PEGDA hydrogels as a replacement for animal tissues in mucoadhesion testing.

Utilization of animal parts in ex-vivo mucoadhesion assays is a common approach that presents many difficulties due to animal rights issues and large variance between animals. This study examines the suitability of two PEGDA (poly(ethylene glycol) diacrylate) based hydrogels to serve as tissue mimetics for mucoadhesion evaluation. One hydrogel, termed PEGDA-QT, was composed of pentaerythritol tetrakis (3-mercaptopropionate) and PEG and contained free thiol groups mimicking those found in natural mucosa. The other hydrogel was formed by UV (ultraviolet) curing of PEGDA and mimicked the mechanical property of mucosa but not its chemical constitute. When ranking different first generation mucoadhesive polymers using a tensile assay, both hydrogels showed good agreement with the ranking achieved for porcine small intestine. However, only PEGDA-QT and porcine small intestine shared a similar displacement curve. The same ranking for PEGDA-QT and porcine small intestine was also observed when comparing a second-generation mucoadhesive polymer, thiolated alginate, to native alginate. Our findings suggest that PEGDA-QT could serve as a replacement for porcine small intestine in both mucoadhesion evaluations using a tensile machine and the flow-through method for first and second-generation mucoadhesive polymers.

Mucoadhesive acrylated block copolymers micelles for the delivery of hydrophobic drugs.

Blockpolymer micelles having acrylated end groups were fabricated for the development of mucoadhesive drug loaded vehicle. The critical micelle concentration (CMC) of Pluronic(®) F127 modified with acrylate end groups (F127DA) was found to be similar to that of the unmodified Pluronic(®) F127 (F127). Small angle X-ray scattering verified existence of micelles with an inner core of 4.9±0.2 and 5.5±0.3 for F127 and F127DA respectively. Indomethacin, a hydrophobic drug, was incorporated into the micelles using the thin-film hydration method. In vitro drug release assay demonstrated that the micelles sustained the release of the drug in comparison with free drug in solution. Several methods were used for mucoadhesion evaluation. Viscosity profiling was performed by shear rate sweep experiment of hydrated commercial mucin, F127 or F127DA, and combination of both mucin and a copolymer. Elevated viscosity was achieved for acrylated micelles with mucin compared to mixtures of non-acrylated micelles with mucin. The mucoadhesivity of the acrylated micelles was further characterized using nuclear magnetic resonance (NMR); data affirmed the Michael type addition reaction occurred between acrylates on the micelles corona and thiols present in the mucin. SAXS scattering data further showed a modification in the scattering of F127DA micelles with the addition of pig gastric mucin. Cryo-transmission electron microscopy (cryo-TEM) and dynamic light scattering (DLS) data detected increase in the aggregates size while using acrylated micelles enhance mucoadhesion. Thus acrylated F127DA micelles were found to be mucoadhesive, and a suitable and preferred candidate for micellar drug delivery to mucosal surfaces.

Effect of sex difference on platelet adhesion, spreading and aggregate formation under flow.

There are clear but poorly understood differences in the etiology and prognosis of thrombotic diseases in men and women. Due to the fact that platelets play a central role in the formation of occlusive thrombi in atherosclerotic coronary arteries, previous studies have examined whether sex differences exist for platelets, and have obtained conflicting results. Additionally, due to the increased use of genetically modified mouse models to explore the molecular mechanisms underlying platelet activation and thrombotic disorders, it is critical to determine if sex is a confounding variable. Our study of the role of sex differences in platelet function was designed to utilise purified platelets from inbred paired female/male littermates in order to minimise genetic and environmental variability. In the current study, we demonstrate that platelet adhesion to and spreading on immobilised fibrinogen, thrombin or collagen was equivalent for both female and male mouse platelets. The ability of the soluble agonist thrombin or convulxin to potentiate platelet P-selectin exposure, fibrinogen binding, or adhesion and spreading on immobilised fibrinogen was equivalent for both female and male mouse platelets. Our data show that an equivalent degree of platelet adhesion and aggregation on collagen or fibrinogen under shear flow was observed for both female and male mouse platelets. In conclusion, our data would argue against an intrinsic difference for female mouse platelets in regulating the major functional platelet responses: platelet adhesion, spreading, or aggregation under flow.

Fibre-optic bacterial biosensors and their application for the analysis of bioavailable Hg and As in soils and sediments from Aznalcollar mining area in Spain.

Fibre-optic biosensors for Hg and As were developed by attaching alginate-immobilised recombinant luminescent Hg- and As-sensor bacteria onto optical fibres. The optimised biosensors (consisting of seven layers of fibre-attached bacteria pre-grown till mid-logarithmic growth phase) enabled quantification of environmentally relevant concentrations of the target analytes: 2.6 microg l-1 of Hg(II) and 141 microg l-1 of As(V) or 18 microg l-1 of As(III). The highest viability and sensitivity for target analyte was obtained when fibre tips were stored in CaCl2 solution at -80 degrees C. Applicability of the fibre-optic biosensors in parallel to the respective non-immobilised sensors was assessed on 10 natural soil and sediment samples from Aznalcollar mining area (Spain). On the average 0.2% of the total Hg and 0.87% of the total As proved bioavailable to fibre-attached bacteria. Interestingly, about 20-fold more Hg and 4-fold more As was available to non-immobilised sensor bacteria indicating the importance of direct cell contact (possible only for non-immobilised cells) for enhanced bioavailability of these metals in solid samples.

Detection of bioavailable heavy metals in EILATox-Oregon samples using whole-cell luminescent bacterial sensors in suspension or immobilized onto fibre-optic tips.

At the EILATox-Oregon Workshop, nine luminescent whole-cell bacterial sensors were used for the determination of bioavailable metals in blind samples (17 synthetic and 3 environmental). A non-inducible luminescent control strain was used to determine sample matrix effects and bacterial toxicity. Whole-cell bacterial sensors capable of determining arsenic, inorganic mercury and its organic derivatives, cadmium, lead or copper were used in suspensions and a bacterial sensor for the detection of inorganic mercury was immobilized onto fibre-optic tips using calcium alginate. Bioavailable amounts of metals were estimated using calibration plots, that were constructed to determine the range of metals giving rise to a linear relationship between luminescence and the amount of metals present in the standard solutions. EILATox-Oregon sample 5, which contained 74 mg l(-1) of Hg, gave a significant response with both formats of the mercury sensor. The bioavailable amounts of mercury according to the measurement of bacterial sensor in suspension and immobilized onto a fibre-optic tip were 76 and 93 mg l(-1), respectively. The bacterial sensor for arsenic and copper showed a response with sample 6 (58 mg l(-1) of As) and sample 8 (400 mg l(-1) of metham sodium), respectively. This study showed that the bacterial sensors in suspension or immobilized onto optical fibres are capable of quantifying bioavailable metals from unknown samples. The measurement protocol of bacterial sensors is simple and possible to perform in the field. Moreover, the samples do not need any pretreatment before analysis. Construction and characterization of the strain for the detection of bioavailable copper are described.