High speed DSC (hyper-DSC) as a tool to measure the solubility of a drug within a solid or semi-solid matrix

Conventional differential scanning calorimetry (DSC) techniques are commonly used to quantify the solubility of drugs within polymeric-controlled delivery systems. However, the nature of the DSC experiment, and in particular the relatively slow heating rates employed, limit its use to the measurement of drug solubility at the drug's melting temperature. Here, we describe the application of hyper-DSC (HDSC), a variant of DSC involving extremely rapid heating rates, to the calculation of the solubility of a model drug, metronidazole, in silicone elastomer, and demonstrate that the faster heating rates permit the solubility to be calculated under non-equilibrium conditions such that the solubility better approximates that at the temperature of use. At a heating rate of 400°C/min (HDSC), metronidazole solubility was calculated to be 2.16 mg/g compared with 6.16 mg/g at 20°C/min. © 2005 Elsevier B.V. All rights reserved.

A solvent-free matrix application method for matrix-assisted laser desorption/ionization imaging of small molecules

Matrix application continues to be a critical step in sample preparation for matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI). Imaging of small molecules such as drugs and metabolites is particularly problematic because the commonly used washing steps to remove salts are usually omitted as they may also remove the analyte, and analyte spreading is more likely with conventional wet matrix application methods. We have developed a method which uses the application of matrix as a dry, finely divided powder, here referred to as dry matrix application, for the imaging of drug compounds. This appears to offer a complementary method to wet matrix application for the MALDI-MSI of small molecules, with the alternative matrix application techniques producing different ion profiles, and allows the visualization of compounds not observed using wet matrix application methods. We demonstrate its value in imaging clozapine from rat kidney and 4-bromophenyl-1,4-diazabicyclo(3.2.2)nonane-4-carboxylic acid from rat brain. In addition, exposure of the dry matrix coated sample to a saturated moist atmosphere appears to enhance the visualization of a different set of molecules.

Matrix changes induced by transglutaminase 2 lead to inhibition of angiogenesis and tumor growth

Administration of active TG2 to two different in vitro angiogenesis assays resulted in the accumulation of a complex extracellular matrix (ECM) leading to the suppression of endothelial tube formation without causing cell death. Matrix accumulation was accompanied by a decreased rate of ECM turnover, with increased resistance to matrix metalloproteinase-1. Intratumor injection of TG2 into mice bearing CT26 colon carcinoma tumors demonstrated a reduction in tumor growth, and in some cases tumor regression. In TG2 knockout mice, tumor progression was increased and survival rate reduced compared to wild-type mice. In wild-type mice, an increased presence of TG2 was detectable in the host tissue around the tumor. Analysis of CT26 tumors injected with TG2 revealed fibrotic-like tissue containing increased collagen, TG2-mediated crosslink and reduced organized vasculature. TG2-mediated modulation of cell behavior via changes in the ECM may provide a new approach to solid tumor therapy.

Inhibition of transglutaminase activity reduces extracellular matrix accumulation induced by high glucose levels in proximal tubular epithelial cells

Diabetic nephropathy affects 30-40% of diabetics leading to end-stage kidney failure through progressive scarring and fibrosis. Previous evidence suggests that tissue transglutaminase (tTg) and its protein cross-link product epsilon(gamma-glutamyl)lysine contribute to the expanding renal tubulointerstitial and glomerular basement membranes in this disease. Using an in vitro cell culture model of renal proximal tubular epithelial cells we determined the link between elevated glucose levels with changes in expression and activity of tTg and then, by using a highly specific site directed inhibitor of tTg (1,3-dimethyl-2[(oxopropyl)thio]imidazolium), determined the contribution of tTg to glucose-induced matrix accumulation. Exposure of cells to 36 mm glucose over 96 h caused an mRNA-dependent increase in tTg activity with a 25% increase in extracellular matrix (ECM)-associated tTg and a 150% increase in ECM epsilon(gamma-glutamyl)lysine cross-linking. This was paralleled by an elevation in total deposited ECM resulting from higher levels of deposited collagen and fibronectin. These were associated with raised mRNA for collagens III, IV, and fibronectin. The specific site-directed inhibitor of tTg normalized both tTg activity and ECM-associated epsilon(gamma-glutamyl)lysine. Levels of ECM per cell returned to near control levels with non-transcriptional reductions in deposited collagen and fibronectin. No changes in transforming growth factor beta1 (expression or biological activity) occurred that could account for our observations, whereas incubation of tTg with collagen III indicated that cross-linking could directly increase the rate of collagen fibril/gel formation. We conclude that Tg inhibition reduces glucose-induced deposition of ECM proteins independently of changes in ECM and transforming growth factor beta1 synthesis thus opening up its possible application in the treatment other fibrotic and scarring diseases where tTg has been implicated.

Use of a solvent-free dry matrix coating for quantitative matrix-assisted laser desorption ionization imaging of 4-bromophenyl-1,4-diazabicyclo(3.2.2)nonane-4-carboxylate in rat brain and quantitative analysis of the drug from laser microdissected tissue regions

A dry matrix application for matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) was used to profile the distribution of 4-bromophenyl-1,4-diazabicyclo(3.2.2)nonane-4-carboxylate, monohydrochloride (BDNC, SSR180711) in rat brain tissue sections. Matrix application involved applying layers of finely ground dry alpha-cyano-4-hydroxycinnamic acid (CHCA) to the surface of tissue sections thaw mounted onto MALDI targets. It was not possible to detect the drug when applying matrix in a standard aqueous-organic solvent solution. The drug was detected at higher concentrations in specific regions of the brain, particularly the white matter of the cerebellum. Pseudomultiple reaction monitoring imaging was used to validate that the observed distribution was the target compound. The semiquantitative data obtained from signal intensities in the imaging was confirmed by laser microdissection of specific regions of the brain directed by the imaging, followed by hydrophilic interaction chromatography in combination with a quantitative high-resolution mass spectrometry method. This study illustrates that a dry matrix coating is a valuable and complementary matrix application method for analysis of small polar drugs and metabolites that can be used for semiquantitative analysis.

A novel extracellular role for tissue transglutaminase in matrix-bound VEGF-mediated angiogenesis

The importance of tissue transglutaminase (TG2) in angiogenesis is unclear and contradictory. Here we show that inhibition of extracellular TG2 protein crosslinking or downregulation of TG2 expression leads to inhibition of angiogenesis in cell culture, the aorta ring assay and in vivo models. In a human umbilical vein endothelial cell (HUVEC) co-culture model, inhibition of extracellular TG2 activity can halt the progression of angiogenesis, even when introduced after tubule formation has commenced and after addition of excess vascular endothelial growth factor (VEGF). In both cases, this leads to a significant reduction in tubule branching. Knockdown of TG2 by short hairpin (shRNA) results in inhibition of HUVEC migration and tubule formation, which can be restored by add back of wt TG2, but not by the transamidation-defective but GTP-binding mutant W241A. TG2 inhibition results in inhibition of fibronectin deposition in HUVEC monocultures with a parallel reduction in matrix-bound VEGFA, leading to a reduction in phosphorylated VEGF receptor 2 (VEGFR2) at Tyr1214 and its downstream effectors Akt and ERK1/2, and importantly its association with b1 integrin. We propose a mechanism for the involvement of matrix-bound VEGFA in angiogenesis that is dependent on extracellular TG2-related activity. © 2013 Macmillan Publishers Limited. All rights reserved.

Using organoclay to promote morphology refinement and co-continuity in high-density polyethylene/polyamide 6 blends - Effect of filler content and polymer matrix composition

We investigate the gradual changes of the microstructure of two blends of high-density polyethylene (HDPE) and polyamide 6 (PA6) at opposite composition filled with increasing amounts of an organomodified clay. The filler locates preferentially inside the polyamide phase, bringing about radical alterations in the micron-scale arrangement of the polymer phases. When the host polyamide represents the major constituent, a sudden reduction of the average sizes of the polyethylene droplets was observed upon addition of even low amounts of organoclay. A morphology refinement was also noticed at low filler contents when the particles distributes inside the minor phase. In this case, however, keep increasing the organoclay content eventually results in a high degree of PA6 phase continuity. Rheological analyses reveal that the filler loading at which the polyamide assembles in a continuous network corresponds to the critical threshold for its rheological transition from a liquid- to a gel-like behaviour, which is indicative of the structuring of the filler inside the host PA6. On the basis of this finding, a schematic mechanism is proposed in which the role of the filler in driving the space arrangement of the polymer phases is discussed. Finally, we show that the synergism between the reinforcing action of the filler and its ability to affect the blend microstructure can be exploited in order to enhance relevant technological properties of the materials, such as their high temperature structural integrity.

Matrix-assisted diffusion-ordered spectroscopy:mixture resolution by NMR using SDSmicelles

Diffusion-ordered spectroscopy (DOSY) is a powerful technique for mixture analysis, but in its basic form it cannot separate the component spectra for species with very similar diffusion coefficients. It has been recently demonstrated that the component spectra of a mixture of isomers with nearly identical diffusion coefficients (the three dihydroxybenzenes) can be resolved using matrix-assisted DOSY (MAD), in which diffusion is perturbed by the addition of a co-solute such as a surfactant [R. Evans, S. Haiber, M. Nilsson, G. A. Morris, Anal. Chem. 2009, 81, 4548-4550]. However, little is known about the conditions required for such a separation, for example, the concentrations and concentration ratios of surfactant and solutes. The aim of this study was to explore the concentration range over whichmatrix-assisted DOSY using the surfactant SDS can achieve diffusion resolution of a simple model set of isomers, the monomethoxyphenols. The results show that the separation is remarkably robust with respect to both the concentrations and the concentration ratios of surfactant and solutes, supporting the idea that MAD may become a valuable tool formixture analysis. © 2010 John Wiley & Sons, Ltd.

A collagen IV matrix is required for guinea pig gastric epithelial cell monolayers to provide an optimal model of the stomach surface for biopharmaceutical screening

The surface epithelial cells of the stomach represent a major component of the gastric barrier. A cell culture model of the gastric epithelial cell surface would prove useful for biopharmaceutical screening of new chemical entities and dosage forms. Primary cultures of guinea pig gastric mucous epithelial cells were grown on filter inserts (Transwells®) for 3 days. Tight-junction formation, assessed by transepithelial electrical resistance (TEER) and permeability of mannitol and fluorescein, was enhanced when collagen IV rather than collagen I was used to coat the polycarbonate filter. TEER for cells grown on collagen IV was close to that obtained with intact guinea pig gastric epithelium in vitro. Differentiation was assessed by incorporation of [ 3H]glucosamine into glycoprotein and by activity of NADPH oxidase, which produces superoxide. Both of these measures were greater for cells grown on filters coated with collagen I than for cells grown on plastic culture plates, but no major difference was found between cells grown on collagens I and IV. The proportion of cells, which stained positively for mucin with periodic acid Schiff reagent, was greater than 95% for all culture conditions. Monolayers grown on membranes coated with collagen IV exhibited apically polarized secretion of mucin and superoxide, and were resistant to acidification of the apical medium to pH 3.0 for 30 min. A screen of nonsteroidal anti-inflammatory drugs revealed a novel effect of diclofenac and niflumic acid in reversibly reducing permeability by the paracellular route. In conclusion, the mucous cell preparation grown on collagen IV represents a good model of the gastric surface epithelium suitable for screening procedures. © 2005 The Society for Biomolecular Screening.

Nanoindentation measurement of surface residual stresses in particle-reinforced metal matrix composites

The surface residual stresses in SiC particle-reinforced Al matrix composites are measured using a recently developed nanoindentation technique. The tensile biaxial residual stress in Al is found to increase with the particle concentration. The stress magnitudes are in reasonable agreement with those from numerical modeling.

Mechanical properties and the evolution of matrix molecules in PTFE upon irradiation with MeV alpha particles

The morphology, chemical composition, and mechanical properties in the surface region of α-irradiated polytetrafluoroethylene (PTFE) have been examined and compared to unirradiated specimens. Samples were irradiated with 5.5 MeV 4He2+ ions from a tandem accelerator to doses between 1 × 106 and 5 × 1010 Rad. Static time-of-flight secondary ion mass spectrometry (ToF-SIMS), using a 20 keV C60+ source, was employed to probe chemical changes as a function of a dose. Chemical images and high resolution spectra were collected and analyzed to reveal the effects of a particle radiation on the chemical structure. Residual gas analysis (RGA) was utilized to monitor the evolution of volatile species during vacuum irradiation of the samples. Scanning electron microscopy (SEM) was used to observe the morphological variation of samples with increasing a particle dose, and nanoindentation was engaged to determine the hardness and elastic modulus as a function of a dose. The data show that PTFE nominally retains its innate chemical structure and morphology at a doses <109 Rad. At α doses ≥109 Rad the polymer matrix experiences increased chemical degradation and morphological roughening which are accompanied by increased hardness and declining elasticity. At  α doses >1010 Rad the polymer matrix suffers severe chemical degradation and material loss. Chemical degradation is observed in ToF-SIMS by detection of ions that are indicative of fragmentation, unsaturation, and functionalization of molecules in the PTFE matrix. The mass spectra also expose the subtle trends of crosslinking within the α-irradiated polymer matrix. ToF-SIMS images support the assertion that chemical degradation is the result of a particle irradiation and show morphological roughening of the sample with increased a dose. High resolution SEM images more clearly illustrate the morphological roughening and the mass loss that accompanies high doses of a particles. RGA confirms the supposition that the outcome of chemical degradation in the PTFE matrix with continuing irradiation is evolution of volatile species resulting in morphological roughening and mass loss. Finally, we reveal and discuss relationships between chemical structure and mechanical properties such as hardness and elastic modulus.

Recovery of glass fibre and carbon fibres from reinforced thermosets by batch pyrolysis and investigation of fibre re-using as reinforcement in LDPE matrix

A semi-batch pyrolysis process was used to recover samples carbon fibre and glass fibre from their respective wastes. The mechanical properties of the recovered fibres were tested and compared to those of virgin fibres, showing good retention of the fibre properties. The recovered fibres were then used to prepare new LDPE composite materials with commercial and laboratory-synthesized compatibilizers. Mild oxidation of the post-pyrolysis recovered fibres and the use of different compatibilizers gave significant improvements in the mechanical properties of the LDPE composites; however some of the manufactured composites made from recovered fibres had properties similar to those made from virgin fibres.