Preparation and characterization of bosentan monohydrate/ε-polycaprolactone nanoparticles obtained by electrospraying

The electrospraying technique provides nano and microparticles that can be used as drug delivery systems. The aims of this study were, firstly, to optimize the influent parameters of electrospraying for the manufacture of a Bosentan (BOS) nanoparticulate platform, and secondly, to evaluate its physicochemical properties and in vitro biopharmaceutical behavior. Particles were characterized by scanning electron microscopy (SEM), powder X-ray diffraction (PXRD), differential scanning calorimetry (DSC), thermogravimetry (TG) and Fourier transformed Infrared spectroscopy (FTIR). Drug loading, encapsulation efficiency and kinetic dissolution were determined. Additionally, Bosentan release assays at 24 and 72 h were performed in vitro to evaluate biopharmaceutical properties of nano-scaffolds by diffusion technique through dialysis bag. The nanostructures had heterogeneous sizes predominantly smaller than 550 nm and they were semicrystalline according to PXRD, indicating a partial amorphization of BOS during the encapsulation in the polymer matrix. FT-IR and DSC showed an absence of chemical interactions between BOS and ε-Polycaprolactone (PCL), suggesting that both components behaved as a physical mixture in these particles. The drug loading was 25.98%, and the encapsulation efficiency was 58.51%. Additionally, the release assays showed an extended and controlled release of BOS, in comparison to non-encapsulated BOS. These data also showed to fit with the Cubic Root kinetic dissolution. As a conclusion, we demonstrate that the use of electrospraying for the manufacture of BOS (or similar drugs) controlled release nanoplatforms would represent an interesting contribution in the development of new therapeutic alternatives for the treatment of pathologies such as pulmonary hypertension and other related diseases. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2748, 2019.     

Comparison of the subcellular distribution of G-proteins in hepatocytes in situ and in primary cultures

The subcellular localization of the heterotrimeric G-proteins in hepatocytes in situ was compared to that in hepatocytes in primary culture. The ability of various ligands to activate adenylyl cyclase (AC) in membrane preparations was also investigated. In hepatocytes in situ the G proteins were mainly localized at the plasma membrane while in hepatocytes in culture they were predominantly cytoplasmic. The localization of the G-proteins in hepatocytes in situ correlates with their role in signal transduction. In homogenates prepared from the cultured cells, ligands which stimulate AC via G_sα were without effect, which was consistent with the localization of G_sα in the cytoplasmic and nuclear compartments. The “relocalization” of the G proteins to the cytoplasm when cells are cultured suggests that transmembrane signalling may be regulated by cell differentiation and cell-cell and cell-extracellular matrix interactions. © 1996 Wiley-Liss, Inc.     

Glucocorticoids regulate transendothelial fluid flow resistance and formation of intercellular junctions

The regulation of transendothelial fluid flow by glucocorticoidswas studied in vitro with use of human endothelial cells cultured fromSchlemm's canal (SCE) and the trabecular meshwork (TM) in conjunctionwith computer-linked flowmeters. After 2-7 wk of 500 nMdexamethasone (Dex) treatment, the following physiological, morphometric, and biochemical alterations were observed: a 3- to 5-foldincrease in fluid flow resistance, a 2-fold increase in therepresentation of tight junctions, a 10- to 30-fold reduction in themean area occupied by interendothelial "gaps" or preferential flow channels, and a 3- to 5-fold increase in the expression of thejunction-associated protein ZO-1. The more resistive SCE cells expressed two isoforms of ZO-1; TM cells expressed only one. To investigate the role of ZO-1 in the aforementioned Dex effects, itsexpression was inhibited using antisense phosphorothioate oligonucleotides, and the response was compared with that observed withthe use of sense and nonsense phosphorothioate oligonucleotides. Inhibition of ZO-1 expression abolished the Dex-induced increase inresistance and the accompanying alterations in cell junctions and gaps.These results support the hypothesis that intercellular junctions arenecessary for the development and maintenance of transendothelial flowresistance in cultured SCE and TM cells and are likely involved in themechanism of increased resistance associated with glucocorticoid exposure.

     

Biochemical, genetic and physiological characterization of venom components from two species of scorpions: Centruroides exilicauda Wood and Centruroides sculpturatus Ewing

Current literature concerning the taxonomic names of two possibly distinct species of scorpions from the genus Centruroides (sculpturatus and/or exilicauda) is controversial. This communication reports the results of biochemical, genetic and electrophysiological experiments conducted with C. exilicauda Wood of Baja California (Mexico) and C. sculpturatus Ewing of Arizona (USA). The chromatographic profile fractionation of the soluble venom from both species of scorpions is different. The N-terminal amino acid sequence for nine toxins of C. exilicauda was determined and compared with those from C. sculpturatus. Lethality tests conducted in mice support the idea that C. exilicauda venom should be expected to be medically less important than C. sculpturatus. Thirteen genes from the venomous glands of the scorpion C. exilicauda were obtained and compared with previously published sequences from genes of the species C. sculpturatus. Genes coding for cytochrome oxidase I and II of both species were also sequenced. A phylogenetic tree was generated with this information showing important differences between them. Additionally, the results of electrophysiological assays conducted with the venom from both species on the Ca(2+)-dependent K(+)-channels, showed significant differences. These results strongly support the conclusion that C. exilicauda and C. sculpturatus are in fact two distinct species of scorpions.