Sungsoo S. Lee

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.

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.

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.

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 β-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.

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.

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 very different polysaccharide binding domains. Binding does not disrupt the filamentous shape of the nanostructures or their internal β-sheet backbone, but must involve accessible adaptive configurations to interact with such different proteins. The glycopeptide nanostructures amplified signaling 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 expected. These super-bioactive nanostructures may enable many therapies in the horizon involving proteins.

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.

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.

Antibody-catalysed glycosyl transfer reactions from in vitro immunization

In vitro immunization using a 5-membered ring iminocyclitol afforded antibodies that catalyse the hydrolysis of p-nitrophenyl glucopyranoside and p-nitrophenyl galactopyranoside with rate enhancements (kcat/kuncat) of greater than 104.