Synergistic regulation of cerebellar Purkinje neuron development by laminin epitopes and collagen on an artificial hybrid matrix construct

Cataloged from PDF version of article.The extracellular matrix (ECM) creates a dynamic environment around the cells in the developing central nervous system, providing them with the necessary biochemical and biophysical signals. Although the functions of many ECM molecules in neuronal development have been individually studied in detail, the combinatorial effects of multiple ECM components are not well characterized. Here we demonstrate that the expression of collagen and laminin-1 (lam-1) are spatially and temporally correlated during embryonic and post-natal development of the cerebellum. These changes in ECM distribution correspond to specific stages of Purkinje neuron (PC) migration, somatic monolayer formation and polarization. To clarify the respective roles of these ECM molecules on PC development, we cultured cerebellar neurons on a hybrid matrix comprised of collagen and a synthetic peptide amphiphile nanofiber bearing a potent lam-1 derived bioactive IKVAV peptide epitope. By systematically varying the concentration and ratio of collagen and the laminin epitope in the matrix, we could demonstrate a synergistic relationship between these two ECM components in controlling multiple aspects of PC maturation. An optimal ratio of collagen and IKVAV in the matrix was found to promote maximal PC survival and dendrite growth, while dendrite penetration into the matrix was enhanced by a high IKVAV to collagen ratio. In addition, the laminin epitope was found to guide PC axon development. By combining our observations in vivo and in vitro, we propose a model of PC development where the synergistic effects of collagen and lam-1 play a key role in migration, polarization and morphological maturation of PCs. This journal is © the Partner Organisations 2014

Self-assembled 2D Free-Standing Janus Nanosheets with Single-Layer Thickness

We report the thermodynamically controlled growth of solution-processable and free-standing nanosheets via peptide assembly in two dimensions. By taking advantage of self-sorting between peptide β-strands and hydrocarbon chains, we have demonstrated the formation of Janus 2D structures with single-layer thickness, which enable a predetermined surface heterofunctionalization. A controlled 2D-to-1D morphological transition was achieved by subtly adjusting the intermolecular forces. These nanosheets provide an ideal substrate for the engineering of guest components (e.g., proteins and nanoparticles), where enhanced enzyme activity was observed. We anticipate that sequence-specific programmed peptides will offer promise as design elements for 2D assemblies with face-selective functionalization

Peptide-based microcapsules obtained by self-assembly and microfluidics as controlled environments for cell culture

Funding for this study was provided by the Portuguese Foundation for Science and Technology (FCT, grant PTDC/EBB-BIO/ 114523/2009). D. S. Ferreira gratefully acknowledges FCT for the PhD scholarship (SFRH/BD/44977/2008)

A designer peptide as a template for growing Au nanoclusters

A peptide was designed to generate a sub-nanometric template that guides the growth of fluorescent gold nanoclusters. The peptide was endorsed with nucleating moieties and a three-dimensional structure that arrests the growth of ultrasmall nanoparticles. The nanoclusters are not cytotoxic and can be found in the cytosol of cells

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. © 2011 Elsevier Ltd

Plasmonic chirality imprinting on nucleobase-displaying supramolecular nanohelices by metal-nucleobase recognition

Supramolecular self-assembly is an important process that enables the conception of complex structures mimicking biological motifs. Herein, we constructed helical fibrils through chiral self-assembly of nucleobase–peptide conjugates (NPCs), where achiral nucleobases are helically displayed on the surface of fibrils, comparable to polymerized nucleic acids. Selective binding between DNA and the NPC fibrils was observed with fluorescence polarization. Taking advantage of metal–nucleobase recognition, we highlight the possibility of deposition/assembly of plasmonic nanoparticles onto the fibrillar constructs. In this approach, the supramolecular chirality of NPCs can be adaptively imparted to metallic nanoparticles, covering them to generate structures with plasmonic chirality that exhibit significantly improved colloidal stability. The self-assembly of rationally designed NPCs into nanohelices is a promising way to engineer complex, optically diverse nucleobase-derived nanomaterials

Reversible helical unwinding transition of a self-assembling peptide amphiphile

A designed peptide amphiphile C16-KKFFVLK self-assembles into nanotubes and helical ribbons in aqueous solution at room temperature. A remarkable unwinding transition, leading to twisted tapes, is observed on heating. Nanotubes and ribbons re-form on cooling

PEGylation affects the self-assembling behaviour of amphiphilic octapeptides

Surfactant-like peptides are a class of amphiphilic macromolecules, which are able to self-assemble in water forming different supramolecular structures. Among them, octapeptides composed of six hydrophobic and two hydrophilic residues have attracted interest since they have a length similar to those of natural phospholipids. Supramolecular structures of different amphiphilic octapeptides have been widely reported, but no study has been performed aimed at investigating the effect of PEGylation on their self-assembling behaviour. The aim of the present work was to synthesize and characterise the self-assembling behaviour of PEGylated alanine- or valine based amphiphilic octapeptides (mPEG1.9kDa-DDAAAAAA and mPEG1.9kDa-DDVVVVVV) in comparison to the non-PEGylated ones (DDAAAAAA and DDVVVVVV). The self-aggregation process in ultrapure water was investigated by fluorescence spectroscopy, small angle neutron scattering (SANS), dynamic light scattering (DLS), while the secondary structure was assessed by circular dichroism. PEGylation markedly affects the self-assembling behaviour of these amphiphilic octapeptides in terms of both critical aggregation concentration (CAC) and shape of the formed supramolecular aggregates. Indeed, PEGylation increases CAC and prevents the self-aggregation into fibrillary supramolecular aggregates (as observed for non-PEGylated peptides), by promoting the formation of micelle-like structures (as demonstrated for valine-based octapeptide). On the other side, the secondary structure of peptides seems not to be affected by PEGylation. Overall, these results suggest that self-assembling behaviour of amphiphilic octapeptides can be modified by PEGylation, with a great potential impact for the future applications of these nanomaterials

Hydrodynamically Guided Hierarchical Self-Assembly of Peptide-Protein Bioinks

Effective integration of molecular self-assembly and additive manufacturing would provide a technological leap in bioprinting. This article reports on a biofabrication system based on the hydrodynamically guided co-assembly of peptide amphiphiles (PAs) with naturally occurring biomolecules and proteins to generate hierarchical constructs with tuneable molecular composition and structural control. The system takes advantage of droplet-on-demand (DoD) inkjet printing to exploit interfacial fluid forces and guide molecular self-assembly into aligned or disordered nanofibers, hydrogel structures of different geometries and sizes, surface topographies and higherordered constructs bound by molecular diffusion. PAs were designed to co-assemble during printing in cell diluent conditions with a range of extracellular matrix (ECM) proteins and biomolecules including fibronectin, collagen, keratin, elastin-like proteins (ELPs) and hyaluronic acid. Using combinations of these molecules, NIH-3T3 and adipose derived stem cells were bioprinted within complex structures while exhibiting high cell viability (> 88 %). By integrating self-assembly with 3D-bioprinting, the study introduces a novel biofabrication platform capable of encapsulating and spatially distributing multiple cell types within tuneable pericellular environments. In this way, the work demonstrates the potential of the approach to generate complex bioactive scaffolds for applications such as tissue engineering, in vitro models, and drug screening