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PubMed1990

Regulation of the formation and water permeability of endosomes from toad bladder granular cells.

Osmotic water permeability (Pf) in toad bladder is regulated by the vasopressin (VP)-dependent movement of vesicles containing water channels between the cytoplasm and apical membrane of granular cells. Apical endosomes formed in the presence of serosal VP have the highest Pf of any biological or artificial membrane (Shi and Verkman. 1989. J. Gen. Physiol. 94:1101-1115). We examine here: (a) the influence of protein kinase A and C effectors on transepithelial Pf (Pfte) in intact bladders and on the number and Pf of labeled endosomes, and (b) whether endosome Pf can be modified physically or biochemically. In paired hemibladder studies, Pfte induced by maximal serosal VP (50 mU/ml, 0.03 cm/s) was not different than that induced by 8-Br-cAMP (1 mM), forskolin (50 microM), VP + 8-Br-cAMP, or VP + forskolin. Pf was measured in endosomes labeled in intact bladders with carboxyfluorescein by a stopped-flow, fluorescence-quenching assay using an isolated microsomal suspension; the number and Pf (0.08-0.11 cm/s, 18 degrees C) of labeled endosomes was not different in bladders treated with VP, forskolin, and 8-Br-cAMP. Protein kinase C activation by 1 microM mucosal phorbol myristate acetate (PMA) induced submaximal bladder Pfte (0.015 cm/s) and endosome Pf (0.022 cm/s) in the absence of VP, but had little effect on maximal Pfte and endosome Pf induced by VP. However, PMA increased by threefold the number of apical endosomes with high Pf formed in response to serosal VP. Pf of endosomes containing the VP-sensitive water channel decreased fourfold by increasing membrane fluidity with hexanol or chloroform (0-75 mM); Pf of phosphatidylcholine liposomes (0.002 cm/s) increased 2.5-fold under the same conditions. Endosome Pf was mildly pH dependent, strongly inhibited by HgCl2, but not significantly altered by GTP gamma S, Ca, ATP + protein kinase A, and phosphatase action. We conclude that: (a) water channels cycled in endocytic vesicles are functional and not subject to physiological regulation, (b) VP and forskolin do not have cAMP-independent cellular actions, (c) activation of protein kinase C stimulates trafficking of water channels, but does not increase the number of apical membrane water channels induced by maximal VP, and (d) water channel function is sensitive to membrane fluidity. By using VP and PMA together, large quantities of endosomes containing the VP-sensitive water channel are labeled with fluid-phase endocytic markers.

Animals↗
PubMed2005

Biocompatible oligochitosans as cationic modifiers of alginate/Ca microcapsules.

In the past, it has been proven that by properly adjusting the molecular mass of the oligochitosan samples, it is possible to optimize the formation of rigid, biocompatible capsules with semipermeable membranes under physiological conditions. In this study, the feasibility of four oligochitosan samples, with varying molar masses (M(n) in range 3-5 kDa), as biocompatible coatings of alginate/Ca capsules was investigated. By selection of appropriate depolymerization and purification methods we obtained oligochitosan samples that appeared to be noncytotoxic for C(2)C(12) myoblasts and did not influence the mammalian cell metabolism especially in relative short time during the process of capsule formation. Furthermore, oligochitosans can be used as a tool to reduce the membrane cut-off of the alginate capsules. However, such reduction, as well as mechanical resistance of formed microcapsules, depend on MM of the cationic polysaccharide and the chemical composition of the alginate (mannuronic/guluronic acid ratio). Here, we address that the use of low molar mass chitosan (< 5000 g/mol) permits the formation of mechanical stable capsules at physiological pH, which represents a strong advantage over other chitosan-based chemistries.

Alginates↗
PubMed2006

Stimulatory effect of polyethylene glycol (PEG) on gene expression in mouse liver following hydrodynamics-based transfection.

BACKGROUND: Rapid intravenous injection of a large volume of plasmid DNA (pDNA), i.e. a transfection procedure based on hydrodynamics, is known to be an efficient and liver-specific method of in vivo gene delivery. However, the gene expression is transient. METHODS: We investigated the effect of addition of polyethylene glycol (PEG) to a solution of naked pDNA (luciferase) on the expression of the gene in mouse liver following transfection by the hydrodynamics-based technique. In addition, the mechanism leading to the enhancement of the gene expression was studied. RESULTS: The addition of 1% (w/v) PEG2000 to the pDNA solution enhanced the resulting gene expression in the liver. Increasing the PEG2000 concentration to more than 1 and up to 10% (w/v) rather diminished the gene expression level. By contrast, increasing the molecular weight of PEG to over 2000 up to 10 000 did not affect the level of gene expression. Histopathological and serum-chemistry examinations indicated that hydrostatic or osmotic pressure increased tissue and hepatocellular damage in a PEG-concentration-dependent manner, and resulted in a decrease in gene expression. Quantitative evaluation showed that the enhanced gene expression resulted from stabilization of the pDNA introduced into the hepatocytes and an enhancement of the transport of intact pDNA to the nucleus. CONCLUSIONS: For most gene therapy applications and gene function studies, sustained expression of the introduced gene(s) is necessary. This simple method to achieve enhanced gene expression in liver may have a great potential for a wide variety of laboratory studies in molecular and cellular biology as well as possibly for future clinical applications in humans.

Animals↗
PubMed2004

Effect of chemical enhancers and iontophoresis on thiocolchicoside permeation across rabbit and human skin in vitro.

The aim of this work was to study the permeation of thiocolchicoside across the skin in vitro. The effect of the chemical enhancer lauric acid and the physical technique of iontophoresis was investigated. Permeation experiments were performed in vitro using rabbit ear skin as barrier. The effect of lauric acid at different concentrations (2% and 4%) and of the vehicle (water, ethanol, or ethanol/water) was investigated. The primary effect of lauric acid was on the partitioning parameter, whereas the diffusive parameter did not change significantly. When human epidermis was used, the permeation parameters were generally lower, although not significantly different from rabbit ear skin. The data obtained with full-thickness human skin indicate that, despite the hydrophilic nature of thiocolchicoside, the resistance to drug transport is not limited to the stratum corneum, but that the underlying dermal tissue can also contribute. Iontophoresis enhanced the flux of thiocolchicoside compared with the passive control. The mechanism by which iontophoresis enhanced thiocolchicoside transport across the skin was electroosmosis. The permeation of thiocolchicoside across the skin can be enhanced using chemical or physical penetration enhancers.

Adjuvants, Pharmaceutic↗
PubMed2005

Influence of asymmetric donor-receiver ion concentration upon transscleral iontophoretic transport.

Recent in vitro and in vivo studies have suggested transscleral iontophoresis as a means for non-invasive drug delivery to the eye. However, there remains a lack of information of the iontophoretic transport behavior of the sclera. The objective of the present study was to investigate the effects of permeant concentration upon transscleral iontophoretic transport. Constant current direct current (DC) iontophoresis was conducted with rabbit sclera in vitro at permeant concentration ranging from 0.015 to 1.0 M in the donor chamber without background electrolyte at 0.4-4 mA (current density: 2-20 mA/cm2). PBS (0.15 M) was the receiver solution. Salicylate (SA) and tetraethylammonium (TEA) were the model ionic permeants, and mannitol was the neutral probe permeant. Conductivity experiments of SA and TEA solutions were performed to determine the effects of ion concentration upon SA and TEA electromobilities. Model simulations were carried out and compared with the experimental data. It was found that the fluxes of the ionic permeants increased linearly with the electric current but were relatively independent of their donor concentrations. Electric field-induced convective solvent flow (electroosmosis) in the sclera was observed to be from the anode to cathode, suggesting that the sclera is net negatively charge at neutral pH. For the studied permeants, electrophoresis was the main transport enhancing mechanism with electroosmosis as a secondary effect. No significant interaction between the permeants and sclera was observed that significantly altered electroosmosis in the membrane. Under the asymmetric donor and receiver conditions, the transference of the permeants could not be predicted by the concentrations of the ions in the donor and receiver chambers with the assumption of constant electric field in the membrane. The membrane ion concentrations were different from those in the chambers due to the requirement of charge neutrality in the membrane.

Administration, Cutaneous↗
PubMed1975

Elementary osmotic pump.

The elementary osmotic pump is a new delivery system for drugs or other active agents; it delivers the agent by an osmotic process at a controlled rate. Control resides in the: (a) water permeation characteristics of a semipermeable membrane surrounding the formulated agent, and (b) osmotic properties of the formulation. In its simplest embodiment, the system is constructed by coating an osmotically activie solid agent with the rate-controlling, semipermeable membrane. This membrane contains an orifice of critical size through which solubilized agent is dispensed. The system can contain the agent in solid form at loading higher than 90% of the total volume, and the agent can be delivered at rates several orders of magnitude higher than can be achieved by solution diffusion through polymeric membranes. The delivery rate, the fraction of total content delivered at zero order, and the system's delivery portal size have been calculated for delivery of a single compound. Experimental work verified the theory. The release rate from the system was found to be independent of outside agitation when the system is not deformed by shaking action, the pH of the environment, and delivery portal size for sizes within a specified range. The delivery rate from this system in vitro and in the GI tract of dogs was found to be equal.

Animals↗
PubMed1983

Elementary osmotic pump for indomethacin.

Based on the principles of an elementary osmotic pump, systems were designed to deliver indomethacin in solution at a constant rate, Z, to contain a total amount of drug, Mt, and to deliver 80% of their content at time t80. To allow selection of the optimal delivery rate into the body, three different prototypes were prepared with respective values for Z, Mt, and t80 of: 7 mg/hr, 85 mg, 11 hr; 9 mg/hr, 85 mg, 8 hr; and 12 mg/hr, 85 mg, 6 hr. These systems were found to deliver 70% of each system's contents at zero-order rates. Delivery rates were independent of pH, method of measurement, and stirring rate. In keeping with these results, the systems in the GI tract of dogs delivered at the same rate as in vitro, which qualifies the in vitro test as a bioanalogous method predictive of the in vivo performance of the dosage forms. Preliminary results in normal volunteers yielded similar urinary recoveries, while plasma profiles were different from each other and distinct from those following conventional capsules.

Animals↗
PubMed1994

A linear theory of transdermal transport phenomena.

A theory of charge, fluid-mass, and solute (including macromolecular) transport through porous media is applied to describe transport phenomena across the external layer of mammalian skin. Linear relationships are derived between transport fluxes and applied fields. These relationships introduce six effective transdermal transport coefficients. Formulas for each of these coefficients are provided. The practical relevance of these parameters is emphasized in the specific context of transdermal drug delivery. By employing typical physiological values for the various geometrical and physicochemical parameters that appear in the formulas for the transdermal transport coefficients, predictions are made for transport rates of charge, fluid mass, and solute species across a uniform-thickness skin sample contained within a diffusion-cell apparatus. These results are used to explore transdermal phenomena involving forced convection, current flow, electroosmosis, iontophoresis, and molecular diffusion (including convective dispersion). Comparisons with existing transdermal drug delivery data are made. On the basis of these comparisons, the theory suggests that transdermal transport in the presence of an electrical field may occur through corneocytes of the stratum corneum. The theory confirms the importance of a shunt route for small ion transport, as well as an intercellular route of transport for passive diffusion of noncharged substances. These latter conclusions, also based on comparisons with experimental data, are consistent with previous statements in the literature. A new form of solute transport enhancement, termed transdermal convective dispersion, is included in the theory, and methods for its measurement are described. Generalizations and future applications of the theory are discussed.

Administration, Cutaneous↗
PubMed1979

Volume flows across gallbladder epithelium induced by small hydrostatic and osmotic gradients.

The hydraulic conductivity of rabbit gallbladder epithelium has been studied using a continuous volumetric method based on capacitance measurements. The time resolution for measuring osmotic flows is in the range of seconds. Volume flows have been induced by osmotic gradients between 0 and 100 mosmol. In this range the flow-force relation is linear and the Pf value is 9.3 X 10(-3) cm/sec. After correction for solute polarization effects, the Pf value amounts to 0.05 cm/sec. The observed flow is constant between 5 sec up to 20 min after a sudden increase in the osmolarity of the mucosal solution. The wet weight of the gallbladder tissue decreases by 22% and increases by 30% during osmotic flows from mucosa to serosa, respectively. Volume flows induced by hydrostatic pressure gradients on the mucosal surface are linearly related to the driving forces between 0 and 40 mbar. The Pf value is 0.15 cm/sec. The volume flows are constant between 2 sec and 15 min after pressure application. The flow-force relation for pressure gradients on the serosal surface is markedly nonlinear for gradients greater than 5 mbar. Below 5 mbar the Pf value is 4.5 cm/sec. From electrical measurements, e.g., resistance and streaming potentials, and from flux studies with inulin and polyethylene glycol 4000, it is concluded that hydrostatic and osmotic gradients are not comparable when they are applied to gallbladder epithelium. They induce volume flows across different pathways, e.g., osmosis predominantly across the cellular route and pressure filtration predominantly across paracellular routes.

Animals↗