aniline-blue and laminaran

Relatively little is known about how liposomal formulations modulate drug delivery to fungal pathogens. We compared patterns of hyphal cell wall binding for empty rhodmine-labeled liposomes and the clinically available amphotericin B-containing liposomal formulation (AmBisome) in Aspergillus fumigatus and Candida albicans. Following 0.5 h of coincubation with A. fumigatus , empty liposomes concentrated primarily in fungal septae along at the surface of the cell wall, suggesting that liposome uptake is concentrated in areas of the cell wall where linear glucan is exposed on the cell surface, which was confirmed by aniline blue staining. Consistent with this hypothesis, pretreatment of liposomes with soluble linear glucan (laminarin) decreased liposome binding in both Aspergillus and Candida fungal hyphae, while growth of Aspergillus hyphae in the presence of an agent that increases fungal cell wall surface exposure of linear β-glucans without cell death (caspofungin) increased liposome uptake throughout the Aspergillus fungal cell wall. Increasing the polyethylene glycol (PEG) concentration in liposomes from 0 to 30% significantly increased fungal uptake of liposomes that was only modestly attenuated when fungal cells were incubated in serum concentrations ranging from 10 to 100%. The presence of β-glucans on the fungal hyphae cell walls of Aspergillus fumigatus is one of the factors responsible for mediating the binding of liposome carriers to the hyphae and could explain possible synergy reported between liposomal amphotericin B and echinocanins.

Topics: Amphotericin B; Aniline Compounds; Antifungal Agents; Aspergillus fumigatus; beta-Glucans; Candida albicans; Caspofungin; Cell Wall; Chemistry, Pharmaceutical; Drug Carriers; Echinocandins; Glucans; Hyphae; Lipopeptides; Liposomes; Models, Molecular; Polyethylene Glycols; Polysaccharides

beta-Glucanases were detected after polyacrylamide gel electrophoresis under native and denaturing conditions using various beta-1,3- and beta-1,4-glucans, including mixed glucans (laminarin, pachyman, carboxymethyl cellulose, lichenan and barley beta-glucan). After electrophoresis and incubation of gels, substrates incorporated into polyacrylamide gels were stained with specific fluorochromes, Sirofluor for beta-1,3 linkages and Calcofluor White M2R for beta-1,4 linkages. Under UV illumination, lysis zones appeared as dark bands against a fluorescent background. Enzymes of bacterial, fungal and plant sources could be revealed sequentially in gles containing mixed beta-(1,3)(1,4)-glucans by staining first with sirofluor followed by staining with Calcofluor White M2R. Active profiles were more diverse when substrates were stained with sirofluor. The use of purified sirofluor at pH 11.5 compared with Aniline Blue at pH 8.6 allowed better detection of beta-1,3-glucanase activities. In gels containing laminarin stained with sirofluor, bands exhibiting a more intense fluorescence than the background fluorescence were observed in addition to dark nonfluorescent bands. It is postulated that these two types of beta-1,3-glucanase activities differ by their enzymatic action (partial versus extensive hydrolysis). Analysis of fungal extracts using denaturing gels embedded with various beta-glucans displayed lysis bands migrating between 32 and 35 kDa.

Topics: Aniline Compounds; Benzenesulfonates; beta-Glucosidase; Electrophoresis, Polyacrylamide Gel; Fluorescent Dyes; Glucan 1,3-beta-Glucosidase; Glucans; Polysaccharides; Protein Denaturation; Staining and Labeling