Surface rheological properties alter aerosol formation from mucus mimetic surfaces.

The effects of surface tension and surface viscoelastic properties on the formation of aerosol droplets generated from mucus-like viscoelastic gels (mucus mimetics) during shearing with a high velocity air stream were investigated. Mucus mimetic samples were formulated with similar composition (94% water and 6% dissolved solids, consisting of mucins, proteins, and ions), surface tension (via the addition of surfactant to the mimetic surface) and bulk viscoelastic properties (via crosslinking of mucin macromolecules in the mimetic) to that of native non-diseased tracheal mucus. The surface tension of the mucus mimetic was decreased by spreading one of two surfactants, dipalmitoyl phosphatidylcholine (DPPC) or calf lung surfactant (Infasurf®), on the mimetic surface. Aerosols were generated from the mimetic surfaces during simulated coughing using an enhanced simulated cough machine (ESCM) operating under controlled environmental conditions. The size distribution of aerosol droplets generated during simulated coughing from the surfactant-coated mimetic surfaces was multimodal, while no droplets were generated from the bare mimetic surface due to its high surface viscoelastic properties and high surface tension. The concentration of aerosols generated from the DPPC-coated mimetic was higher than that of the Infasurf®-coated mimetic, even though the surface tension of the two interfaces was the same. The experimental results suggest that a balance of surface elastic behavior and surface viscous behavior is required for the generation of aerosols from the viscoelastic surfaces.

Tensiometric and Phase Domain Behavior of Lung Surfactant on Mucus-like Viscoelastic Hydrogels.

Lung surfactant has been observed at all surfaces of the airway lining fluids and is an important contributor to normal lung function. In the conducting airways, the surfactant film lies atop a viscoelastic mucus gel. In this work, we report on the characterization of the tensiometric and phase domain behavior of lung surfactant at the air-liquid interface of mucus-like viscoelastic gels. Poly(acrylic acid) hydrogels were formulated to serve as a model mucus with bulk rheological properties that matched those of tracheobronchial mucus secretions. Infasurf (Calfactant), a commercially available pulmonary surfactant derived from calf lung extract, was spread onto the hydrogel surface. The surface tension lowering ability and relaxation of Infasurf films on the hydrogels was quantified and compared to Infasurf behavior on an aqueous subphase. Infasurf phase domains during surface compression were characterized by fluorescence microscopy and phase shifting interferometry. We observed that increasing the bulk viscoelastic properties of the model mucus hydrogels reduced the ability of Infasurf films to lower surface tension and inhibited film relaxation. A shift in the formation of Infasurf condensed phase domains from smaller, more spherical domains to large, agglomerated, multilayer structures was observed with increasing viscoelastic properties of the subphase. These studies demonstrate that the surface behavior of lung surfactant on viscoelastic surfaces, such as those found in the conducting airways, differs significantly from aqueous, surfactant-laden systems.

Expression of TWISTED DWARF1 lacking its in-plane membrane anchor leads to increased cell elongation and hypermorphic growth.

Plant growth is achieved predominantly by cellular elongation, which is thought to be controlled on several levels by apoplastic auxin. Auxin export into the apoplast is achieved by plasma membrane efflux catalysts of the PIN-FORMED (PIN) and ATP-binding cassette protein subfamily B/phosphor-glycoprotein (ABCB/PGP) classes; the latter were shown to depend on interaction with the FKBP42, TWISTED DWARF1 (TWD1). Here by using a transgenic approach in combination with phenotypical, biochemical and cell biological analyses we demonstrate the importance of a putative C-terminal in-plane membrane anchor of TWD1 in the regulation of ABCB-mediated auxin transport. In contrast with dwarfed twd1 loss-of-function alleles, TWD1 gain-of-function lines that lack a putative in-plane membrane anchor (HA-TWD1-Ct ) show hypermorphic plant architecture, characterized by enhanced stem length and leaf surface but reduced shoot branching. Greater hypocotyl length is the result of enhanced cell elongation that correlates with reduced polar auxin transport capacity for HA-TWD1-Ct . As a consequence, HA-TWD1-Ct displays higher hypocotyl auxin accumulation, which is shown to result in elevated auxin-induced cell elongation rates. Our data highlight the importance of C-terminal membrane anchoring for TWD1 action, which is required for specific regulation of ABCB-mediated auxin transport. These data support a model in which TWD1 controls lateral ABCB1-mediated export into the apoplast, which is required for auxin-mediated cell elongation.

Rapid auxin-induced root growth inhibition requires the TIR and AFB auxin receptors.

We investigated the relation between auxin-induced gene expression and the rapid auxin-induced growth inhibition in Arabidopsis thaliana roots. The natural auxin indole-3-acetic acid (IAA) induced a strong activation of gene expression as visualized by the DR5rev::GFP reporter gene technique. This effect was specific for active auxins and was abolished in knockout mutants of the F-box auxin receptors. We measured the IAA-induced growth inhibition at high time resolution and show that the F-box auxin receptor mutants failed to display this effect. We conclude that the F-box auxin receptors are needed for the response. In hypocotyls, auxin induces an increase in elongation growth, and this effect has been earlier shown to be independent of the F-box receptors. Based on these findings, we discuss differences in the growth control modes in roots and shoots. We demonstrate that the rapid auxin-induced root growth inhibition, unlike the induction of growth in hypocotyls, requires the presence of the F-box auxin receptors.

Identification of critical process parameters and its interplay with nanosuspension formulation prepared by top down media milling technology--a QbD perspective.

The purpose of this study was to optimize the process parameters of a poorly soluble drug by top down media milling process using different polymer systems. Process parameters including agitation rate (RPM), size of grinding media and drug content were studied through a Quality by Design (QbD) approach, using three different polymeric stabilizers (HPMC 3 cps, PVP K-30 and HPC-EXF) with the addition of Vitamin E TPGS as a surface active agent. From the statistical analysis, the RPM of the media milling was determined to be the most significant process parameter with respect to influence on particle size. The effects of varying the size of grinding media or drug content were not found to be as significant as the effects of RPM. Finally, the polymeric stabilizer played an important role in the production of nanoparticles. Among the different polymers, HPMC stabilized systems demonstrated superior results with regards to the consistency in producing successful nanoparticles and inhibition of crystal growth during storage. This study established the interplay among the formulation parameters in order to select the design space, which helped us in the identification and rank ordering of critical and noncritical variables related to the quality attributes of nanosuspension formulation during the early phase of product development.

Optimization of formulation and process parameters for the production of nanosuspension by wet media milling technique: effect of Vitamin E TPGS and nanocrystal particle size on oral absorption.

The purpose of this study was to develop nanosuspension formulations of a poorly soluble drug using a wet media milling technique. The milling process was optimized by studying the effects of critical process parameters on the size of nanoparticles using a factorial design approach. During the design of experiments (DOEs) study, different concentrations of Vitamin E TPGS in the suspensions were used to evaluate its influence on the stabilization of a nanosuspension. Once the final formulation was optimized, a pharmacokinetic study was performed in beagle dogs to investigate the effect of different ranges of particle size of nanocrystals on the plasma profile. A significant increase in AUC and C(max) was observed when the drug substance was converted into nanocrystals, likely due to the increase in dissolution rate. Results also revealed that the nanosuspension formulation (consists of nanocrystals with narrow size distribution, having a mean particle size<300 nm) produced less variability with regards to the individual plasma concentrations in the dogs when compared an alternate nanocrystal formulation (consists of nanocrystals with broad size distribution having a mean particle size<750 nm). This type of observation can be explained due to the Ostwald ripening phenomena between the nanocrystals when the particle size distribution was very broad (higher poly dispersity index). Surprisingly, the un-micronized suspension containing Vitamin E TPGS did not show any significant impact on pharmacokinetic parameters.

Application of spray granulation for conversion of a nanosuspension into a dry powder form.

The in vivo effect of particle agglomeration after drying of nanoparticles has not been extensively studied till date based on current literature review. The purpose of this research was to evaluate the feasibility of spray granulation as a processing method to convert a nanosuspension of a poorly water soluble drug into a solid dosage form and to evaluate the effect of the transformation into a solid powder on the in vivo exposure in beagle dogs. Formulation variables like the level of stabilizer in the nanosuspension formulation, granulation substrate and drug loading in the granulation were evaluated. The granules were characterized for moisture content, drug content, particle size, crystallinity and in vitro dissolution rate. Granulations with 10% drug loading showed dissolution profiles comparable to the nanosuspension, slightly slower dissolution profiles were observed at 20% drug loading. This can be attributed to an increase in the surface hydrophobicity at a higher drug loading and the formation of agglomerates that were harder to disintegrate, thereby compromising the dissolution rate. An in vivo PK study in beagle dogs showed an 8-fold increase and a 6-fold increase in the AUC(0-48) from the nanosuspension and dried nanosuspension formulations respectively compared to the coarse suspension. Also, the nanosuspension and dried nanosuspension formulations showed a 12-fold and 8-fold increase in the C(max) respectively compared to the coarse suspension. This shows the feasibility of using spray granulation as a processing method to convert a nanosuspension into a solid dosage form with improved in vivo exposure compared to the coarse suspension formulation.

Exploring the link between auxin receptors, rapid cell elongation and organ tropisms.

Auxin receptor F-box proteins of the TIR1/AFB family are known to regulate auxin-induced gene expression. We could demonstrate that rapid auxin-induced hypocotyl elongation, the most classical auxin response, is only mildly affected in Arabidopsis plants in which most of the receptor genes have been knocked out, while gene expression is almost completely abolished. Here we test the same receptor mutant plants for their gravitropic and phototropic responsiveness, generally considered to base on auxin gradients across the hypocotyl.

The diageotropica mutation of tomato disrupts a signalling chain using extracellular auxin binding protein 1 as a receptor.

The diageotropica ( dgt) mutant of tomato ( Lycopersicon esculentum Mill.) is known to lack a number of typical auxin responses. Here we show that rapid auxin-induced growth of seedling hypocotyls is completely abolished by the mutation over the full range of auxin concentrations tested, and also in early phases of the time course. Protoplasts isolated from wild-type hypocotyls respond to auxin by a rapid increase in cell volume, which we measured by image analysis at a high temporal resolution. A similar swelling could be triggered by antibodies directed against a part of the putative auxin-binding domain (box-a) of the auxin-binding protein 1 (ABP1). Induction of swelling both by auxin and by the antibody was not observed in the protoplasts isolated from the dgt mutant. However, dgt protoplasts are able to respond to the stimulator of the H(+)-ATPase, fusicoccin, with normal swelling. We propose that dgt is a signal-transduction mutation interfering with an auxin-signalling pathway that uses ABP1 as a receptor.