Charles M. Rubert Pérez

Hierarchical assembly of collagen peptide triple helices into curved disks and metal ion-promoted hollow spheres.

A 27 amino acid collagen-based peptide (Hbyp3) was designed to radially display nine hydrophobic bipyridine moieties from a triple helical scaffold. Self-assembly of such functionalized triple helices led to the formation of micrometer-scaled disks with a curved morphology, presumably mediated by aromatic interactions, with a height that is in the range of the length of the triple helical peptide. Higher order assembly of these curved disks into micrometer-sized hollow spheres was accomplished through metal-ligand interactions between bipyridine groups of the disks and metal ions such as Fe(II), Co(II), Zn(II) and Cu(II). The thickness of the shell of these hollow spheres corresponds well with the thickness of the collagen peptide-based triple helix and the corresponding self-assembled disks. Addition of a metal ion chelator was found to reverse the assembly of the hollow spheres back to the curved disk structures. These data support the formation of the hollow spheres from the self-assembled disks of Hbyp3 upon addition of metal ions.

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.

Metal-mediated tandem coassembly of collagen peptides into banded microstructures.

The ability to recapitulate the features of natural collagen at the micro- and nanoscale with novel biopolymers has the potential to lead to improved biomaterials. Herein we describe stimuli-responsive collagen-based peptides (IdaCol and HisCol) that together form higher order assemblies in the presence of added metal ions. SEM and TEM imaging of these assemblies revealed microscale petal-like and intertwined fiber morphologies, each with periodic banding on the nanometer scale. The observed banding is consistent with tandem coassembly of alternating IdaCol and HisCol triple helical blocks that may laterally associate either in or out of register to form higher order structures, and mimics the banding found in natural collagen fibers.

A collagen peptide-based physical hydrogel for cell encapsulation.

Collagen peptide-based hydrogels are prepared and characterized for application in 3D cell growth. Physical hydrogels are formed by covalently linking a collagen-based peptide to an 8-arm poly(ethylene glycol) star polymer. The resulting viscoelastic hydrogels have the ability to melt into a liquid-like state near the melting temperature of the collagen triple helix and reform back into an elastic-state at room temperature, adding a thermoresponsive feature to the material. In addition, the hydrogels possess desirable stiffness, as well as a highly cross-linked network of pores where cells are found to reside, making the hydrogels promising scaffolds for the culture of hMSCs.

Tissue-Factor Targeted Peptide Amphiphile Nanofibers as an Injectable Therapy To Control Hemorrhage

Noncompressible torso hemorrhage is a leading cause of mortality in civilian and battlefield trauma. We sought to develop an i.v.-injectable, tissue factor (TF)-targeted nanotherapy to stop hemorrhage. Tissue factor was chosen as a target because it is only exposed to the intravascular space upon vessel disruption. Peptide amphiphile (PA) monomers that self-assemble into nanofibers were chosen as the delivery vehicle. Three TF-binding sequences were identified (EGR, RLM, and RTL), covalently incorporated into the PA backbone, and shown to self-assemble into nanofibers by cryo-transmission electron microscopy. Both the RLM and RTL peptides bound recombinant TF in vitro. All three TF-targeted nanofibers bound to the site of punch biopsy-induced liver hemorrhage in vivo, but only RTL nanofibers reduced blood loss versus sham (53% reduction, p < 0.05). Increasing the targeting ligand density of RTL nanofibers yielded qualitatively better binding to the site of injury and greater reductions in blood loss in vivo (p < 0.05). In fact, 100% RTL nanofiber reduced overall blood loss by 60% versus sham (p < 0.05). Evaluation of the biocompatibility of the RTL nanofiber revealed that it did not induce RBC hemolysis, did not induce neutrophil or macrophage inflammation at the site of liver injury, and 70% remained intact in plasma after 30 min. In summary, these studies demonstrate successful binding of peptides to TF in vitro and successful homing of a TF-targeted PA nanofiber to the site of hemorrhage with an associated decrease in blood loss in vivo. Thus, this therapeutic may potentially treat noncompressible hemorrhage.

Inhibitors of anthrax lethal factor based upon N-oleoyldopamine.

The structural features of an anthrax lethal factor inhibitor, N-oleoyldopamine (OLDA, 1) have been probed. The oleic acid moiety is critical, but, more interestingly, the presence of the double bond and its geometry were found to play an essential role. One compound, 5, was found to be an uncompetitive inhibitor of lethal factor (LF) with a K(i) value of 2.2microM and a cell-based IC(50) value of 4.3microM.

Injectable biomimetic liquid crystalline scaffolds enhance muscle stem cell transplantation

Significance Most research aiming to achieve muscle regeneration focuses on the biology of “muscle stem cells,” but delivery methods that enhance transplantation efficiency of these cells are at early stages. We report on a liquid crystalline scaffold that encapsulates the cells and gels upon injection in vivo without requiring an external stimulus. As a unique structural feature, the scaffold contains nanofibers that align preferentially with surrounding natural muscle fibers. The biomimetic scaffold can have a stiffness that matches that of muscle, has great ability to retain growth factors, and has a biodegradation rate that is compatible with regeneration time scales. Most importantly, the scaffold enhances engraftment efficiency of the cells in injured muscle, and without injury when combined with growth factors. Muscle stem cells are a potent cell population dedicated to efficacious skeletal muscle regeneration, but their therapeutic utility is currently limited by mode of delivery. We developed a cell delivery strategy based on a supramolecular liquid crystal formed by peptide amphiphiles (PAs) that encapsulates cells and growth factors within a muscle-like unidirectionally ordered environment of nanofibers. The stiffness of the PA scaffolds, dependent on amino acid sequence, was found to determine the macroscopic degree of cell alignment templated by the nanofibers in vitro. Furthermore, these PA scaffolds support myogenic progenitor cell survival and proliferation and they can be optimized to induce cell differentiation and maturation. We engineered an in vivo delivery system to assemble scaffolds by injection of a PA solution that enabled coalignment of scaffold nanofibers with endogenous myofibers. These scaffolds locally retained growth factors, displayed degradation rates matching the time course of muscle tissue regeneration, and markedly enhanced the engraftment of muscle stem cells in injured and noninjured muscles in mice.

Small Molecule Inhibitors of Anthrax Toxin–induced Cytotoxicity Targeted Against Protective Antigen

Two molecular scaffolds were designed using the CAVEAT molecular design package to inhibit the oligomerization of protective antigen (PA63), a key protein component of anthrax toxin. The inhibitors were designed to prevent heptamerization of PA63 by mimicking key residues of PA63 needed for the intermolecular interactions that stabilize the heptamer. Using the scaffolds identified by CAVEAT, seven candidate inhibitors were synthesized and tested for their ability to inhibit anthrax toxin–induced cytotoxicity, with three of the agents demonstrating modest inhibition in murine J774A.1 macrophage cells.

Mimicking the Bioactivity of Fibroblast Growth Factor-2 Using Supramolecular Nanoribbons

Fibroblast growth factor (FGF-2) is a multifunctional growth factor that has pleiotropic effects in different tissues and organs. In particular, FGF-2 has a special role in angiogenesis, an important process in development, wound healing, cell survival, and differentiation. Therefore, incorporating biological agents like FGF-2 within therapeutic biomaterials is a potential strategy to create angiogenic bioactivity for the repair of damaged tissue caused by trauma or complications that arise from age and/or disease. However, the use of growth factors as therapeutic agents can be costly and does not always bring about efficient tissue repair due to rapid clearance from the targeted site. An alternative would be a stable supramolecular nanostructure with the capacity to activate the FGF-2 receptor that can also assemble into a scaffold deliverable to tissue. We report here on peptide amphiphiles that incorporate a peptide known to activate the FGF-2 receptor and peptide domains that drive its self-assembly into supramolecular nanoribbons. These FGF2-PA nanoribbons displayed the ability to increase the proliferation and migration of the human umbilical vein endothelial cells (HUVECs) in vitro to the same extent as the native FGF-2 protein at certain concentrations. We confirmed that this activity was specific to the FGFR1 signaling pathway by tracking the phosphorylation of downstream signaling effectors such ERK1/2 and pH3. These results indicated the specificity of FGF2-PA nanoribbons in activating the FGF-2 signaling pathway and its potential application as a supramolecular scaffold that can be used in vivo as an alternative to the encapsulation and delivery of the native FGF-2 protein.

Peptide Amphiphile Nanostructures for Targeting of Atherosclerotic Plaque and Drug Delivery

Coassembled peptide amphiphile nanofibers designed to target atherosclerotic plaque and enhance cholesterol efflux are shown to encapsulate and deliver a liver X receptor agonist to increase efflux from murine macrophages in vitro. Fluorescence microscopy reveals that the nanofibers, which display an apolipoprotein‐mimetic peptide, localize at plaque sites in low density lipoprotein receptor knockout (LDLR KO) mice with or without the encapsulated molecule, while nanofibers displaying a scrambled, nontargeting peptide sequence do not demonstrate comparable binding. These results show that nanofibers functionalized with apolipoprotein‐mimetic peptides may be effective vehicles for intravascular targeted drug delivery to treat atherosclerosis.