A peptidomimetic targeting white fat causes weight loss and improved insulin resistance in obese monkeys.

Obesity, defined as body mass index greater than 30, is a leading cause of morbidity and mortality and a financial burden worldwide. Despite significant efforts in the past decade, very few drugs have been successfully developed for the treatment of obese patients. Biological differences between rodents and primates are a major hurdle for translation of anti-obesity strategies either discovered or developed in rodents into effective human therapeutics. Here, we evaluate the ligand-directed peptidomimetic CKGGRAKDC-GG-(D)(KLAKLAK)(2) (henceforth termed adipotide) in obese Old World monkeys. Treatment with adipotide induced targeted apoptosis within blood vessels of white adipose tissue and resulted in rapid weight loss and improved insulin resistance in obese monkeys. Magnetic resonance imaging and dual-energy x-ray absorptiometry confirmed a marked reduction in white adipose tissue. At experimentally determined optimal doses, monkeys from three different species displayed predictable and reversible changes in renal proximal tubule function. Together, these data in primates establish adipotide as a prototype in a new class of candidate drugs that may be useful for treating obesity in humans.

Dynamic in vivo biocompatibility of angiogenic peptide amphiphile nanofibers.

Biomaterials that promote angiogenesis have great potential in regenerative medicine for rapid revascularization of damaged tissue, survival of transplanted cells, and healing of chronic wounds. Supramolecular nanofibers formed by self-assembly of a heparin-binding peptide amphiphile and heparan sulfate-like glycosaminoglycans were evaluated here using a dorsal skinfold chamber model to dynamically monitor the interaction between the nanofiber gel and the microcirculation, representing a novel application of this model. We paired this model with a conventional subcutaneous implantation model for static histological assessment of the interactions between the gel and host tissue. In the static analysis, the heparan sulfate-containing nanofiber gels were found to persist in the tissue for up to 30 days and revealed excellent biocompatibility. Strikingly, as the nanofiber gel biodegraded, we observed the formation of a de novo vascularized connective tissue. In the dynamic experiments using the dorsal skinfold chamber, the material again demonstrated good biocompatibility, with minimal dilation of the microcirculation and only a few adherent leukocytes, monitored through intravital fluorescence microscopy. The new application of the dorsal skinfold model corroborated our findings from the traditional static histology, demonstrating the potential use of this technique to dynamically evaluate the biocompatibility of materials. The observed biocompatibility and development of new vascularized tissue using both techniques demonstrates the potential of these angiogenesis-promoting materials for a host of regenerative strategies.

Bioactive nanofibers instruct cells to proliferate and differentiate during enamel regeneration.

During tooth development, ectoderm-derived ameloblast cells create enamel by synthesizing a complex protein mixture serving to control cell to matrix interactions and the habit of hydroxyapatite crystallites. Using an in vitro cell and organ culture system, we studied the effect of artificial bioactive nanostructures on ameloblasts with the long-term goal of developing cell-based strategies for tooth regeneration. We used branched peptide amphiphile molecules containing the peptide motif Arg-Gly-Asp, or "RGD" (abbreviated BRGD-PA), known to self-assemble in physiologic environments into nanofibers that display on their surfaces high densities of this biological signal. Ameloblast-like cells (line LS8) and primary enamel organ epithelial (EOE) cells were cultured within PA hydrogels, and the PA was injected into the enamel organ epithelia of mouse embryonic incisors. The expression of amelogenin, ameloblastin, integrin alpha 5, and integrin alpha 6 was detected by quantitative real-time PCR and immunodetection techniques. We performed cell proliferation assay using BrdU labeling and a biomineralization assay using Alizarin red S staining with quantitative Ca(2+) measurements. In the cell culture model, ameloblast-like cells (LS8) and primary EOE cells responded to the BRGD-PA nanostructures with enhanced proliferation and greater amelogenin, ameloblastin, and integrin expression levels. At the site of injection of the BRGD-PA in the organ culture model, we observed EOE cell proliferation with differentiation into ameloblasts as evidenced by their expression of enamel specific proteins. Ultrastructural analysis showed the nanofibers within the forming extracellular matrix, in contact with the EOE cells engaged in enamel formation and regeneration. This study shows that BRGD-PA nanofibers present with enamel proteins participate in integrin-mediated cell binding to the matrix with delivery of instructive signals for enamel formation.

Nitric oxide and nanotechnology: a novel approach to inhibit neointimal hyperplasia.

OBJECTIVE: Nitric oxide (NO) has been shown to inhibit neointimal hyperplasia after arterial interventions in several animal models. To date, however, NO-based therapies have not been used in the clinical arena. Our objective was to combine nanofiber delivery vehicles with NO chemistry to create a novel, more potent NO-releasing therapy that can be used clinically. Thus, the aim of this study was to evaluate the perivascular application of spontaneously self-assembling NO-releasing nanofiber gels. Our hypothesis was that this application would prevent neointimal hyperplasia. METHODS: Gels consisted of a peptide amphiphile, heparin, and a diazeniumdiolate NO donor (1-[N-(3-Aminopropyl)-N-(3-ammoniopropyl)]diazen-1-ium-1,2-diolate [DPTA/NO] or disodium 1-[(2-Carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate [PROLI/NO]). Nitric oxide release from the gels was evaluated by the Griess reaction, and scanning electron microscopy confirmed nanofiber formation. Vascular smooth muscle cell (VSMC) proliferation and cell death were assessed in vitro by (3)H-thymidine incorporation and Personal Cell Analysis (PCA) system (Guava Technologies, Hayward, Calif). For the in vivo work, gels were modified by reducing the free-water content. Neointimal hyperplasia after periadventitial gel application was evaluated using the rat carotid artery injury model at 14 days (n = 6 per group). Inflammation and proliferation were examined in vivo with immunofluorescent staining against CD45, ED1, and Ki67 at 3 days (n = 2 per group), and graded by blinded observers. Endothelialization was assessed by Evans blue injection at 7 days (n = 3 per group). RESULTS: Both DPTA/NO and PROLI/NO, combined with the peptide amphiphile and heparin, formed nanofiber gels and released NO for 4 days. In vitro, DPTA/NO inhibited VSMC proliferation and induced cell death to a greater extent than PROLI/NO. However, the DPTA/NO nanofiber gel only reduced neointimal hyperplasia by 45% (intima/media [I/M] area ratio, 0.45 +/- 0.07), whereas the PROLI/NO nanofiber gel reduced neointimal hyperplasia by 77% (I/M area ratio, 0.19 +/- 0.03, P < .05) vs control (injury alone I/M area ratio, 0.83 +/- 0.07; P < .05). Both DPTA/NO and PROLI/NO nanofiber gels significantly inhibited proliferation in vivo (1.06 +/- 0.30 and 0.19 +/- 0.11 vs injury alone, 2.02 +/- 0.20, P < .05), yet had minimal effect on apoptosis. Only the PROLI/NO nanofiber gel inhibited inflammation (monocytes and leukocytes). Both NO-releasing nanofiber gels stimulated re-endothelialization. CONCLUSIONS: Perivascular application of NO-releasing self-assembling nanofiber gels is an effective and simple therapy to prevent neointimal hyperplasia after arterial injury. Our study demonstrates that the PROLI/NO nanofiber gel most effectively prevented neointimal hyperplasia and resulted in less inflammation than the DPTA/NO nanofiber gel. This therapy has great clinical potential to prevent neointimal hyperplasia after open vascular interventions in patients.

Sorting carbon nanotubes by electronic structure using density differentiation.

The heterogeneity of as-synthesized single-walled carbon nanotubes (SWNTs) precludes their widespread application in electronics, optics and sensing. We report on the sorting of carbon nanotubes by diameter, bandgap and electronic type using structure-discriminating surfactants to engineer subtle differences in their buoyant densities. Using the scalable technique of density-gradient ultracentrifugation, we have isolated narrow distributions of SWNTs in which >97% are within a 0.02-nm-diameter range. Furthermore, using competing mixtures of surfactants, we have produced bulk quantities of SWNTs of predominantly a single electronic type. These materials were used to fabricate thin-film electrical devices of networked SWNTs characterized by either metallic or semiconducting behaviour.

Presentation of RGDS epitopes on self-assembled nanofibers of branched peptide amphiphiles.

Branched peptide amphiphile (PA) molecules bearing biological epitopes were designed and synthesized using orthogonal protecting group chemistry on amine groups at lysine residues. These molecules self-assemble into high-aspect-ratio cylindrical nanofibers, and their branched architecture enhances accessibility of epitopes for protein binding and also allows the presentation of more than one epitope in a single molecule. The RGDS cell adhesion epitope was used as a model bioactive signal on PA molecules for potential biomedical applications. Aggregation of the branched PA molecules into nanofibers was demonstrated by TEM and through shifts in the protonation profiles of peripheral amines. These systems also formed self-supporting gels in the presence of physiological fluids and other biologically relevant macromolecules such as synovial fluid and DNA, an important property for their potential use in medicine. Fluorescence anisotropy measurements on the PAs with tryptophan residues were performed to examine the effect of branching on packing and mobility of the peptides in the self-assembled nanofibers. The mobility of tryptophan residues was observed to be restricted upon packing of PA molecules into nanofibers. However, relative to linear analogues, branched molecules retain more mobility in the supramolecular aggregates.

Heparin binding nanostructures to promote growth of blood vessels.

Controlling new blood vessel formation is of interest in regenerative medicine and cancer treatment. Heparin, a biopolymer that binds to angiogenic growth factors, was used to nucleate the self-assembly of nanostructures from designed peptide amphiphile molecules. This process yields rigid nanofibers that display heparin chains to orient proteins for cell signaling. In vivo, the nanostructures stimulated extensive new blood vessel formation using nanogram amounts of growth-factor proteins that by themselves did not induce any detectable angiogenesis.

Presentation and recognition of biotin on nanofibers formed by branched peptide amphiphiles.

A branched peptide amphiphile system was designed for enhanced recognition of biotin on nanofibers formed by self-assembly of these molecules. Branching at a lysine residue was used to design peptide amphiphiles that are capable of presenting more than one epitope per molecule. We found that biotinylated branched structures form nanofibers that enhance recognition by the avidin protein receptor relative to similar nanostructures formed by linear peptide analogues. Biotin-avidin binding to the supramolecular nanofibers was characterized by measurement of fluorescence from nanofibers incubated with chromophore-conjugated avidin.

Self-assembly and luminescence of oligo(p-phenylene vinylene) amphiphiles.

We have synthesized a series of amphiphilic molecules consisting of oligo(phenylene vinylene) (OPV) asymmetrically end-substituted with a hydrophilic poly(ethylene glycol) (PEG) segment and a hydrophobic alkyl chain. This amphiphilic structure induces self-assembly into both thermotropic and lyotropic lamellar liquid crystalline (LC) phases. The molecules form strongly fluorescent, self-supporting gels in both water and polar organic solvents, even at high concentrations on the order of 30 wt %. These self-assembled structures have been characterized by small-angle X-ray scattering (SAXS), differential scanning calorimetry (DSC), and polarized optical microscopy (POM). Photoluminescence (PL) is influenced by the structure of the material, with enhanced emission in the LC state due to assembly of the chromophore in confined two-dimensional layers. Self-assembly controlling molecular aggregation at the nanoscale could significantly improve the performance of OPV-based materials in optoelectronic devices.

Synthesis, self-assembly, and characterization of supramolecular polymers from electroactive dendron rodcoil molecules.

We report here the synthesis and self-assembly of a series of three molecules with dendron rodcoil architecture that contain conjugated segments of oligo(thiophene), oligo(phenylene-vinylene), and oligo(phenylene). Despite their structural differences, all three molecules yield similar self-assembled structures. Electron and atomic force microscopy reveals the self-assembly of the molecules into high aspect ratio ribbon-like nanostructures which at low concentrations induce gelation in nonpolar solvent. Self-assembly results in a blue-shifted absorption spectrum and a red-shifted, quenched fluorescence spectrum, indicating aggregation of the conjugated segments within the ribbon-like structures. The assembly of these molecules into one-dimensional nanostructures is a route to pi-pi stacked supramolecular polymers for organic electronic functions. In the oligo(thiophene) derivative, self-assembly leads to a 3 orders of magnitude increase in the conductivity of iodine-doped films due to self-assembly. We also found that electric field alignment of these supramolecular assemblies can be used to create arrays of self-assembled nanowires on a device substrate.