Hyaluronan (HA)-inspired glycopolymers as molecular tools for studying HA functions.

Hyaluronic acid (HA), the only non-sulphated glycosaminoglycan, serves numerous structural and biological functions in the human body, from providing viscoelasticity in tissues to creating hydrated environments for cell migration and proliferation. HA is also involved in the regulation of morphogenesis, inflammation and tumorigenesis through interactions with specific HA-binding proteins. Whilst the physicochemical and biological properties of HA have been widely studied for decades, the exact mechanisms by which HA exerts its multiple functions are not completely understood. Glycopolymers offer a simple and precise synthetic platform for the preparation of glycan analogues, being an alternative to the demanding synthetic chemical glycosylation. A library of homo, statistical and alternating HA glycopolymers were synthesised by reversible addition-fragmentation chain transfer polymerisation and post-modification utilising copper alkyne-azide cycloaddition to graft orthogonal pendant HA monosaccharides (N-acetyl glucosamine: GlcNAc and glucuronic acid: GlcA) onto the polymer. Using surface plasmon resonance, the binding of the glycopolymers to known HA-binding peptides and proteins (CD44, hyaluronidase) was assessed and compared to carbohydrate-binding proteins (lectins). These studies revealed potential structure-binding relationships between HA monosaccharides and HA receptors and novel HA binders, such as Dectin-1 and DEC-205 lectins. The inhibitory effect of HA glycopolymers on hyaluronidase (HAase) activity was also investigated suggesting GlcNAc- and GlcA-based glycopolymers as potential HAase inhibitors.

Nano-to-meso structure of cellulose nanocrystal phases in ethylene-glycol-water mixtures.

The self-assembly and phase behavior of cellulose nanocrystals (CNCs) in binary liquid mixtures of ethylene-glycol (EG):water was investigated. Our findings indicate that a small fraction of water delays the onset of colloidal jammed states previously reported in water-free organic solvents. Here the full phase diagram of CNCs evolves, including the chiral nematic phase (N*), characterized by long-range orientational order and non-isotropic macroscopic properties. Furthermore, the effect of the solvent-mixture composition on the properties of the CNC mesophases is found to be scale-dependent: the micron-size pitch of the N* phase decreases as the dielectric constant (εr) of the solvent mixture is reduced (higher EG content). Yet the nanometric inter-particle spacing of the CNC rods (measured using SAXS and cryo-TEM) is almost independent on the EG content. Also, unlike theoretical predictions, the transition to the biphasic regime is not sensitive to εr of the solvent mixtures and takes place at a higher CNC volume fraction than in aqueous suspensions. These observations may be rationalized by hypothesizing that vicinal water, adsorbed at the CNC surface, prevents kinetic arrest, and dictates the local dielectric constant and thus the effective diameter of the rods (via the Debye length), while εr of the liquid-mixture dominates the pitch length (micron scale) and the optical properties. These findings indicate that the water content of EG:water mixtures may be used for engineering colloidal inks where delayed kinetic arrest and jamming of the CNCs enable printing and casting of tunable, optically-active thin films and coatings.

Effect of the C-terminal amino acid of the peptide on the structure and mechanical properties of alginate-peptide hydrogels across length-scales.

Alginate is a natural anionic polysaccharide that exhibits excellent biocompatibility and biodegradability. Alginate hydrogels have many different applications in the field of regenerative medicine especially when peptides are conjugated to the alginate backbone. Here, we systematically investigate the effect of six arginine-glycine-aspartic acid (RGD)-containing peptides, G6KRGDY/S, A6KRGDY/S and V6KRGDY/S, on the macroscopic and microscopic physical properties and spatial organization of alginate-peptides hydrogels. Using rheology, small angle X-ray scattering and nanoindentation measurements we show a strong correlation between the macroscopic-bulk properties and the microscopic-local properties of the alginate-peptide hydrogels. Furthermore, our results indicate that the identity of the amino acid at the C-terminal of the peptide plays a major role in determining the structure and mechanical properties of the hydrogel across length-scales, where the presence of tyrosine (Y) terminated peptides introduce more junction-zones and consequently larger stiffness than those terminated with serine (S).

Physico-chemical characteristics of the sulfated polysaccharides of the red microalgae Dixoniella grisea and Porphyridium aerugineum.

The sulfated polysaccharides of red microalgae have attracted increasing attention in recent years due to their unique rheological and bioactivities. Todate, most studies are devoted to the polysaccharide of the marine species Porphyridium sp., with limited information about that of the brackish water- Dixoniella grisea and the freshwater- Porphyridium aerugineum. We therefore conducted a comparative study of the two less explored sulfated polysaccharides, emphasizing their similarities and differences in composition, physical properties and biocompatibility. Both polysaccharides were found to be composed of 6-8 monosaccharides, predominantly xylose. Sulfur content was 0.8% for P. aerugineum and 1.6% for D. grisea. Solutions of both polysaccharides were highly viscous and exhibited shear thinning and weak gel behavior. Both were found to be stable in an alkaline environment, whereas in an acidic environment the viscosity of the polysaccharide of the brackish water species increased while that of the freshwater species decreased. Both exhibited a similar morphology, having a porous fibrous structure with a rough amorphous surface. By complementing previous studies on the Porphyridium sp. polysaccharide, we have established a sound basis for elucidating the structure/function relationships that in turn, will promote the development of innovative applications for the biotech industries for pharmaceutics, food and drug-delivery.

Short and Soft: Multidomain Organization, Tunable Dynamics, and Jamming in Suspensions of Grafted Colloidal Cylinders with a Small Aspect Ratio.

The yet virtually unexplored class of soft colloidal rods with a 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 the nematic order, these short and soft nanocylinders alter their organization with increasing concentration from isotropic liquid with random orientation to small domains with preferred local orientation and eventually a multidomain arrangement with a local orientational order. The latter gives rise to a kinetically suppressed state akin to structural glass with detectable terminal relaxation, which, on further 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-solidlike transition with a structural relaxation time that grows by 4 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 a multidomain 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 a large aspect ratio) and provide insights for fundamentally understanding the physics in this intermediate soft colloidal regime and for tailoring the flow properties of nonspherical soft colloids.

Tuning the mechanical properties of alginate-peptide hydrogels.

Alginate, a polysaccharide that gels in the presence of divalent ions, has been used in the field of regenerative medicine to facilitate cell growth in impaired tissues by providing an artificial bio-surrounding similar to the natural extra cellular matrix (ECM). Here, we present a systematic investigation of the effect of three arginine-glycine-aspartic acid (RGD)-containing peptides, G6KRGDY, A6KRGDY and V6KRGDY, on the physical properties of alginate-peptide hydrogels. Rheology measurements showed that the storage modulus of the alginate-A6KRGDY and alginate-V6KRGDY gels is an order of magnitude higher than that of the alginate-G6KRGDY gel. Small angle X-ray scattering (SAXS) measurements suggest that the difference in the mechanical properties of the gels is due to the formation of larger peptide junction zones in addition to the ones formed by calcium ions. These findings indicate that the peptides' ability to self-assemble in aqueous solution is a significant factor in tuning the stiffness of the alginate/peptide hybrid hydrogels.

RGD-presenting peptides in amphiphilic and anionic ß-sheet hydrogels for improved interactions with cells.

The interest in developing functional biomaterials based on designed peptides has been increasing in recent years. The amphiphilic and anionic ß-sheet peptide Pro-Asp-(Phe-Asp)5-Pro, denoted FD, was previously shown to assemble into a hydrogel that induces adsorption of calcium and phosphate ions and formation of the bone mineral hydroxyapatite. In this study the integrin binding peptide, Arg-Gly-Asp (RGD), was incorporated into the hydrogel to assess its influence on an osteoblast culture. In solutions and in hydrogels FD fibrils dominated the assembly structures for up to 25 mol% FD-RGD incorporation. The cellular density of osteoblasts cultured in hydrogels composed of 25 mol% FD-RGD in FD was higher than that of only FD hydrogel cultures. These results demonstrate that RGD and possibly other cell binding motifs can be combined into amphiphilic and anionic ß-sheet hydrogels, using the design principles of FD and FD-RGD systems, to enhance interactions with cells.

Enzymatic activation of cell-penetrating peptides in self-assembled nanostructures triggers fibre-to-micelle morphological transition.

We report here a proof-of-concept design of a multi-domain cell-penetrating peptide amphiphile (CPPA) which can self-assemble into fibrous nanostructures and transform into spherical micelles upon enzymatic degradation by matrix metalloproteinase-2 (MMP-2) up-regulated in the tumour environment. Concomitant with this morphological transition, the cell-penetrating peptide (CPP), which was previously buried inside the CPPA fibers, could be presented on the surface of the CPPA micelles, enhancing their cell-penetrating ability. These multifunctional and enzyme-responsive CPP nanostructures hold potential as nanocarriers for tumour-targeted intracellular delivery of therapeutic and diagnostic agents.

The sulfated polysaccharide from a marine red microalga as a platform for the incorporation of zinc ions.

The cell-wall sulfated polysaccharide of the marine red microalga Porphyridium sp. is a high molecular weight biopolymer that has potential for use as a platform for metal complexation for various applications. This paper describes the structural and rheological characterization and antibacterial activity of the polysaccharide in combination with Zn(2+) (Zn-PS). SAXS and rheology studies indicate that with the addition of ZnCl2 to the sulfated polysaccharide the only change was the increase in viscosity in the entangled regime. The antibacterial activity of Zn-PS solutions was more potent than that of the native polysaccharide against Gram-negative and Gram-positive bacteria. The synergy between the bioactivities of Zn(2+) (which is a key player in wound healing and is active against variety of pathogens) and the unique bioactivities of the polysaccharide (e.g., anti-inflammatory) indicates promising potential for the development of novel products for the pharmaceutical and cosmetics industries.

The effect of covalently linked RGD peptide on the conformation of polysaccharides in aqueous solutions.

Covalently modified polysaccharides are routinely used in tissue engineering due to their tailored biofunctionality. Understanding the effect of single-chain level modification on the solution conformation of the single chain, and more importantly on the self-assembly and aggregation of the ensemble of chains is expected to improve our ability to control network topology and the properties of the resulting gels. Attaching an RGD peptide to a polysaccharide backbone is a common procedure used to promote cell adhesion in hydrogel scaffolds. Recently it has been shown that the spatial presentation of the RGD sequences affects the cell behavior; thus, understanding the effects of grafted RGD on the conformational properties of the solvated polysaccharide chains is a prerequisite for rational design of polysaccharide-peptide based biomaterials. Here we investigate the effect of covalently linked G4RGDS on the conformational state of the individual chain and chain assemblies of alginate, chitosan, and hyaluronic acid (HA) in aqueous solutions. Two peptide fractions were studied using small-angle X-ray scattering (SAXS) and rheology. In all cases, upon peptide conjugation structural differences were observed. Analysis of the scattering data shows evidence of clustering for a higher fraction of bound peptide. Moreover for all three polysaccharides the typical shear thinning behavior of the natural polysaccharide solutions is replaced by a Newtonian fluid behavior for the lower fraction conjugated peptide while a more pronounced shear thinning behavior is observed for the higher fraction. These results indicate that the fraction of the bounded peptide, determines the behavior of a polysaccharide-peptide conjugates in solution, regardless of the specific nature of the polysaccharide.