Differentiation of induced pluripotent stem cells into definitive endoderm on Activin A-functionalized gradient surfaces.

Activin A plays a central role in the differentiation of stem cells into definitive endoderm, the first step in embryonic development and function development in many organ systems. The aims of this study were to induce controlled and fine-tuned cell differentiation using a gradient nanotechnology and compare this with a classic protocol and to investigate how induced pluripotent stem cells differentiated depending on the gradual increase of Activin A. The density difference was tested by attaching Activin A to a gold nanoparticle gradient for high-precision density continuity. Cells expressed the definitive endoderm markers SRY-box transcription factor 17 and transcription factor GATA-4 to different extents along the gradient, indicating a density-dependent cell response to Activin A. In both the gradient and the classic differentiation setups, the protein expression increased from days 1 to 5, but a significant increase already on day 3 was found only in the gradient-based setup. By utilizing the gradient technology to present the right amount of active biomolecules to cells in vitro, we were able to find an optimal setting for differentiation into definitive endoderm. The use of gradient surfaces for differentiation allows for improvements, such as efficiency and faster differentiation, compared with a classic protocol.

ToF-SIMS imaging of dual biomolecular monolayer gradients.

Precise characterization of a monolayer of two different biomolecules in a gradient pattern on a glass surface puts high demand on the method used. Some techniques can detect protein monolayers but not on a glass surface. Others can distinguish between different proteins but not identify a gradient pattern. Here, we used ToF-SIMS to validate the complete surface composition, checking all the necessary boxes. As these types of surfaces can dictate sensitive cell behaviors, the precision on a nanolevel is crucial, and to visualize and determine the molecular distribution become essential. The dual monolayer consisted of laminin 521 and one of three other biomolecules of different sizes, epidermal growth factor, growth differentiation factor 5, or bovine serum albumin, creating opposing gradient patterns. The resulting ToF-SIMS imaging and line scan data provided detailed information on the distribution of the adsorbed proteins.

Cubic and sponge phases in ether lipid-solvent-water ternary systems: phase behavior and NMR characterization.

The phase behavior of 1-glyceryl monoleyl ether (GME) in mixtures of water and the solvents 1,5-pentanediol (POL) or N-methyl-2-pyrrolidone (NMP) was investigated by ocular inspection, polarization microscopy, and small-angle X-ray diffraction (SAXD). Phase diagrams were constructed based on analyses of more than 200 samples prepared using the two different solvents at 20 °C. The inverse hexagonal phase formed by GME in excess of water was transformed into the cubic and sponge phase with the increasing amount of each solvent. Particularly POL allowed for the formation of an extended sponge phase area in the phase diagram, comprising up to 70% POL-water mixture. The phase behavior using NMP was found to be similar to the earlier investigated solvent propylene glycol. The extended sponge phase for the POL system was attributed to POLs strong surface/interfacial activity with the potential to stabilize the polar/apolar interface of the sponge phase. The cubic and sponge phases formed using POL were further studied by NMR in order to measure the partitioning of POL between the lipid and aqueous domains of the phases. The domain partition coefficient K (lipid domain/aqueous domain) for POL in cubic and sponge phases was found to be 0.78 ± 0.14 and constant for the two phases.

In vivo study of an instantly formed lipid-water cubic phase formulation for efficient topical delivery of aminolevulinic acid and methyl-aminolevulinate.

We demonstrate a rapidly formed cubic liquid crystalline phase, i.e. typically 1g cubic phase in less than 1 min confirmed by X-ray diffraction, consisting of an ether lipid, 1-glyceryl monooleyl ether (GME), an aprotic solvent (propylene glycol or pentane-1,5-diol) and water. The efficacy of the cubic formulation was tested in vivo by administrating formulations containing 3% (w/w) of the HCl salts of δ-aminolevulinic acid (ALA) or methylaminolevulinate (MAL) to hairless mice. The endogenous formation of protoporphyrin IX (PpIX) was monitored spectrophotometrically as a marker for cellular uptake of active compound. As reference, a commercial product containing 16% (w/w) MAL in an oil-in-water emulsion (Metvix(®)), and a cubic phase based on an ester lipid (glyceryl monooleate, GMO), previously shown to facilitate topical delivery of both ALA and MAL, were applied. It was found that in general the cubic phases gave rise to higher fluorescence levels than the mice exposed to the commercial product. The instantly formed cubic formulations based on GME demonstrated the same efficiency as the GMO based formulations. The results imply that instantly formed cubic formulations opens up new opportunities, particularly for transdermal drug delivery of substances subject to stability problems in, e.g. aqueous environments.

Design and characterization of a novel amphiphilic chitosan nanocapsule-based thermo-gelling biogel with sustained in vivo release of the hydrophilic anti-epilepsy drug ethosuximide.

Thermo-gelling injectable nanogels, with no burst release of loaded drug, were prepared by a simple route by combining self assembled nanocapsules of amphiphilically modified chitosan with glycerophosphate di-sodium salt and glycerol. The potential as a depot drug delivery system was demonstrated in vivo through the therapeutic effect of ethosuximide (ESM) loaded nanogels, suppressing spike wave discharges (SWDs) in Long Evan rat model. Simultaneously clearance of gels from the site of administration was monitored non-invasively using MRI. The gel structure was characterized using TEM and SEM, confirming the gels to be an assembly of nanocapsules and using two-photon microscopy to visualize the network structure. In vitro drug release studies using ESM revealed that the nanogels exhibited extended, mostly Fickian release. Finally, all investigated formulations displayed excellent cytotoxicity data determined by MTT assay using human retinal pigmented epithelium cells. All presented properties are highly desirable for injectable depot gels for drug delivery.

Effect of pentane-1,5-diol and propane-1,2-diol on percutaneous absorption of terbinafine.

The aim of this study was to compare pentane-1,5-diol and propane-1,2-diol used as absorption enhancers for cutaneously administered terbinafine. Fresh human skin samples were placed in a continuous flow diffusion cell with a gel containing terbinafine on top of the skin. Receptor fluid samples were analysed using high - performance liquid chromatography. The quantity of gel remaining on the skin surface after completion of each test was weighed and the amount of drug in the skin was analysed. Addition of pentane-1,5-diol or propane-1,2-diol to the gel increased the percutaneous absorption of the drug. The most efficient absorption enhancer in this comparison was 5% pentane-1,5-diol.

Effects of calcium, pH, and blockiness on kinetic rheological behavior and microstructure of HM pectin gels.

The kinetic behavior during gel formation and the microstructure of 0.75% high methoxyl (HM) pectin gels in 60% sucrose have been investigated by oscillatory measurements and transmission electron microscopy for three comparable citrus pectin samples differing in their degree of blockiness (DB). Ca2+ addition at pH 3.0 resulted in faster gel formation and a lower storage modulus after 3 h for gels of the blockwise pectin A. For gels of the randomly esterified pectin B, the Ca2+ addition resulted in faster gel formation and a higher storage modulus at pH 3.0. At pH 3.5, both pectins A and B were reinforced by the addition of Ca2+. In the absence of Ca2+, the shortest gelation time was obtained for the sample with the highest DB. Microstructural characterization of the gel network, 4 and 20 h after gel preparation, showed no visible changes on a nanometer scale. The microstructure of pectins A and B without Ca2+ was similar, whereas the presence of Ca2+ in pectin A resulted in an inhomogeneous structure.