Supramolecular Nanofibers Enhance Growth Factor Signaling by Increasing Lipid Raft Mobility.

The nanostructures of self-assembling biomaterials have been previously designed to tune the release of growth factors in order to optimize biological repair and regeneration. We report here on the discovery that weakly cohesive peptide nanostructures in terms of intermolecular hydrogen bonding, when combined with low concentrations of osteogenic growth factor, enhance both BMP-2 and Wnt mediated signaling in myoblasts and bone marrow stromal cells, respectively. Conversely, analogous nanostructures with enhanced levels of internal hydrogen bonding and cohesion lead to an overall reduction in BMP-2 signaling. We propose that the mechanism for enhanced growth factor signaling by the nanostructures is related to their ability to increase diffusion within membrane lipid rafts. The phenomenon reported here could lead to new nanomedicine strategies to mediate growth factor signaling for translational targets.

Nucleation and Growth of Ordered Arrays of Silver Nanoparticles on Peptide Nanofibers: Hybrid Nanostructures with Antimicrobial Properties.

Silver nanoparticles have been of great interest as plasmonic substrates for sensing and imaging, catalysts, or antimicrobial systems. Their physical properties are strongly dependent on parameters that remain challenging to control such as size, chemical composition, and spatial distribution. We report here on supramolecular assemblies of a novel peptide amphiphile containing aldehyde functionality in order to reduce silver ions and subsequently nucleate silver metal nanoparticles in water. This system spontaneously generates monodisperse silver particles at fairly regular distances along the length of the filamentous organic assemblies. The metal-organic hybrid structures exhibited antimicrobial activity and significantly less toxicity toward eukaryotic cells. Metallized organic nanofibers of the type described here offer the possibility to create hydrogels, which integrate the useful functions of silver nanoparticles with controllable metallic content.

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

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

Advances in cryogenic transmission electron microscopy for the characterization of dynamic self-assembling nanostructures.

Elucidating the structural information of nanoscale materials in their solvent-exposed state is crucial, as a result, cryogenic transmission electron microscopy (cryo-TEM) has become an increasingly popular technique in the materials science, chemistry, and biology communities. Cryo-TEM provides a method to directly visualize the specimen structure in a solution-state through a thin film of vitrified solvent. This technique complements X-ray, neutron, and light scattering methods that probe the statistical average of all species present; furthermore, cryo-TEM can be used to observe changes in structure over time. In the area of self-assembly, this tool has been particularly powerful for the characterization of natural and synthetic small molecule assemblies, as well as hybrid organic-inorganic composites. In this review, we discuss recent advances in cryogenic TEM in the context of self-assembling systems with emphasis on characterization of transitions observed in response to external stimuli.

Peptide-siRNA Supramolecular Particles for Neural Cell Transfection.

Small interfering ribonucleic acid (siRNA)-based gene knockdown is an effective tool for gene screening and therapeutics. However, the use of nonviral methods has remained an enormous challenge in neural cells. A strategy is reported to design artificial noncationic modular peptides with amplified affinity for siRNA via supramolecular assembly that shows efficient protein knockdown in neural cells. By solid phase synthesis, a sequence that binds specifically double-stranded ribonucleic acid (dsRNA) with a self-assembling peptide for particle formation is integrated. These supramolecular particles can be further functionalized with bioactive sequences without affecting their biophysical properties. The peptide carrier is found to silence efficiently up to 83% of protein expression in primary astroglial and neuronal cell cultures without cytotoxicity. In the case of neurons, a reduction in electrical activity is observed once the presynaptic protein synaptophysin is downregulated by the siRNA-peptide particles. The results demonstrate that the supramolecular particles offer an siRNA delivery platform for efficient nonviral gene screening and discovery of novel therapies for neural cells.

Instructing cells with programmable peptide DNA hybrids.

The native extracellular matrix is a space in which signals can be displayed dynamically and reversibly, positioned with nanoscale precision, and combined synergistically to control cell function. Here we describe a molecular system that can be programmed to control these three characteristics. In this approach we immobilize peptide-DNA (P-DNA) molecules on a surface through complementary DNA tethers directing cells to adhere and spread reversibly over multiple cycles. The DNA can also serve as a molecular ruler to control the distance-dependent synergy between two peptides. Finally, we use two orthogonal DNA handles to regulate two different bioactive signals, with the ability to independently up- or downregulate each over time. This enabled us to discover that neural stem cells, derived from the murine spinal cord and organized as neurospheres, can be triggered to migrate out in response to an exogenous signal but then regroup into a neurosphere as the signal is removed.

Sulfated glycopeptide nanostructures for multipotent protein activation.

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

Super-resolution microscopy reveals structural diversity in molecular exchange among peptide amphiphile nanofibres.

The dynamic behaviour of supramolecular systems is an important dimension of their potential functions. Here, we report on the use of stochastic optical reconstruction microscopy to study the molecular exchange of peptide amphiphile nanofibres, supramolecular systems known to have important biomedical functions. Solutions of nanofibres labelled with different dyes (Cy3 and Cy5) were mixed, and the distribution of dyes inserting into initially single-colour nanofibres was quantified using correlative image analysis. Our observations are consistent with an exchange mechanism involving monomers or small clusters of molecules inserting randomly into a fibre. Different exchange rates are observed within the same fibre, suggesting that local cohesive structures exist on the basis of ß-sheet discontinuous domains. The results reported here show that peptide amphiphile supramolecular systems can be dynamic and that their intermolecular interactions affect exchange patterns. This information can be used to generate useful aggregate morphologies for improved biomedical function.

The powerful functions of peptide-based bioactive matrices for regenerative medicine.

In an effort to develop bioactive matrices for regenerative medicine, peptides have been used widely to promote interactions with cells and elicit desired behaviors in vivo. This paper describes strategies that utilize peptide-based molecules as building blocks to create supramolecular nanostructures that emulate not only the architecture but also the chemistry of the extracellular matrix in mammalian biology. After initiating a desired regenerative response in vivo, the innate biodegradability of these systems allow for the natural biological processes to take over in order to promote formation of a new tissue without leaving a trace of the nonnatural components. These bioactive matrices can either bind or mimic growth factors or other protein ligands to elicit a cellular response, promote specific mechano-biological responses, and also guide the migration of cells with programmed directionality. In vivo applications discussed in this review using peptide-based matrices include the regeneration of axons after spinal cord injury, regeneration of bone, and the formation of blood vessels in ischemic muscle as a therapy in peripheral arterial disease and cardiovascular diseases.

Gel scaffolds of BMP-2-binding peptide amphiphile nanofibers for spinal arthrodesis.

Peptide amphiphile (PA) nanofibers formed by self-assembly can be customized for specific applications in regenerative medicine through the use of molecules that display bioactive signals on their surfaces. Here, the use of PA nanofibers with binding affinity for the bone promoting growth factor BMP-2 to create a gel scaffold for osteogenesis is reported. With the objective of reducing the amount of BMP-2 used clinically for successful arthrodesis in the spine, amounts of growth factor incorporated in the scaffolds that are 10 to 100 times lower than that those used clinically in collagen scaffolds are used. The efficacy of the bioactive PA system to promote BMP-2-induced osteogenesis in vivo is investigated in a rat posterolateral lumbar intertransverse spinal fusion model. PA nanofiber gels displaying BMP-2-binding segments exhibit superior spinal fusion rates relative to controls, effectively decreasing the required therapeutic dose of BMP-2 by 10-fold. Interestingly, a 42% fusion rate is observed for gels containing the bioactive nanofibers without the use of exogenous BMP-2, suggesting the ability of the nanofiber to recruit endogenous growth factor. Results obtained here demonstrate that bioactive biomaterials with capacity to bind specific growth factors by design are great targets for regenerative medicine.

Esterase-activated release of naproxen from supramolecular nanofibres.

Nanofibre forming peptide amphiphiles were conjugated to naproxen through an esterase-sensitive linker. The amount of naproxen released, in the presence of enzymes, was influenced by the linker conjugating the drug to the supramolecular assembly. In vitro studies showed the anti-inflammatory activity of the released drug was maintained.

A comparative evaluation of factors influencing osteoinductivity among scaffolds designed for bone regeneration.

Due to differing compositions, synthetic scaffolds developed for bone regeneration vary widely in efficacy. To quantify the impact of such differences on osteoinductivity, numerous parameters were examined. Absorbable collagen sponge (ACS), three ceramic-based carriers (#1-3) of varying compositions, mineralized allograft chips, and an experimental phosphoserine-rich nanofiber scaffold [S(P) gel] were compared in their ability to promote cell adhesion, proliferation/survival, growth factor binding/release, and osteogenic gene expression. Human preosteoblasts were found to adhere most efficiently to the S(P) gel, and the growth/survival was greatest on the S(P) and ACS scaffolds, with minimal growth seen on the allograft and Ceramic #3. In bone morphogenetic protein-2 (BMP-2) binding/release assays, ACS demonstrated a burst release pattern, whereas the allograft and the ceramics inefficiently released BMP-2. The S(P) gel showed the most ideal rates of growth factor binding and release. QPCR analyses showed significant differences in the CXCL12, CXCR4, and RANKL transcripts among the cells grown on these various scaffolds. Although some scaffolds showed an advantage over others in individual parameters, the nanofiber gel appears to provide the optimal balance in the factors important to osteoinductivity evaluated here.

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.