Mucin coated protein-polyphenol microcarriers for daidzein delivery.

Daidzein, an isoflavone found abundantly in legumes, may benefit from bypassing upper gut absorption to reach the colon where it can be metabolized into the potent estrogen equol by the gut microbiome. To achieve this, we developed mucin coated protein-tannin multilayer microcarriers. Highly porous functionalized calcium carbonate (FCC) microparticles efficiently absorbed daidzein from a dimethyl sulfoxide solution, with a loading capacity of 21.6 ± 1.8 wt% as measured by ultra-high pressure liquid chromatography - mass spectrometry (UPLC-MS). Daidzein-containing FCC microparticles were then coated with a bovine serum albumin (BSA)-tannin n-layer film terminated with mucin ((BSA-TA)n-mucin) by layer-by-layer deposition from corresponding aqueous solutions followed by FCC decomposition with HCl. Raman spectroscopy confirmed mucin-tannin complexation involving both hydrophobic interactions and hydrogen bonding. The resulting multilayer microcarriers contained 54 wt% of nanocrystalline daidzein as confirmed by X-ray diffraction and UPLC-MS. Preliminary screening of several types of mucin coatings using an in vitro INFOGEST digestion model demonstrated that mucin type III from porcine stomach provided the highest protection against upper intestinal digestion. (BSA-TA)8-mucin and (BSA-TA)4-mucin microcarriers retained 71 ± 16.4% and 68 ± 4.6% of daidzein, respectively, at the end of the small intestinal phase. Mucin-free (BSA-TA)8 retained a lower daidzein amount of 46%. Daidzein release and further conversion into equol were observed during in vitro colonic studies with fecal microbiota from a healthy non-equol-producing donor and Slackia equolifaciens. The developed approach has potential for encapsulating other hydrophobic nutraceuticals or therapeutics, enhancing their bioaccessibility in the colon.

Stable pH responsive layer-by-layer assemblies of partially hydrolysed poly(2-ethyl-2-oxazoline) and poly(acrylic acid) for effective prevention of protein, cell and bacteria surface attachment.

Polyoxazolines have received increasing attention as low-fouling materials with good stability and ease of functional group incorporation. We investigated layer-by-layer (LbL) assembly of poly(2-ethyl-2-oxazoline) (PEOX) with poly(acrylic acid) (PAA) to incorporate PEOX into thin conformal coatings with controllable thicknesses ranging from the nano- to micron range. Partial hydrolysis of PEOX (to form PEOX-I) was used to introduce secondary amine groups that enable post-assembly multilayer stabilization by heat-induced crosslinking. While as-assembled multilayers dissolve in aqueous solutions at pH 5 and above, thermally crosslinked multilayers were stable against film loss and instead exhibit pH responsive swelling. The anti-fouling properties of crosslinked coatings were assessed by evaluating the resistance of PEOX-I containing multilayers to fouling by proteins, cells and bacteria. Our study of multilayers with thicknesses ranging from ∼12nm to ∼1.5µm revealed thickness dependence of surface fouling resistance to BSA. Crosslinked multilayers of ∼220nm were found to be highly effective in suppressing surface adsorption of bovine serum albumin (BSA), while thinner or thicker layers were increasingly susceptible to BSA adsorption. We further found that coatings of ∼220nm and above were all highly effective at preventing surface attachment of fibroblasts, gram-positive (S. aureus) and gram-negative (E. coli) bacteria.

ASPECTS discrepancies between CT and MR imaging: analysis and implications for triage protocols in acute ischemic stroke.

BACKGROUND: Optimal imaging triage for intervention for large vessel occlusions remains unclear. MR-based imaging provides ischemic core volumes at the cost of increased imaging time. CT Alberta Stroke Program Early CT Score (ASPECTS) estimates are faster, but may be less sensitive. OBJECTIVE: To assesses the rate at which MRI changed management in comparison with CT imaging alone. METHODS: Retrospective analysis of patients with acute ischemic stroke undergoing imaging triage for endovascular therapy was performed between 2008 and 2013. Univariate and multivariate analyses were performed. Multivariate logistic regression was used to evaluate the effect of time on disagreement in MRI and CT ASPECTS scores. RESULTS: A total of 241 patients underwent both diffusion-weighted imaging (DWI) and CT. Six patients with DWI ASPECTS ≥6 and CT ASPECTS <6 were omitted, leaving 235 patients. For 47 patients, disagreement between the two modalities resulted in different treatment recommendations. The estimated probability of disagreement was 20.0% (95% CI 15.4% to 25.6%). In a multivariate logistic regression, CT ASPECTS >7 (p=0.004) and admission National Institutes of Health Stroke Scale (NIHSS) score <16 (p=0.008) were simultaneously significant predictors of agreement in ASPECTS. The time between modalities was a marginally significant predictor (p=0.080). CONCLUSIONS: The study suggests that patients with NIHSS scores at admission of <16 and patients with CT ASPECTS >7 have a higher likelihood of agreement between CT and DWI based on an ASPECTS cut-off value of 6. Additional MRI for triage in patients with NIHSS at admission of >16, and ASPECTS of 6 or 7 may be more likely to change management. Unsurprisingly, patients with low CT ASPECTS had good correlation with MRI ASPECTS.

Individually addressable patterned multilayer microchambers for site-specific release-on-demand.

Patterned arrays of light-responsive microchambers are suggested as candidates for site-specific release of chemicals in small and precisely defined quantities on demand. A composite film is made of poly(allylammonium)-poly(styrene sulfonate) multilayers and gold nanoparticles incorporated between subsequent stacks of polyelectrolytes. The film shaped as microchambers is loaded with colloid particles or oil-soluble molecules. The microchambers are sealed onto a glass slide precoated with an adhesive poly(diallyldimethylammonium)-poly(styrene sulfonate) multilayer film. A focused laser beam is used for remote addressing the individual microchambers and site-specific release of the loaded cargo.

Adhesion of polyelectrolyte multilayers: sealing and transfer of microchamber arrays.

Polyelectrolyte multilayer (PEM) films with array of responsive microchambers are promising candidates for site-specific release of chemicals in small and precisely defined quantities on demand. It requires effective sealing of the microchambers toward a support to prevent leakage of a cargo. In this paper, we study the pressure-induced adhesion of poly(allylammonium)-poly(4-styrenesulfonate) (PAH-PSS) multilayers assembled on different templates toward the poly(4-styrenesulfonate)-poly(diallyldimethylammonium) multilayer. The tensile bond strength increases from 0.4 to 3.5 MPa upon the increase of PAH-PSS bilayers from 10 to 40, if assembled on a silicon template. Weaker tensile bond strength of 0.35 MPa between the PAH-PSS multilayer and a poly(methylmethacrylate) (PMMA) template results in adhesive break at this interface and allows mechanical removal of the template. The successful PEM transfer is demonstrated for templates of various geometrical patterns, while the tensile break of a multilayer film happens for the others.

Peculiarities of polyelectrolyte multilayer assembly on patterned surfaces.

The layer-by-layer assembly of poly(diallyldimethylammonium chloride) and poly(sodium 4-styrenesulfonate) is studied on templates with imprinted arrays of microwells ranging from 2 to 25 µm and different aspect ratios. The thickness and microstructure of polyelectrolyte multilayers (PEMs) are measured using scanning electron microscopy. At 0.2 M ionic strength, the PEM film evenly coats the template both inside and outside the microwells. If the film is thinner than the critical value of about 400 nm, PEM microstructures collapse upon dissolving the template. Euler's model of critical stress is used to describe the collapse. At 2 M ionic strength, a substantially thinner PEM film is assembled inside the 25 µm wells than outside. If the well diameter is reduced to 7 and 2 µm, a much thicker PEM film is formed inside the microwells. These observations have been attributed to the changing of polyelectrolyte conformation in the solutions.

Amyloid beta interaction with receptor for advanced glycation end products up-regulates brain endothelial CCR5 expression and promotes T cells crossing the blood-brain barrier.

How circulating T cells infiltrate into the brain in Alzheimer disease (AD) remains unclear. We previously reported that amyloid beta (Abeta)-dependent CCR5 expression in brain endothelial cells is involved in T cell transendothelial migration. In this study, we explored the signaling pathway of CCR5 up-regulation by Abeta. We showed that inhibitors of JNK, ERK, and PI3K significantly decreased Abeta-induced CCR5 expression in human brain microvascular endothelial cells (HBMECs). Chromatin immunoprecipitation assay revealed that Abeta-activated JNK, ERK, and PI3K promoted brain endothelial CCR5 expression via transcription factor Egr-1. Furthermore, neutralization Ab of receptor for advanced glycation end products (RAGE; an Abeta receptor) effectively blocked Abeta-induced JNK, ERK, and PI3K activation, contributing to CCR5 expression in HBMECs. Abeta fails to induce CCR5 expression when truncated RAGE was overexpressed in HBMECs. Transendothelial migration assay showed that the migration of MIP-1alpha (a CCR5 ligand)-expressing AD patients' T cells through in vitro blood-brain barrier model was effectively blocked by anti-RAGE Ab, overexpression of truncated RAGE, and dominant-negative PI3K, JNK/ERK, or Egr-1 RNA interference in HBMECs, respectively. Importantly, blockage of intracerebral RAGE abolished the up-regulation of CCR5 on brain endothelial cells and the increased T cell infiltration in the brain induced by Abeta injection in rat hippocampus. Our results suggest that intracerebral Abeta interaction with RAGE at BBB up-regulates endothelial CCR5 expression and causes circulating T cell infiltration in the brain in AD. This study may provide a new insight into the understanding of inflammation in the progress of AD.

Peripheral T cells overexpress MIP-1alpha to enhance its transendothelial migration in Alzheimer's disease.

It is unclear how circulating T cells cross the blood-brain barrier (BBB) and participate in the inflammation process in Alzheimer's disease (AD). Here we showed significantly higher macrophage inflammatory protein-1alpha (MIP-1alpha) expression in peripheral T lymphocytes of AD patients than age-matched controls. T cells crossing of the human brain microvascular endothelial cells (HBMECs) which constitute the BBB, were almost completely abrogated by anti-MIP-1alpha antibody. MIP-1alpha induced the expression of CCR5, a potential MIP-1alpha receptor, on HBMECs. HBMECs tranfected with CCR5 resulted in increased T cells transendothelial migration. CCR5 antagonist (2D7 mAb) blocked the T cells transmigration. The MIP-1alpha-CCR5 interaction promoted T cells transendothelial migration via ROCK (Rho kinase). Furthermore, Abeta injection into rats' hippocampus induced MIP-1alpha overexpression accompanied with increased T lymphocytes occurrence in the brain cortex and this enhanced T cells entry was effectively blocked by anti-MIP-1alpha antibody. These data are the first to suggest that the interaction between MIP-1alpha overexpressed by T cells and CCR5 on HBMECs is involved in AD patients' T cells migrating from blood to brain.