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.

Growth and survival of the fish pathogenic bacterium, Flavobacterium columnare, in tilapia mucus and porcine gastric mucin.

Flavobacterium columnare, an economically important Gram-negative bacterium of freshwater farmed fish, colonizes the skin and gills in the initial steps of pathogenesis. The surface of fish is coated with mucus made up of high molecular weight glycoproteins. Limited studies have described the ability of bacterial pathogens to grow in fish mucus. Our objective was to determine if F. columnare isolates could grow and survive in formulated water (FW) containing autoclaved tilapia mucus or porcine gastric mucin. We demonstrated the ability of F. columnare genomovars I, II, II-B and III to replicate (2-3 logs) and survive (21 to >100 days) in FW containing tilapia mucus. In a second experiment, genomovar I and II isolates were found to replicate in FW containing tilapia mucus or porcine mucin but not in FW only. From a practical standpoint, fish handling and/or hauling results in stress that leads to mucus sloughing often with subsequent F. columnare infection. Flavobacterium columnare utilizes fish mucus as a nutrient source, and studies are underway to determine if growth in mucus or mucin results in differential protein expression and/or increased virulence of F. columnare towards fish.

Particle tracking microrheology of purified gastrointestinal mucins.

The rheological characteristics of gastric and duodenal mucin solutions, the building blocks of the mucus layer that covers the epithelia of the two organs, were investigated using particle tracking microrheology. We used biochemically well characterized purified porcine mucins (MUC5AC and MUC2) as models for human mucins, to probe their viscoelasticity as a function of mucin concentration and pH. Furthermore, we used both reducing (dithiothreitol, DTT) and chaotropic agents (guanidinium chloride and urea) to probe the mesoscopic forces that mediate the integrity of the polymer network. At neutral pH both gastric and duodenal mucins formed self-assembled semi-dilute networks above a certain critical mucin concentration (c*) with the viscosity (η) scaling as η∼c(0.53±0.08) for MUC5AC and η∼c(0.53±0.06) for MUC2, where c is the mucin concentration. Above an even higher mucin concentration threshold (ce , the entanglement concentration) reptation occurs and there is a dramatic increase in the viscosity scaling, η∼c(3.92±0.38) for MUC5AC and η∼c(5.1±0.8) for MUC2. The dynamics of the self-assembled comb polymers is examined in terms of a scaling model for flexible polyelectrolyte combs. Both duodenum and gastric mucin are found to be pH switchable gels, gelation occurring at low pHs. There is a hundred-fold increase in the elastic shear modulus once the pH is decreased. The addition of DTT, guanidinium chloride and urea disassembles both the semi-dilute and gel structures causing a large increase in the compliance (decrease in their shear moduli). Addition of the polyphenol EGCG has a reverse effect on mucin viscoelasticity, that is, it triggers a sol-gel transition in semi-dilute mucin solutions at neutral pH.

Human gastric mucins differently regulate Helicobacter pylori proliferation, gene expression and interactions with host cells.

Helicobacter pylori colonizes the mucus niche of the gastric mucosa and is a risk factor for gastritis, ulcers and cancer. The main components of the mucus layer are heavily glycosylated mucins, to which H. pylori can adhere. Mucin glycosylation differs between individuals and changes during disease. Here we have examined the H. pylori response to purified mucins from a range of tumor and normal human gastric tissue samples. Our results demonstrate that mucins from different individuals differ in how they modulate both proliferation and gene expression of H. pylori. The mucin effect on proliferation varied significantly between samples, and ranged from stimulatory to inhibitory, depending on the type of mucins and the ability of the mucins to bind to H. pylori. Tumor-derived mucins and mucins from the surface mucosa had potential to stimulate proliferation, while gland-derived mucins tended to inhibit proliferation and mucins from healthy uninfected individuals showed little effect. Artificial glycoconjugates containing H. pylori ligands also modulated H. pylori proliferation, albeit to a lesser degree than human mucins. Expression of genes important for the pathogenicity of H. pylori (babA, sabA, cagA, flaA and ureA) appeared co-regulated in response to mucins. The addition of mucins to co-cultures of H. pylori and gastric epithelial cells protected the viability of the cells and modulated the cytokine production in a manner that differed between individuals, was partially dependent of adhesion of H. pylori to the gastric cells, but also revealed that other mucin factors in addition to adhesion are important for H. pylori-induced host signaling. The combined data reveal host-specific effects on proliferation, gene expression and virulence of H. pylori due to the gastric mucin environment, demonstrating a dynamic interplay between the bacterium and its host.

Experimental and theoretical studies on the binding of epigallocatechin gallate to purified porcine gastric mucin.

Binding of epigallocatechin gallate (EGCG) to highly purified short side-chain porcine gastric mucin similar to human MUC6 type has been studied by ultraviolet-visible absorption spectroscopy (UV-vis), ultrafiltration isothermal titration microcalorimetry (ITC) and transmission electron microscopy (TEM). The thermodynamic equilibrium of EGCG binding to mucin has been quantitatively determined using ultrafiltration and high-performance liquid chromatography (HPLC)-UV/vis. The relationship suggests multilayer binding rather than simple Langmuir monolayer binding of EGCG. By combining the ultrafiltration and ITC data, the thermodynamic parameters of EGCG binding to mucin have been obtained. The binding constant for the first layer is about an order of magnitude higher than that of the consecutive multilayers. Negative entropy indicates multilayer of EGCG formed. Hydrogen bonding may be responsible for the multilayer formation. Increasing temperature resulted in a decrease in the binding affinity, further suggesting that hydrogen bonds dominated the interaction energy. A TEM micrograph of the EGCG-mucin complex revealed a monodispersion of blobs similar to pure mucin solution but with relatively bigger size (about twice). It is proposed that the EGCG-mucin binding process occurs by single and/or cluster of EGCG molecules driven to the surface of the two hydrophobic globules of mucin by hydrophobic interaction followed by hydrogen bond interaction between EGCG and mucin. Further adsorption of EGCG molecules onto bound EGCG molecules to form multilayers can also occur. This fits well with the observations that EGCG-mucin interaction followed a multilayer adsorption isotherm, the energy released is dominated by hydrogen bonds, and no large aggregates were formed.

The characterization of the first anti-mouse Muc6 antibody shows an increased expression of the mucin in pancreatic tissue of Cftr-knockout mice.

Gel-forming mucins are large high-molecular weight secreted O-glycoproteins responsible for the gel-properties of the mucus blanket. Five orthologous gel-forming mucins have been cloned in human and mouse. Among them, the mucin MUC6 has been less studied, particularly in rodents and no anti rodent-Muc6 antibody has been reported yet. In order to further study Muc6 in mice, our aims were to obtain a specific Muc6 antibody, to validate it and to test it in Cftr deficient mice. A polyclonal serum named CP4 was isolated from a rabbit immunized by a mouse Muc6 peptide. In Western blot experiments, the antibody detected a high-molecular weight molecule secreted by the gastric tissue. Using immunohistochemistry, we showed that the antibody reacted strongly with deep glands of duodenum and ileum and mucous neck cells of gastric body. CP4 also recognized Muc6 protein secreted at the surface of the stomach and renal collecting tubules. The centroacinar cells of pancreatic tissue also reacted with the antibody. Cftr-/- mice showed a higher expression of Muc6 at both protein and RNA levels compared with their control Cftr+/+ littermates suggesting that as in the human disease, Muc6 may contribute to the formation of materials that block pancreatic acini and ducts in mouse models of cystic fibrosis. The rabbit anti-mouse Muc6 polyclonal antibody seems highly specific to the mouse mucin and will be useful to study pancreatic pathology in cystic fibrosis.

PFG-NMR diffusometry: a tool for investigating the structure and dynamics of noncommercial purified pig gastric mucin in a wide range of concentrations.

For the first time, Pulsed Field Gradient-Nuclear Magnetic Resonance, a powerful noninvasive tool for studying the dynamics and structure of complex gels, has been used to measure diffusion of probe molecules in aqueous solutions/gels of noncommercial purified pig gastric mucin (PGM), in a concentration range up to 5 wt %. Complementary data were obtained from rheology measurements. The combination of techniques revealed a strong pH dependency of the structure of the PGM samples while changes in concentration, ionic strength, and temperature appeared to induce less pronounced alterations. Viscosity was found to vary in a nonmonotonous way with pH, with the more viscous solutions found at intermediate pH. We propose that this finding is due to a reduced charge density at lower pH, which is expected to continuously increase the relative importance of hydrophobic associations. The results suggest a loose network of expanded fully charged PGM molecules with considerable mobility at neutral pH (pH 7.4). At intermediate pH (pH 4), a three-dimensional expanded network is favored. At pH 1, the charge density is low and microphase separation occurs since hydrophobic associations prevail. This leads to the formation of clusters concentrated in PGM molecules separated by regions depleted in PGM. The results obtained increase our knowledge about the gastric mucosal layer, which in vivo contains mucin in the same concentration range as that of the samples investigated here.

Presencia de sulfomucinas en gastritis crónica asociada a Helicobacter pylori / Sulfomucins in Helicobacter pylori-associated chronic gastritis

La gastritis crónica asociada a Helicobacter pylori (HP)ha sido relacionada a úlcera péptica, gastritis atrófica ymetaplasia intestinal (MI), estas dos últimas íntimamentevinculadas con el desarrollo de carcinoma y linfoma gástrico.En un estudio reciente sobre gastritis crónica con HP enniños se hallaron signos incipientes de metaplasia intestinal.El objetivo de nuestro trabajo es identificar depósitos desulfomucinas ácidas en pacientes jóvenes con gastritis crónica.En este estudio se evaluaron 28 pacientes con gastritiscrónica asociada a HP sin evidencias histológicas de MI. Laedad media fue 30 años con predominio femenino. Las sulfomucinasácidas se tiñen con PAS-azul alcian (PAS-AA) apH 1,0. Esta coloración demostró sulfomucinas ácidas enforma difusa en todos los casos. La mucosa gástrica normalcontiene mucinas neutras que se tiñen con PAS pero no conAA. La presencia de sulfomucinas ácidas es identificada enla MI incompleta (colónica o tipo III). Hay estudios recientesque demuestran que la MI podría comenzar en la niñez.En nuestro grupo de estudio observamos un alto porcentajede células conteniendo sulfomucinas ácidas que podrían serla primera evidencia de MI incompleta en gastritis crónicaasociada a HP, precediendo a los cambios histológicoscaracterísticos de la MI o representar un cambio transitorioasociado a la infección por HP

Chronic gastritis associated with Helicobacter pylori(HP) has been related to peptic ulcer, intestinal metaplasia(IM) and atrophic gastritis, these two last intimately implicatedin gastric carcinoma and lymphoma development. Ina recent study on chronic gastritis with HP in children incipientsigns of IM were found. The aim of this work is toidentify deposits of acid sulfomucins in young patients withchronic gastritis. For this study 28 patients with chronic gastritisassociated with HP with no histological evidence ofIM were evaluated. The average age was 30 years withfemale predominance. The acid sulfomucins were stainedwith PAS-alcian blue (PAS-AA) to pH 1.0. This stain revealedacid sulfomucins in a diffuse way in all the cases. Normalgastric mucosa contains neutral mucins that are stainedwith PAS but not with AA. The acid sulfomucins are identifiedin the incomplete IM (colonic or type III). Recent studiesdemonstrate that IM could begin in childhood. In ourgroup we observed a high percentage of cells containingacid sulfomucins that could be the first evidence of IM inHP associated chronic gastritis, preceding IM histologicalcharacteristic changes or representing a transitory changeassociated with HP infection

Extraction, isolation, and SDS-PAGE analysis of purified gastric mucin in a patient with Menetrier's disease.

Menetrier's disease is a rare condition characterized by marked proliferation of gastric mucosa with variable mucus secretion and achlorhydria. Although crude mucus secretion and gastric aspirates have been evaluated in this disease for output of dry matter, hexosamine, fucose, protein content, and transforming growth factor alpha activity, we report for the first time the isolation, purification, and gel electrophoresis of mucin from crude mucus scrapings. The fragmentation pattern of mucin in Menetrier's disease demonstrated less large polymeric mucin than the control. There was also a band of approximately 55-65 kd M, on polyacrylamide gel electrophoresis similar to that found in gastric carcinoma or peptic ulcer, but absent in the control specimens.

Disulfide-bound proteolytic fragments of gastric mucin are 100- and 140-kDa proteins.

Pig gastric mucus was tested for its autodegradative proteolytic degradation at pH 7.0, in the presence or absence of proteinase inhibitors and SDS. Samples of crude mucus were incubated at room temperature for 48 and 96 h in sodium azide stabilized buffer, pH 7. 0, and urea-extracted mucin was purified. Electrophoretically homogenic mucin preparation was reduced and alkylated with iodo[(14)C]acetamide, and analyzed for labeled products. On 7.5% SDS/PAGE protein bands at 80 and 120 kDa were noted, but radioactivity was incorporated into 100- and 140-kDa bands, with increasing intensity from T(0) to T(96), and into high molecular mass mucin subunits. The results confirmed the autodegradative properties of gastric mucin and demonstrated that the 100- and 140-kDa fragments are the main proteolytical products of pig gastric mucin and are disulfide bound with the rest of the molecule.

Deglycosylated mucin as a substrate in enzymatic O-glycosylation in vitro.

To obtain the apomucin as a substrate for glycosyltransferases activity testing, the pig gastric mucin deglycosylation by chemical method was carried out. Resulted apomucin, rich in Ser, Thr and Pro residues, was as good carbohydrate acceptor in enzymatic O-glycosylation in vitro, as synthetic peptide, analogue of MUC2 mucin tandem repeats sequence.

Low molecular mass products of depolymerization of purified mucin--attempts at isolation and characterization.

Samples of crude mucin were incubated at room temperature for 48 and 96 h in a sodium azide containing buffer, pH 7.0. Then each sample was purified, reduced and alkylated with iodo[14C]acetamide. Electrophoretic analysis demonstrated that radioactivity was incorporated into the mucin subunits and proteins of 100 and 140 kDa. The results of our experiments suggest that the released proteins can be a part of mucin molecule, cleaved by proteolysis and reduction of disulfide bridges.

Preparation of gastric mucin "STP-domains" using pronase digestion and chemical deglycosylation.

In this work we present a method of mucin protein backbone fragments preparation, which are rich in serine, threonine and proline amino acid residues. The purified native gastric mucin was reduced, the pronase digested and chemically deglycosylated. The obtained apomucin preparations contained slight quantities of residual carbohydrates (0-1%). In O-glycosylation reaction in vitro, with the participation of GalNAc-transferase, the amounts of incorporated [14C]GalNAc to apomucin fractions were about twofold greater in relation to the deglycosylated non-digested mucin subunits. We also demonstrate the usefulness of immunoblotting technique of apomucin preparations detection.

Factors affecting solubility and penetration of clarithromycin through gastric mucus.

BACKGROUND: Factors influencing the pharmacokinetics of clarithromycin in gastric mucus are poorly defined. AIM: To determine: (i) whether the clinical formulation of clarithromycin (Biaxin granules and powdered Biaxin tablets) affects the water solvency of the antibiotic or changes the barrier properties of pig gastric mucus (PGM), thereby influencing the penetration of clarithromycin through the gastric mucus layer; and (ii) whether topically active anti-ulcer agents affect clarithromycin penetration through gastric mucus. METHODS: Solubility of clarithromycin in aqueous solution was studied at pH 7. PGM viscosities were determined using a falling ball microviscometer. Permeability of clarithromycin through PGM with and without added anti-ulcer drugs at pH 7 was monitored using a microfiltration device and an agar diffusion bioassay. RESULTS: Clarithromycin showed the poorest solubility at pH 7, whereas both Biaxin formulations demonstrated identical solubility of their antibiotic ingredient. Clarithromycin and both Biaxin formulations markedly increased mucin viscosity over the pH range 2-7. PGM markedly retarded the penetration of clarithromycin: unformulated clarithromycin and Biaxin tablets penetrated more rapidly through mucus than Biaxin granules. Pre-treatment of PGM with aluminium-magnesium-containing antacids (Riopan and Talcid preparations) decreased the rate of clarithromycin penetration, whereas Carafate and Peptobismol had no significant effect on mucus penetration of clarithromycin. CONCLUSIONS: The availability of clarithromycin in gastric mucus is significantly influenced by its clinical formulation, which affects its solubility as well as the viscous properties of mucus. Pulverized Biaxin tablets provide better local distribution of clarithromycin in mucus than Biaxin granules. Pre-treatment of mucus with anti-ulcer medications does not increase the penetration of clarithromycin through mucus.

Comparison of four monoclonal antibodies reacting with gastric gland mucous cell-derived mucins of rat and frog.

Features of four monoclonal antibodies (MAbs), PGM36, PGM37, PGM38 and HIK1083, reacting with the mucin derived from rat gastric gland mucous cells were compared. By applying enzyme linked immunosorbent assay, all of these MAbs reacted not only with the mucins purified from both rat and frog stomach, but also with the oligosaccharides obtained from the antigenic mucins by alkaline borohydride treatment. These MAbs could be characterized as distinct MAbs due to the immunohistochemical observation of rat cecal mucosa and the reactivity to paranitrophenyl derivatives of monosaccharides. These MAbs might be useful tools to compare the gastric gland-type mucins in different species of vertebrates and to investigate the heterogeneity of the carbohydrate structure of the mucin molecules of various origins.

Gastric mucin secretion from cultured rat epithelial cells.

In order to establish the measurement of gastric mucin secreted from cultured mucous cells, rat gastric mucin was purified from secreted mucus with Sepharose CL-4B column chromatography. Gastric mucin was measured by dot blot analysis using an enzyme-linked lectin (soybean agglutinin) assay in a good concentration-dependent manner. Surface epithelial cells were dispersed by limited digestion of a rat everted stomach and collected by density gradient centrifugation with Percoll. These cells were inoculated onto gelled collagen dishes, then cultured in a medium supplemented with 10% fetal calf serum under a 5% CO2 atmosphere in air. Changing the medium after a 2-d culture, the cells were cultured for another 3 d. During the culture, the numbers of cells each day were almost equal, but mucin contents in the cells increased, and then dropped at day 5 after inoculation. At that time, the edge of the cell layer peeled off and the cells adhered to each other. Using 2-d cultured cells, the effects of some secretagogues on mucin secretion were investigated. Carbachol, secretin, CCK-8 and prostaglandin E2 (PGE2) strongly stimulated mucin secretion, and gastrin I weakly did. However, histamine offered no stimulation.

An improved method for chemical deglycosylation of gastric mucus glycoprotein.

We describe an improved method for chemical deglycosylation of gastric mucin which involves: reduction, alkylation, desialylation, periodate oxidation with beta-elimination and two-steps of TFMSA/anisole treatment. The product was 96% deglycosylated protein with amino acid composition similar to purified mucin with apparent molecular weight of 90 kDa.

The effects of reductive dissociation of gastric mucin isolated in the presence of proteinase inhibitors.

Mucin was purified by the gel filtration method on columns with high porous molecular sives in buffers with SDS and proteinase inhibitors. The addition of proteinase inhibitors distinctly inhibited proteolytic activity. It was found that the obtained mucin, after disulphide-bound reduction, is dissociated to mucin subunits and N-glycosylated glycoprotein of molecular weight about 75 kDa. This protein has carbohydrate and amino acid composition different from high molecular fraction. The 75 kDa protein is strongly associated with high molecular mass mucin subunits and can be separated either during electrophoresis or fractionation in buffers with 2-mercaptoethanol.

Alterations in porcine gastric mucin during the development of experimental ulceration.

Bile duct ligation in the pig results in ulceration of the pars oesophagea (oesophagogastric junction) within 48 h with 100% reproducibility. This work describes novel observations made during development of such ulcers using an endoscope introduced at intervals postoperatively via a Thomas gastric cannula. Macroscopic and histological changes were recorded and compared with quantitative and qualitative changes in crude mucus scrapings and purified mucins. Crude mucus scrapings of the cardiac gland region had a higher protein content in the ulcerated states than in the normals. After bile duct ligation, the (degraded) mucin glycopeptide/total protein ratio was higher in partially purified mucus from pre-ulcerated and ulcerated stomachs as compared with normal samples. The quantity of purified mucin was less in samples from ulcerated stomachs, and the N-acetylgalactosamine and fucose contents were also decreased. It is possible that these changes resulted in the failure of the mucus barrier and the development of oesophagogastric junction ulceration.

Mouse gastric mucin: cloning and chromosomal localization.

Mucins protect gastric epithelium by maintaining a favourable pH gradient and preventing autodigestion. The purpose of this study was to clone a mouse gastric mucin which would provide a foundation for analysis of mucin gene regulation. Mucin was purified from the glandular portion of gastric specimens and deglycosylated by HF solvolysis. Antibodies against native and deglycosylated mouse gastric mucin (MGM) were raised in chickens. Screening of a mouse stomach cDNA library with the anti-(deglycosylated MGM) antibody yielded partial clones containing a 48 bp tandem repeat and 768 bp of non-repetitive sequence. The 16-amino-acid tandem repeat has a consensus sequence of QTSSPNTGKTSTISTT with 25% serine and 38% threonine. The MGM tandem repeat sequence bears no similarity to previously identified mucins. The MGM non-repetitive region shares sequence similarity with human MUC5AC and, to a lesser extent, human MUC2 and rat intestinal mucin. Northern blot analysis reveals a polydisperse message beginning at 13.5 kb in mouse stomach with no expression in oesophagus, trachea, small intestine, large intestine, caecum, lung or kidney. Immunoreactivity of antibodies against deglycosylated MGM and against a synthetic MGM tandem repeat peptide was restricted to superficial mucous cells, antral glands and Brunner's glands in the pyloric-duodenal region. DNA analysis shows that MGM recognizes mouse and rat DNA but not hamster, rabbit or human DNA. The MGM gene maps to a site on mouse chromosome 7 homologous to the location of a human secretory mucin gene cluster on human chromosome 11p15. Due to sequence similarity and predominant expression in the stomach, the MGM gene may be considered a MUC5AC homologue and named Muc5ac.