Shantanu Sur

Bone regeneration with low dose BMP-2 amplified by biomimetic supramolecular nanofibers within collagen scaffolds

Bone morphogenetic protein-2 (BMP-2) is a potent osteoinductive cytokine that plays a critical role during bone regeneration and repair. In the extracellular environment, sulfated polysaccharides anchored covalently to glycoproteins such as syndecan and also non-covalently to fibronectin fibers have been shown to bind BMP-2 through a heparin-binding domain and regulate its bioactivity. We report here on a synthetic biomimetic strategy that emulates biological BMP-2 signaling through the use of peptide amphiphile nanofibers designed to bind heparin. The supramolecular nanofibers, which integrate the biological role of syndecan and fibronectin, were allowed to form gel networks within the pores of an absorbable collagen scaffold by simply infiltrating dilute solutions of the peptide amphiphile, heparan sulfate, and BMP-2. The hybrid biomaterial enhanced significantly bone regeneration in a rat critical-size femoral defect model using BMP-2 amounts that are one order of magnitude lower than required for healing in this animal model. Using micro-computed tomography, we also showed that the hybrid scaffold was more effective at bridging within the gap relative to a conventional scaffold of the type used clinically based on collagen and BMP-2. Histological evaluation also revealed the presence of more mature bone in the new ossified tissue when the low dose of BMP-2 was delivered using the biomimetic supramolecular system. These results demonstrate how molecularly designed materials that mimic features of the extracellular environment can amplify the regenerative capacity of growth factors.

Aligned neurite outgrowth and directed cell migration in self-assembled monodomain gels

Regeneration of neural tissues will require regrowth of axons lost due to trauma or degeneration to reestablish neuronal connectivity. One approach toward this goal is to provide directional cues to neurons that can promote and guide neurite growth. Our laboratory previously reported the formation of aligned monodomain gels of peptide amphiphile (PA) nanofibers over macroscopic length scales. In this work, we modified these aligned scaffolds specifically to support neural cell growth and function. This was achieved by displaying extracellular matrix (ECM) derived bioactive peptide epitopes on the surface of aligned nanofibers of the monodomain gel. Presentation of IKVAV or RGDS epitopes enhanced the growth of neurites from neurons encapsulated in the scaffold, while the alignment guided these neurites along the direction of the nanofibers. After two weeks of culture in the scaffold, neurons displayed spontaneous electrical activity and established synaptic connections. Scaffolds encapsulating neural progenitor cells were formed in situ within the spinal cord and resulted in the growth of oriented processes in vivo. Moreover, dorsal root ganglion (DRG) cells demonstrated extensive migration inside the scaffold, with the direction of their movement guided by fiber orientation. The bioactive and macroscopically aligned scaffold investigated here and similar variants can potentially be tailored for use in neural tissue regeneration.

Cell death versus cell survival instructed by supramolecular cohesion of nanostructures

Many naturally occurring peptides containing cationic and hydrophobic domains have evolved to interact with mammalian cell membranes and have been incorporated into materials for non-viral gene delivery, cancer therapy, or treatment of microbial infections. Their electrostatic attraction to the negatively charged cell surface and hydrophobic interactions with the membrane lipids enable intracellular delivery or cell lysis. While the effects of hydrophobicity and cationic charge of soluble molecules on the cell membrane are well known, the interactions between materials with these molecular features and cells remain poorly understood. Here we report that varying the cohesive forces within nanofibres of supramolecular materials with nearly identical cationic and hydrophobic structure instruct cell death or cell survival. Weak intermolecular bonds promote cell death through disruption of lipid membranes, while materials reinforced by hydrogen bonds support cell viability. These findings provide new strategies to design biomaterials that interact with the cell membrane.

A hybrid nanofiber matrix to control the survival and maturation of brain neurons

Scaffold design plays a crucial role in developing graft-based regenerative strategies, especially when intended to be used in a highly ordered nerve tissue. Here we describe a hybrid matrix approach, which combines the structural properties of collagen (type I) with the epitope-presenting ability of peptide amphiphile (PA) nanofibers. Self-assembly of PA and collagen molecules results in a nanofibrous scaffold with homogeneous fiber diameter of 20-30 nm, where the number of laminin epitopes IKVAV and YIGSR can be varied by changing the PA concentrations over a broad range of 0.125-2 mg/ml. Granule cells (GC) and Purkinje cells (PC), two major neuronal subtypes of cerebellar cortex, demonstrate distinct response to this change of epitope concentration. On IKVAV hybrid constructs, GC density increases three-fold compared with the control collagen substrate at a PA concentration of ≥0.25 mg/ml, while PC density reaches a maximum (five-fold vs. control) at 0.25 mg/ml of PA and rapidly decreases at higher PA concentrations. In addition, adjustment of the epitope number allowed us to achieve fine control over PC dendrite and axon growth. Due to the ability to modulate neuron survival and maturation by easy manipulation of epitope density, our design offers a versatile test bed to study the extracellular matrix (ECM) contribution in neuron development and the design of optimal neuronal scaffold biomaterials.

Photodynamic Control of Bioactivity in a Nanofiber Matrix

Self-assembling peptide materials have been used extensively to mimic natural extracellular matrices (ECMs) by presenting bioactive epitopes on a synthetic matrix. Although this approach can facilitate a desired response from cells grown in the matrix, it lacks the capacity for spatial or temporal regulation of the presented signals. We describe here a photoresponsive, synthetic ECM using a supramolecular platform composed of peptide amphiphiles (PAs) that self-assemble into cylindrical nanofibers. A photocleavable nitrobenzyl ester group was included in the peptide backbone using a novel Fmoc-amino acid that is compatible with microwave-assisted solid-phase peptide synthesis. The placement of the photolabile group on the peptide backbone enabled efficient removal of the ECM-derived cell adhesion epitope RGDS from PA molecules upon exposure to light (half-life of photolysis ~1.9 min) without affecting the nanofiber assembly. Fibroblasts cultured on RGDS-presenting PA nanofiber substrates demonstrated increased cell spreading and more mature focal adhesions compared with unfunctionalized and control (RGES-presenting) surfaces, as determined by immunostaining and cell morphological analysis. Furthermore, we observed an arrest in fibroblast spreading on substrates containing a cleavable RGDS epitope when the culture was exposed to light; in contrast, this dynamic shift in cell response was absent when the RGDS epitope was attached to the PA molecule by a light-insensitive control linker. Light-responsive bioactive materials can contribute to the development of synthetic systems that more closely mimic the dynamic nature of native ECM.

Bioactive DNA-peptide nanotubes enhance the differentiation of neural stem cells into neurons

We report the construction of DNA nanotubes covalently functionalized with the cell adhesion peptide RGDS as a bioactive substrate for neural stem cell differentiation. Alteration of the Watson–Crick base pairing program that builds the nanostructures allowed us to probe independently the effect of nanotube architecture and peptide bioactivity on stem cell differentiation. We found that both factors instruct synergistically the preferential differentiation of the cells into neurons rather than astrocytes.

Self‐assembly of Cytotoxic Peptide Amphiphiles into Supramolecular Membranes for Cancer Therapy

Peptide amphiphiles (PAs) provide a versatile platform for the design of complex and functional material constructs for biomedical applications. The hierarchical self-assembly of PAs with biopolymers is used to create robust hybrid membranes with molecular order on the micron scale. Fabrication of membranes by assembling hyaluronic acid with positively charged PA nanostructures containing anti-cancer PAs bearing a (KLAKLAK)(2) peptide sequence is reported here. Changes in membrane microstructure as the positively charged PA nanostructures vary from cylindrical nanofibers to spherical aggregates are characterized. Results indicate that formation of highly aligned fibrous membranes requires a threshold concentration of nanofibers in solution. Additionally, variation of PA nanostructure morphology from spherical aggregates to cylindrical nanofibers allows membranes to act either as reservoirs for sustained release of cytotoxicity upon enzymatic degradation or as membranes with surface-bound cytotoxicity, respectively. Thus, the self-assembly processes of these PA-biopolymer membranes can be potentially used to design delivery platforms for anti-cancer therapeutics.

Self-assembling glucagon-like peptide 1-mimetic peptide amphiphiles for enhanced activity and proliferation of insulin-secreting cells

Current treatment for type 1 diabetes mellitus requires daily insulin injections that fail to produce physiological glycemic control. Islet cell transplantation has been proposed as a permanent cure but is limited by loss of β-cell viability and function. These limitations could potentially be overcome by relying on the activity of glucagon-like peptide 1 (GLP-1), which acts on β-cells to promote insulin release, proliferation and survival. We have developed a peptide amphiphile (PA) molecule incorporating a peptide mimetic for GLP-1. This GLP-1-mimetic PA self-assembles into one-dimensional nanofibers that stabilize the active secondary structure of GLP-1 and can be cross-linked by calcium ions to form a macroscopic gel capable of cell encapsulation and three-dimensional culture. The GLP-1-mimetic PA nanofibers were found to stimulate insulin secretion from rat insulinoma (RINm5f) cells to a significantly greater extent than the mimetic peptide alone and to a level equivalent to that of the clinically used agonist exendin-4. The activity of the GLP-1-mimetic PA is glucose-dependent, lipid-raft dependent and partially PKA-dependent consistent with native GLP-1. The GLP-1-mimetic PA also completely abrogates inflammatory cytokine-induced cell death to the level of untreated controls. When used as a PA gel to encapsulate RINm5f cells, the GLP-1-mimetic PA stimulates insulin secretion and proliferation in a cytokine-resistant manner that is significantly greater than a non-bioactive PA gel containing exendin-4. Due to its self-assembling property and bioactivity, the GLP-1-mimetic PA can be incorporated into previously developed islet cell transplantation protocols with the potential for significant enhancement of β-cell viability and function.

Post-Assembly Functionalization of Supramolecular Nanostructures with Bioactive Peptides and Fluorescent Proteins by Native Chemical Ligation

Post-assembly functionalization of supramolecular nanostructures has the potential to expand the range of their applications. We report here the use of the chemoselective native chemical ligation (NCL) reaction to functionalize self-assembled peptide amphiphile (PA) nanofibers. This strategy can be used to incorporate specific bioactivity on the nanofibers, and as a model, we demonstrate functionalization with the RGDS peptide following self-assembly. Incorporation of bioactivity is verified by the observation of characteristic changes in fibroblast morphology following NCL-mediated attachment of the signal to PA nanofibers. The NCL reaction does not alter the PA nanofiber morphology, and biotinylated RGDS peptide was found to be accessible on the nanofiber surface after ligation for binding with streptavidin-conjugated gold nanoparticles. In order to show that this strategy is not limited to short peptides, we utilized NCL to conjugate yellow fluorescent protein and/or cyan fluorescent protein to self-assembled PA nanofibers. Förster resonance energy transfer and fluorescence anisotropy measurements are consistent with the immobilization of the protein on the PA nanofibers. The change in electrophoretic mobility of the protein upon conjugation with PA molecules confirmed the formation of a covalent linkage. NCL-mediated attachment of bioactive peptides and proteins to self-assembled PA nanofibers allows the independent control of self-assembly and bioactivity while retaining the biodegradable peptide structure of the PA molecule and thus can be useful in tailoring design of biomaterials.

Supramolecular Assembly of Multifunctional Maspin-Mimetic Nanostructures as a Potent Peptide-Based Angiogenesis Inhibitor

Aberrant angiogenesis plays a large role in pathologies ranging from tumor growth to macular degeneration. Anti-angiogenic proteins have thus come under scrutiny as versatile, potent therapeutics but face problems with purification and tissue retention. We report here on the synthesis of supramolecular nanostructures that mimic the anti-angiogenic activity of maspin, a class II tumor suppressor protein. These maspin-mimetic nanostructures are formed via self-assembly of small peptide amphiphiles containing the g-helix motif of maspin. Using tubulogenesis assays with human umbilical vein endothelial cells, we demonstrate that maspin-mimetic nanostructures show anti-angiogenic activity at concentrations that are significantly lower than those necessary for the g-helix peptide. Furthermore, in vivo assays in the chick chorioallantoic membrane show maspin-mimetic nanostructures to be effective over controls at inhibiting angiogenesis. Thus, the nanostructures investigated here offer an attractive alternative to the use of anti-angiogenic recombinant proteins in the treatment of cancer or other diseases involving abnormal blood vessel formation.