipulated, sutured, and is self-healing with the addition of excess PA to a defect. These sacs can be prepared with other solutions or cells on their interior. Human MSCs cultured in gels within the sac structure remained viable for as long as four weeks and also demonstrated the ability to differentiate into a chondrogenic phenotype within the sac when cultured in chondrogenic media, indicating the ability of proteins and soluble signals to pass through the membrane. These unique hierarchical structures hold promise for a number of applications in regenerative medicine, from cell scaffolds to drug delivery vehicles. Control over mechanical properties and permeability are likely to play important roles in their functionality. Membrane inflation and osmotic swelling techniques were used to understand how factors such as HA concentration and membrane maturation time affected the mechanical properties and water permeability of these structures.[79a] Results showed that both the area modulus and water permeability could be controlled by varying the amount of HA or the incubation time of the sac. Longer incubation times resulted in thicker membranes with higher area moduli. Area modulus was also increased by using a higher concentration of HA. Alternatively, water permeability was reduced by longer incubation times or through the use of lower HA concentrations. The kinetics, structure, and properties of membrane formation could also be altered by applying an electric field, with thickness varying by up to twofold depending on the field orientation.[80] The specific nanostructure present in the PA solution at the time of membrane formation also governs the microstructure of the formed membrane, as evidenced by dramatic changes in microstructure and function when a cytotoxic PA that made spherical micelles was incorporated instead of a nanofiber-forming PA.[79b] This strategy to prepare hierarchically structured PAbiopolymer hybrid materials was also used to prepare membranes that promote angiogenesis.[81] By replacing the generic positively charged PA used in previous work with a PA previously designed to bind heparin, HBPA, and including heparin into the HA biopolymer solution, the interfacial planar membrane formed with heparin incorporated into the structure. The addition of heparin to the biopolymer solution also led to a more deformable membrane structure, indicated by a lower area modulus. Cells were viable when cultured on these membranes. The incorporation of heparin into this membrane allowed for the binding and slow release of angiogenic growth factors FGF-2 and VEGF from the membrane structure. When evaluated in an in vivo chicken embryo angiogenesis model, membranes prepared with heparin and growth

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Figure 9. A) A schematic representation of a method to form a self-sealing sac by dropping a biopolymer solution into an oppositely charged PA solution. B) Open and C) closed sac formation. D) Self-assembled sacs of varying sizes. E) An SEM of the cross section of the sac membrane, showing the characteristic layers of the complex assembly process; region 1 is an amorphous layer formed on the side of the membrane with biopolymer, region 2 is a parallel fiber layer rapidly formed at the interface between the two solutions, and region 3 is a perpendicular fiber layer that grows as biopolymer diffuses into the PA compartment. F) Schematic representation of polymer stubs (red) penetrating the diffusion barrier formed at the interface between the two solutions. G) Subsequent self-assembly of nanofibers (blue) initiated by the stubs. H) Growth of the nanofibers perpendicular to the interface over time, as the biopolymer diffuses into the PA compartment. Adapted with permission from Capito et al.[8e]  2008 AAAS.

factors incorporated into their structure prompted a stronger angiogenic response on the vasculature of the chorioallantoic membrane of the embryo. Such technology could enable custom-fit bioactive wound dressings that could promote granulation and healing for chronic wounds. The formation of an encapsulated membrane upon interfacial complexation of PA molecules with a droplet of biopolymer inspired efforts to miniaturize these assemblies into micro-sized spherical particles.[8f] Using a nebulizing spray device, picoliter droplets of alginate were formed and sprayed into a bath containing PA as well as calcium. It is well known that calcium can cross-link alginate and lead to hydrogel formation. The gelled droplets of alginate could then be coated by filamentous PA nanofibers, since the positively charged PA chosen for this work could complex with the negatively charged alginate. 550

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The resultant spherical particles were approximately 25 mm in diameter, with a high density of PA nanofiber filaments on their surface that could afford a dense surface for bioactive signaling along with controlled release of bioactive components from the gelled core.

5. Summary and Outlook We have described our recent progress on the study of peptide amphiphile supramolecular assemblies and materials, and their potential in novel therapies for regenerative medicine. From a fundamental standpoint, the challenges ahead for these systems will be to understand the synergy of all interactions instructing self-assembly and predict their structure through theory and computational chemistry. Two other basic challenges are the exploration

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of the internal dynamics and their relation to bioactivity, and methods for greater control over structural features on the surfaces and internally as well as length control. It is also of interest to expand their chemistry beyond peptides and alkylated hydrophobic segments and to impart in these systems elements that are active, responsive, and adaptive through supramolecular structure. There are also many opportunities in the exploration of hierarchical systems, especially in the form of hybrid structures. With regard to their use in regenerative medicine, there are still many targets beyond those described here that could be explored, especially in therapies that combine both peptide amphiphiles and cells or biologics. A basic understanding of these supramolecular systems will be critical to generate robust systems with highly predictable bioactivity and strong efficacy in therapeutic models. Bioactivity, and thus therapeutic efficacy, in these systems is rooted in their supramolecular structure and not simply in molecular structure of the peptide amphiphile, and thus a deep understanding of their behavior as assemblies in physiological environments is critical. The broad translation of PAs from the pre-clinical in vivo models described here to clinical use requires consideration of several issues. In studies performed so far, these supramolecular materials have demonstrated biocompatibility, and immune response or chronic inflammation has not been observed. However, for specific peptide sequences, polyvalent display on self-assembled nanostructures could lead to an immune response.[82] For clinical translation it is also of course necessary that molecules can be produced reproducibly with acceptable purity, in large scale, and under sterile conditions. In this respect there is already precedent that synthetic peptides can be produced for clinical translation, as evidenced by clinical use of Enfuvirtide and Eptifibatide, which are peptide therapies for HIV and cardiac ischemia, respectively.[83] An important factor for translation is the fact that PAs and other “supramolecular therapies” require self-assembly of molecules for efficacy. This implies that not only molecular but also supramolecular reproducibility is essential for consistent function. To address this issue, one must ensure that well-defined self-assembly protocols are followed to create the bioactive supramolecular structures, a process that begins with molecular synthesis and continues through purification, sterilization, storage, reconstitution, and delivery of the therapy to biological tissues. It may be that all emerging therapies based on peptides, nucleic acids, and polysaccharides naturally have a supramolecular component that cannot be overlooked in optimizing their efficacy. PAs are specifically designed to self-assemble into nanostructures and this is the form in which they should be bioactive; thus awareness of self-assembly pathways is critical. This supramolecular issue is a new axis in emerging therapies that deviates from the traditional pharmaceutical ap-

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proach of using small organic molecules that are primarily designed to have efficacy as single molecules. An exciting direction is the design of peptide-based nanostructures that can be delivered systemically and targeted to specific tissues or organs in the body to promote regenerative processes or deliver therapies. This includes developing materials that cross the blood-brain barrier, which could make therapies for the brain much less invasive. Well-programmed systemic targeting of nanostructures could be especially useful in the treatment of metastatic cancers, as nearly all cancer deaths are attributed to metastasis. In addition to systemic targeting, another interesting aspect would be intracellular targeting. PAs evaluated to date have seen broadest application interacting with cells from the extracellular compartment. Building nanostructures that could interact with targets in the intracellular milieu such as transcription factors, complexes that control protein translation, and enzymes that induce post-translational modifications could be highly useful. Following targeting, the next fertile area would be to produce molecules that could undergo tissue-specific assembly or dynamic changes in nanostructure morphology. This could enable the injection of small molecules that could organize and assemble into insoluble structures at a specific tissue site, or alternatively, the injection of assemblies that could disassemble to deliver an encapsulated payload of drugs or proteins once they reach their targeted locations. The development of dynamic systems would also add an additional level of biomimicry to PA nanostructures. A number of self-assembled systems in biology undergo dynamic structural changes, such as the reorganization that occurs among actin or tubulin assemblies, the conformational changes that result in the exposure of cryptic sites within collagen, and natural on/off states that exist for enzymes, receptors, and membrane transport protein complexes. As such, adding dynamic structure/function capabilities to self-assembled peptide amphiphile systems could further advance their therapeutic impact. Another promising area within regenerative medicine is the use of stem or progenitor cells to assist in regenerating or healing tissue. The momentum behind this work has been fueled by recent research into embryonic and induced pluripotent stem cells as well as populations of adult stem cells. One interesting area that will see advancement in the coming years is the development of materials to interface with, support, and instruct these cells in their therapeutic duty. Preliminary studies have examined the support and delivery of potent therapeutic cells using PAs. Certainly, future directions will evaluate the use of PAs to facilitate stem cell therapies as well as to control the differentiation and lineage commitment of these cells. Looking even further forward, PA-based materials could be envisioned that could promote dedifferentiation or transdifferentiation of committed cells in

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order to generate other lineages with functional impact on damaged tissues. As mentioned in the introduction to our work preparing hierarchically ordered materials from assembled PA nanofibers, much can be learned from the way nature uses multiple levels of organization to build structure and perform function. The work we have demonstrated, especially for highly aligned PA fiber bundles and for enclosed PA-polymer sac structures, could see future application in a host of therapeutic roles. For example, a highly aligned scaffold could be useful in promoting regeneration and directionality in aligned tissues such as spinal cord and muscle fiber. The hybrid PA-polymer sac structure has potential on multiple levels to signal cells using PAs presented on its surface, provide a more structured and slower degrading membrane material for use as a wound dressing or cell scaffold, or be used for the delivery or slow release of encapsulated cells or cargo. Furthermore, miniaturization of the technology by promoting the assembly on a smaller length scale could enable materials that signal cells on the cellular level or traverse blood vessels in a way similar to erythrocytes. Such a hierarchically ordered system could have multiple axes of bioactivity, preparing an even more biomimetic therapy that could function in multiple capacities, reminiscent of a cell. The work we have highlighted in this review points to the potential of therapies rooted in supramolecular selfassembly to provide function in advanced therapies with highly biomimetic structures. Our hope is that the exciting findings demonstrated thus far with peptide amphiphile systems will contribute to the next generation of sophisticated therapies using supramolecular chemistry.

[4] [5] [6]

[7]

[8]

[9]

[10]

[11] [12]

Acknowledgements Experimental work performed in the Stupp laboratory was made possible through support from the National Institutes of Health award number 5R01DE015920 from the National Institute of Dental and Craniofacial Research (NIDCR) and award number 5R01EB003806 from the National Institute of Biomedical Imaging and Bioengineering (NIBIB).

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 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Received: May 20, 2013 Accepted: July 11, 2013 Published online: August 8, 2013

Isr. J. Chem. 2013, 53, 530 – 554

Supramolecular Nanofibers of Peptide Amphiphiles for Medicine.

Peptide nanostructures are an exciting class of supramolecular systems that can be designed for novel therapies with great potential in advanced medic...
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