Structure-properties Relationships of Multicomponent Polysaccharide-peptide Hydrogels

Over the past two decades, the potential of hydrogels as scaffolds for tissue regeneration has been widely explored due to their similarity to native extra cellular matrices (ECMs) and the ability to easily adjust both their physicochemical properties as well as their mechanical properties to meet the demands for tissue scaffolds and cell encapsulation. To better emulate the functionality of the natural ECM, current effort in the engineering of synthetic extracellular matrixes has focused on installing molecular features (proteins and bio-interactive polymers) within insoluble scaffolds, either by self-assembly or through covalent modifications of polymer or biopolymer networks. Polysaccharides (e.g hyaluronic acid, alginate, and chitosan) – being non-toxic, hemocompatible, and relatively cheap– possess many of the favorable properties required for biomaterials. Combining polysaccharides and peptides for creating hydrogels for tissue engineering is of particular interest, due to the complementary properties of both molecules: Bio-functionality of the peptides on one hand and similarity to the natural ECM of the polysaccharides on the other. Apart from their direct role in cell interaction, peptide sequences may affect the hierarchical structural organization and mechanical properties of the resulting hydrogel, thus indirectly affecting the cellular response. The overall aim of our study is to develop a fundamental understanding of the structure-mechanical properties relations of multicomponent polysaccharide hydrogels used in tissue engineering applications Here we present a systematic investigation of the effect of RGD- containing peptides on the hierarchical structure of polysaccharide-peptide hybrids (solutions and gels). Polysaccharide type, ligand incorporation method (covalently attached or self-assembled) as well as peptide nanostructure and amount were tested using advanced tools including small angle X-ray scattering (SAXS), electron microscopy and rheology. Our results show that the fraction of the covalently bounded peptide, determines the behavior of a polysaccharide-peptide conjugates in solution, regardless of the specific nature of the polysaccharide. And that the peptides\u27 ability to self-assemble in aqueous solution affects the spatial organization of the polysaccharide and the mechanical properties of the polysaccharide /peptide hybrid hydrogel, both when the peptide is covalently attached to the polysaccharide backbone and when peptide and polymer solutions are simply mixed together. These results indicate the importance of possible intermolecular interactions between the peptide and the polymer in determining the hydrogel\u27s properties. Our findings suggest that elucidating key factors involved in the structure-property relationships of these systems will improve our ability to design and prepare tailor-made scaffolds for a variety of applications

Polysaccharide Hydrogels Cross–Linked Via Peptide–Dendrimers

Dendritic and other highly branched structures are of importance in biomaterials since their topological features may lead to useful properties, including substantially improved resistance to proteolysis of branched peptides compared to linear ones. As crosslinkers, dendrimers offer the advantage of versatility in terms of number and chemical composition of surface functionalities; a versatility that can be translated to gels with easily tunable structure and properties. Here we present gels prepared from hyaluronic acid (HA) and dendritic elastin-like peptides (denELPs) of generations 1, 2, and 3 (G1, 2, and 3) as crosslinking units. The physical properties of these hydrogels were investigated by rheology, scanning electron microscopy (SEM), Small angle x-ray scattering (SAXS), and model drug loading and release assays. Hydrogels made with different generation denELPs exhibited different structures and physical properties, demonstrating the importance of the multivalency effect in determining hydrogel characteristics. Moreover, upon rehydration after lyophilization, a gradual decrease in hydrogel stiffness was observed. The rate of this decrease is also correlated to the denELP generation, highlighting the role that dendritic peptides can play in making new biomaterials

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

Genetic Manipulation of Iron Biomineralization Enhances MR Relaxivity in a Ferritin-M6A Chimeric Complex

Ferritin has gained significant attention as a potential reporter gene for in vivo imaging by magnetic resonance imaging (MRI). However, due to the ferritin ferrihydrite core, the relaxivity and sensitivity for detection of native ferritin is relatively low. We report here on a novel chimeric magneto-ferritin reporter gene – ferritin-M6A – in which the magnetite binding peptide from the magnetotactic bacteria magnetosome-associated Mms6 protein was fused to the C-terminal of murine h-ferritin. Biophysical experiments showed that purified ferritin-M6A assembled into a stable protein cage with the M6A protruding into the cage core, enabling magnetite biomineralisation. Ferritin-M6A-expressing C6-glioma cells showed enhanced (per iron) r2 relaxivity. MRI in vivo studies of ferritin-M6A-expressing tumour xenografts showed enhanced R2 relaxation rate in the central hypoxic region of the tumours. Such enhanced relaxivity would increase the sensitivity of ferritin as a reporter gene for non-invasive in vivo MRI-monitoring of cell delivery and differentiation in cellular or gene-based therapies

Short and soft: multi-domain organization, tunable dynamics and jamming in suspensions of grafted colloidal cylinders with small aspect ratio

The yet virtually unexplored class of soft colloidal rods with small aspect ratio is investigated and shown to exhibit a very rich phase and dynamic behavior, spanning from liquid to nearly melt state. Instead of nematic order, these short and soft nanocylinders alter their organization with increasing concentration from isotropic liquid with random orientation to one with preferred local orientation and eventually a multi-domain arrangement with local orientational order. The latter gives rise to a kinetically suppressed state akin to structural glass with detectable terminal relaxation, which, on increasing concentration reveals features of hexagonally packed order as in ordered block copolymers. The respective dynamic response comprises four regimes, all above the overlapping concentration of 0.02 g/ml: I) from 0.03 to 0.1 g/mol the system undergoes a liquid-to-solid like transition with a structural relaxation time that grows by four orders of magnitude. II) from 0.1 to 0.2 g/ml a dramatic slowing-down is observed and is accompanied by an evolution from isotropic to multi-domain structure. III) between 0.2 and 0.6 g/mol the suspensions exhibit signatures of shell interpenetration and jamming, with the colloidal plateau modulus depending linearly on concentration. IV) at 0.74 g/ml in the densely jammed state, the viscoelastic signature of hexagonally packed cylinders from microphase-separated block copolymers is detected. These properties set short and soft nanocylinders apart from long colloidal rods (with large aspect ratio) and provide insights for fundamentally understanding the physics in this intermediate soft colloidal regime, as well as and for tailoring the flow properties of non-spherical soft colloids

Cation Diffusion Facilitators Transport Initiation and Regulation Is Mediated by Cation Induced Conformational Changes of the Cytoplasmic Domain

Cation diffusion facilitators (CDF) are part of a highly conserved protein family that maintains cellular divalent cation homeostasis in all domains of life. CDF's were shown to be involved in several human diseases, such as Type-II diabetes and neurodegenerative diseases. In this work, we employed a multi-disciplinary approach to study the activation mechanism of the CDF protein family. For this we used MamM, one of the main ion transporters of magnetosomes - bacterial organelles that enable magnetotactic bacteria to orientate along geomagnetic fields. Our results reveal that the cytosolic domain of MamM forms a stable dimer that undergoes distinct conformational changes upon divalent cation binding. MamM conformational change is associated with three metal binding sites that were identified and characterized. Altogether, our results provide a novel auto-regulation mode of action model in which the cytosolic domain's conformational changes upon ligand binding allows the priming of the CDF into its transport mode

Defect-Free Carbon Nanotube Coils

Carbon nanotubes are promising building blocks for various nanoelectronic components. A highly desirable geometry for such applications is a coil. However, coiled nanotube structures reported so far were inherently defective or had no free ends accessible for contacting. Here we demonstrate the spontaneous self-coiling of single-wall carbon nanotubes into defect-free coils of up to more than 70 turns with identical diameter and chirality, and free ends. We characterize the structure, formation mechanism, and electrical properties of these coils by different microscopies, molecular dynamics simulations, Raman spectroscopy, and electrical and magnetic measurements. The coils are highly conductive, as expected for defect-free carbon nanotubes, but adjacent nanotube segments in the coil are more highly coupled than in regular bundles of single-wall carbon nanotubes, owing to their perfect crystal momentum matching, which enables tunneling between the turns. Although this behavior does not yet enable the performance of these nanotube coils as inductive devices, it does point a clear path for their realization. Hence, this study represents a major step toward the production of many different nanotube coil devices, including inductors, electromagnets, transformers, and dynamos

The dual role of MamB in magnetosome membrane assembly and magnetite biomineralization

Magnetospirillum gryphiswaldense MSR‐1 synthesizes membrane‐enclosed magnetite (Fe$_3$O$_4$) nanoparticles, magnetosomes, for magnetotaxis. Formation of these organelles involves a complex process comprising key steps which are governed by specific magnetosome‐associated proteins. MamB, a cation diffusion facilitator (CDF) family member has been implicated in magnetosome‐directed iron transport. However, deletion mutagenesis studies revealed that MamB is essential for the formation of magnetosome membrane vesicles, but its precise role remains elusive. In this study, we employed a multi‐disciplinary approach to define the role of MamB during magnetosome formation. Using site‐directed mutagenesis complemented by structural analyses, fluorescence microscopy and cryo‐electron tomography, we show that MamB is most likely an active magnetosome‐directed transporter serving two distinct, yet essential functions. First, MamB initiates magnetosome vesicle formation in a transport‐independent process, probably by serving as a landmark protein. Second, MamB transport activity is required for magnetite nucleation. Furthermore, by determining the crystal structure of the MamB cytosolic C‐terminal domain, we also provide mechanistic insight into transport regulation. Additionally, we present evidence that magnetosome vesicle growth and chain formation are independent of magnetite nucleation and magnetic interactions respectively. Together, our data provide novel insight into the role of the key bifunctional magnetosome protein MamB, and the early steps of magnetosome formation

Effects of Non-Ionic Micelles on the Acid-Base Equilibria of a Weak Polyelectrolyte

Weak polyelectrolytes (WPEs) are widely used as pH-responsive materials, pH modulators and charge regulators in biomedical and technological applications that involve multi-component fluid environments. In these complex fluids, coupling between (often weak) interactions induced by micelles, nanoparticles and molecular aggregates modify the pKa as compared to that measured in single component solutions. Here we investigated the effect of coupling between hydrogen bonding and excluded volume interactions on the titration curves and pKa of polyacrylic acid (PAA) in solutions comprising PEO-based micelles (Pluronics and Brij-S20) of different size and volume fraction. Titration experiments of dilute, salt-free solutions of PAA (5 kDa, 30 kDa and 100 kDa) at low degree of polymer ionization (α < 0.25) drive spatial re-organization of the system, reduce the degree of ionization and consequentially increase the pKa by up to ~0.7 units. These findings indicate that the actual degree of ionization of WPEs measured in complex fluids is significantly lower (at a given pH) than that measured in single-component solutions

Distribution of guest molecules in Pluronic micelles studied by double electron electron spin resonance and small angle X-ray scattering

Ruthstein S, Raitsimring AM, Bitton R, Frydman V, Godt A, Goldfarb D. Distribution of guest molecules in Pluronic micelles studied by double electron electron spin resonance and small angle X-ray scattering. PHYSICAL CHEMISTRY CHEMICAL PHYSICS. 2009;11(1):148-160.Pulse double electron-electron spin resonance (DEER) measurements were applied to characterize the distribution and average number of guest-molecules (in the form of spin-probes) in Pluronic P123 micelles. Two types of spin-probes were used, one of which is a spin-labeled P123 (P123-NO), which is similar to the micelles constituent molecules, and the other is spin-labeled Brij56 (Brij56-NO) which is significantly different. Qualitative information regarding the relative location of the spin-labels within the micelles was obtained from the isotropic hyperfine coupling and the correlation times, determined from continuous wave EPR measurements. In addition, complementary small angle X-ray scattering (SAXS) measurements on the P123 micellar solutions, with and without the spin-probes, were carried out for an independent determination of the size of the core and corona of the micelles and to ensure that the spin-probes do not alter the size or shape of the micelles. Two approaches were used for the analysis of the DEER data. The first is model free, which is based on the determination of the leveling of value of the DEER kinetics. This provided good estimates of the number of radicals per micelle (low limit) which, together with the known concentration of the P123 molecules, gave the aggregation number of the P123 micelles. In addition, it provided an average distance between radicals which is within the range expected from the micelles' size determined by SAXS. The second approach was to analyze the full kinetic form which is model dependent. This analysis showed that both spin-labels are not homogeneously distributed in either a sphere or a spherical shell, and that large distances are preferred. This analysis yielded a slightly larger occupation volume within the micelle for P123-NO than for Brij56-NO, consistent with their chemical character