Incorporation of FGF-2 into Pharmaceutical Grade Fucoidan/Chitosan Polyelectrolyte Multilayers.

Biopolymer polyelectrolyte multilayers are a commonly studied soft matter system for wound healing applications due to the biocompatibility and beneficial properties of naturally occurring polyelectrolytes. In this work, a popular biopolymer, chitosan, was combined with the lesser known polysaccharide, fucoidan, to create a multilayer film capable of sequestering growth factor for later release. Fucoidan has been shown to act as a heparin-mimic due to similarities in the structure of the two molecules, however, the binding of fibroblast growth factor-2 to fucoidan has not been demonstrated in a multilayer system. This study assesses the ability of fucoidan to bind fibroblast growth factor-2 within a fucoidan/chitosan polyelectrolyte multilayer structure using attenuated total internal reflectance infrared spectroscopy and quartz crystal microbalance with dissipation monitoring. The fibroblast growth factor-2 was sequestered into the polyelectrolyte multilayer as a cationic layer in the uppermost layers of the film structure. In addition, the diffusion of fibroblast growth factor-2 into the multilayer has been assessed.

The influence of polyanion molecular weight on polyelectrolyte multilayers at surfaces: protein adsorption and protein-polysaccharide complexation/stripping on natural polysaccharide films on solid supports.

Two different fucoidan polymers (unfractionated Fucus vesiculosus fucoidan, and fractionated low molecular weight Fucus vesiculosus fucoidan) have been used to create substrates for protein adsorption studies. Polyelectrolyte multilayers were formed using the fucoidans (polyanions) with chitosan as the corresponding polycation. Multilayer formation was studied using zeta potential measurements, quartz crystal microbalance with dissipation monitoring (QCM-D) and attenuated total reflectance (ATR) FTIR spectroscopy. The formation studies reveal that the low molecular weight (LMW) fucoidan produces a less hydrated multilayer, with a significantly increased adsorbed mass, and with fucoidan as the diffusing species during formation. Protein adsorption studies using bovine serum albumin (BSA) were undertaken for solution conditions designed to mimic biological conditions, and to minimise the role of electrical double layer forces in influencing adsorption. Under these conditions, and as revealed by ATR FTIR spectroscopy, BSA is seen to adsorb less substantially to multilayers formed with the LMW fucoidan, and to cause extraction/stripping of the LMW fucoidan from the multilayer. FTIR spectra reveal that the protein adopts a different conformation when adsorbed to the LMW fucoidan multilayer, both relative to the protein in solution and when adsorbed at the surface of the multilayer formed from unfractionated fucoidan.

The influence of polyanion molecular weight on polyelectrolyte multilayers at surfaces: elasticity and susceptibility to saloplasticity of strongly dissociated synthetic polymers at fluid-fluid interfaces.

We studied the interfacial mechanical properties of polyelectrolyte multilayer assemblies of poly(diallylamine hydrochloride) (PAH) and poly(4-styrenesulfonate)sodium salt (PSS) at the air-water interface using axisymmetric drop shape analysis (ADSA) during hydrostatic inflation as a function of aqueous salt concentration and two different polyanion molecular weights (Mw ∼ 13 and 70 kDa). Surface elastic moduli (Gs) ranged from 50 to 300 mN m-1. Using the measured film thickness, the bulk moduli (G) ranged from 10 to 90 MPa consistent with elastomeric solids. This solid-like interface was evidenced by a systematic departure of the inflated shape from the Young-Laplace equation, which assumes a liquid-like interface. Surface elastic moduli increased and bulk elastic moduli decreased with increasing nanomembrane transverse dimension, and multilayers with the lower molecular weight anion were more transversely compact than those of higher molecular weight and displayed a larger elastic modulus. The bulk moduli of both types of multilayer assemblies asymptotically approach a constant value for films with more than two bilayers of polyelectrolyte, consistent with the observed transition from a 'glassy' to 'rubbery' state. Both types of multilayer assemblies displayed plasticization with increasing sodium chloride concentration in the adjoining aqueous phase, i.e. saloplasticity, and exhibited a transition from elastic to plastic response to deformation. The restored mobility of the polyelectrolyte resulting from the shift from intrinsic to extrinsic charge complexation, restores fluidity to the interface and is evidenced by experimental observation of a liquid-like interface when loaded. The higher molecular weight polyanion multilayers plasticized at lower salt concentrations suggesting that the lower melting point of the higher molecular weight polyanion assembly is attributable to a lesser extent of electrostatic cross-linking underscoring the unconventional dependence of molecular weight on saloplasticity in strongly dissociated polyelectrolytes.

Lysozyme uptake into pharmaceutical grade fucoidan/chitosan polyelectrolyte multilayers under physiological conditions.

Polyelectrolyte multilayers composed of pharmaceutical grade fucoidan and chitosan have been assembled and studied in terms of their response to physiological solution conditions and the presence of lysozyme. The influence of phosphate buffered saline (PBS) solution on the multilayer was interrogated using attenuated total reflectance (ATR) Fourier transform infrared (FTIR) spectroscopy and atomic force microscopy (AFM). The combination of the techniques reveal that the polyelectrolyte multilayers swell when exposed to PBS after build-up and may include a small degree of mass loss as the film swells. The degree of swelling was influenced by the terminating layer of the multilayer. Upon exposure to lysozyme, it was observed that some deswelling occurred, as the enzyme adsorbed onto and permeated into the multilayer. The behaviour of the multilayer as a potential reservoir for lysozyme contrasts with the interaction with bovine serum albumin, which did not penetrate into the multilayer, indicating either exclusion by size or due to the overall net negative charge of the film.

Odd-even effects on hydration of natural polyelectrolyte multilayers: An in situ synchrotron FTIR microspectroscopy study.

HYPOTHESIS: Odd-even effects in polysaccharide polyelectrolyte multilayers influence their hydration content and the chemical environment of the water within them. EXPERIMENTS: Polysaccharide polyelectrolyte multilayers (PEMs) composed of pharmaceutical grade fucoidan and chitosan were studied under confinement using synchrotron FTIR microspectroscopy at increasing pressure, in order to isolate and measure infrared spectra of water within the PEM, without interference from bulk water. Complementary studies of the PEMs were carried out using lab-based in situ attenuated total reflectance Fourier transform spectroscopy (ATR FTIR) and quartz crystal microbalance with dissipation monitoring (QCM-D), as well as zeta potential measurements, to determine the quantity of adsorbed polymer, hydration content, film thickness, viscoelastic properties and surface charge during layer-by-layer deposition. FINDINGS: The hydration of the PEM followed a saw-tooth profile, known as the odd-even effect, where the film increased hydration with fucoidan adsorption and dehydrated/densified with chitosan adsorption. The water structure within the film showed a lower degree of hydrogen bonding than water in the bulk electrolyte. However, the water structure/environment was independent of the terminating layer of the PEM, in spite of the alteration in percentage hydration water, indicating only a partial proof of the initial hypothesis for this multilayer system (hydration amount changes, hydration water environment does not).

A Novel Soft Contact Piezo-Controlled Liquid Cell for Probing Polymer Films under Confinement using Synchrotron FTIR Microspectroscopy.

Soft polymer films, such as polyelectrolyte multilayers (PEMs), are useful coatings in materials science. The properties of PEMs often rely on the degree of hydration, and therefore the study of these films in a hydrated state is critical to allow links to be drawn between their characteristics and performance in a particular application. In this work, we detail the development of a novel soft contact cell for studying hydrated PEMs (poly(sodium 4-styrenesulfonate)/poly(allylamine hydrochloride)) using FTIR microspectroscopy. FTIR spectroscopy can interrogate the nature of the polymer film and the hydration water contained therein. In addition to reporting spectra obtained for hydrated films confined at the solid-solid interface, we also report traditional ATR FTIR spectra of the multilayer. The spectra (microspectroscopy and ATR FTIR) reveal that the PEM film build-up proceeds as expected based on the layer-by-layer assembly methodology, with increasing signals from the polymer FTIR peaks with increasing bilayer number. In addition, the spectra obtained using the soft contact cell indicate that the PEM film hydration water has an environment/degree of hydrogen bonding that is affected by the chemistry of the multilayer polymers, based on differences in the spectra obtained for the hydration water within the film compared to that of bulk electrolyte.

Formation and enzymatic degradation of poly-l-arginine/fucoidan multilayer films.

A polyelectrolyte multilayer (PEM) system based on biopolymers has been constructed and studied in its formation and enzymatic breakdown. The multilayer is composed of fucoidan (a proven antimicrobial/anti-inflammatory seaweed-based polysaccharide) and poly-l-arginine (a polypeptide that can be readily degraded with trypsin to yield arginine, a known NO donor), thus making the multilayer a potential dual action surface treatment for wound dressings. Studies on the formation of the multilayer revealed that the film built-up in the expected stepwise manner with consistent reversal of the zeta potential upon the adsorption of each subsequent polyion. The completed film (8 bilayers) was seen to have low hydration (30% water), as determined by H2O/D2O solvent replacement studies using the quartz crystal microbalance, with an adsorbed mass (without hydration water) of approx. 4.8µgcm-2, as determined by quantitative attenuated total reflectance Fourier transform infrared (ATR FTIR) spectroscopy. The enzymatic breakdown of the film in response to exposure to trypsin was also investigated, and the film was seen to release both polymers over time, with a projected complete film removal period of approximately 24h. Critically, this information was determined using ATR FTIR spectroscopy experiments, which allowed unambiguous deconvolution of the removal rates of the two polyions, which is information that cannot be obtained from other methodologies used to study enzymatic breakdown of surface films.

Synergistic effect of field enhanced sample injection on micelle to solvent stacking in capillary electrophoresis.

Injection of a sodium dodecyl sulfate micellar plug prior to the sample solution induced transient micellar phase extraction of cationic drugs in capillary electrophoresis. Micelle to solvent stacking mechanism was utilized by preparing the sample in aqueous organic solvent. Synergism was achieved with field enhanced sample injection that allowed larger sample loads. Micelle to solvent stacking occurred concurrently with the enhanced injection after the fast moving analytes electrophoretically migrated into the micellar plug. This was different from stacking combinations with field enhancement where the electric field strength difference was involved in analyte focusing. Using a sample diluent that had one-tenth the conductivity of the background solution, the strategy afforded thousands-fold improvements in peak height and LODs (S/N=3) of as low as 1.1 ng/mL. The results were repeatable and linear. Adaptability to real sample analysis was evaluated using spiked urine sample after minimal sample clean-up.