Responsive core−shell latex particles as colloidosome microcapsule membranes

Responsive core-shell latex particles are used to prepare colloidosome microcapsules using thermal annealing and internal cross-linking of the shell, allowing production of the microcapsules at high concentrations. The core-shell particles are composed of a polystyrene core and a shell of poly[2-(dimethylamino)ethyl methacrylate]-b-poly[methyl methacrylate] (PDMA-b-PMMA) chains adsorbed onto the core surface, providing steric stabilisation. The PDMA component of adsorbed polymer shell confers the latex particle thermal and pH responsive characteristics, it also provides glass transitions at lower temperatures than that of the core and reactive amine groups. These features facilitate the formation of stable Pickering emulsion droplets and the immobilisation of the latex particle monolayer on these droplets to form colloidosome microcapsules. The immobilisation is achieved through thermal annealing or cross-linking of the shell at mild conditions feasible for large scale economic production. We demonstrate here that it is possible to anneal the particle monolayer on the emulsion drop surface at 75-86 ºC by using the lower glass transition temperature of the shell compared to that of the polystyrene cores (~108 ºC). The colloidosome microcapsules formed have a rigid membrane basically composed of a monolayer of particles. Chemical cross-linking has also been successfully achieved by confining a cross-linker within the disperse droplet. This approach leads to the formation of single-layered stimulus-responsive soft colloidosome membranes and provides the advantage of working at very high emulsion concentrations since inter-droplet cross-linking is thus avoided. The porosity and mechanical strength of microcapsules are also discussed here in terms of the observed structure of the latex particle monolayers forming the capsule membrane.

Human single-donor composite skin substitutes based on collagen and polycaprolactone copolymer

The development and characterization of an enhanced composite skin substitute based on collagen and poly(e-caprolactone) are reported. Considering the features of excellent biocompatibility, easy-manipulated property and exempt from cross-linking related toxicity observed in the 1:20 biocomposites, skin substitutes were developed by seeding human single-donor keratinocytes and fibroblasts alone on both sides of the 1:20 biocomposite to allow for separation of two cell types and preserving cell signals transmission via micro-pores with a porosity of 28.8 ± 16.1 µm. The bi-layered skin substitute exhibited both differentiated epidermis and fibrous dermis in vitro. Less Keratinocyte Growth Factor production was measured in the co-cultured skin model compared to fibroblast alone condition indicating a favorable microenvironment for epidermal homeostasis. Moreover, fast wound closure, epidermal differentiation, and abundant dermal collagen deposition were observed in composite skin in vivo. In summary, the beneficial characteristics of the new skin substitutes exploited the potential for pharmaceutical screening and clinical application.

Pituitary adenylate cyclase-activating polypeptide type 1 receptor (PAC1) gene is suppressed by transglutaminase 2 activation

Pituitary adenylate cyclase-activating polypeptide (PACAP) functions as a neuroprotective factor through the PACAP type 1 receptor, PAC1. In a previous work, we demonstrated that nerve growth factor augmented PAC1 gene expression through the activation of Sp1 via the Ras/MAPK pathway. We also observed that PAC1 expression in Neuro2a cells was transiently suppressed during in vitro ischemic conditions, oxygen-glucose deprivation (OGD). Because endoplasmic reticulum (ER) stress is induced by ischemia, we attempted to clarify how ER stress affects the expression of PAC1. Tunicamycin, which induces ER stress, significantly suppressed PAC1 gene expression, and salubrinal, a selective inhibitor of the protein kinase RNA-like endoplasmic reticulum kinase signaling pathway of ER stress, blocked the suppression. In luciferase reporter assay, we found that two Sp1 sites were involved in suppression of PAC1 gene expression due to tunicamycin or OGD. Immunocytochemical staining demonstrated that OGD-induced transglutaminase 2 (TG2) expression was suppressed by salubrinal or cystamine, a TG activity inhibitor. Further, the OGD-induced accumulation of cross-linked Sp1 in nuclei was suppressed by cystamine or salubrinal. Together with cystamine, R283, TG2-specific inhibitor, and siRNA specific for TG2 also ameliorated OGD-induced attenuation of PAC1 gene expression. These results suggest that Sp1 cross-linking might be crucial in negative regulation of PAC1 gene expression due to TG2 in OGD-induced ER stress. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.

Celiac disease patient IgA antibodies induce endothelial adhesion and cell polarization defects via extracellular transglutaminase 2

We have recently found that celiac disease patient serum-derived autoantibodies targeted against transglutaminase 2 interfere with several steps of angiogenesis, including endothelial sprouting and migration, though the mechanism involved remained to be fully characterized. This study now investigated the processes underlying the antiangiogenic effects exerted by celiac disease patient antibodies on endothelial cells, with particular regard to the adhesion, migration, and polarization signaling pathway. We observed that celiac IgA reduced endothelial cell numbers by affecting adhesion without increasing apoptosis. Endothelial cells in the presence of celiac IgA showed weak attachment, a high susceptibility to detach from fibronectin, and a disorganized extracellular matrix due to a reduction of protein cross-links. Furthermore, celiac patient IgA led to secretion of active transglutaminase 2 from endothelial cells into the culture supernatants. Additionally, cell surface transglutaminase 2 mediated integrin clustering in the presence of celiac IgA was coupled to augmented expression of ß1-integrin. We also observed that celiac patient IgA-treated endothelial cells had migratory defects and a less polarized phenotype when compared to control groups, and this was associated with the RhoA signaling pathway. These biological effects mediated by celiac IgA on endothelial cells were partially influenced but not completely abolished by R281, an irreversible extracellular transglutaminase 2 enzymatic activity inhibitor. Taken together, our results imply that celiac patient IgA antibodies disturb the extracellular protein cross-linking function of transglutaminase 2, thus altering cell-extracellular matrix interactions and thereby affecting endothelial cell adhesion, polarization, and motility. © 2013 Springer Basel.

Transglutaminase 2 interaction with small heat shock proteins mediate cell survival upon excitotoxic stress

Transglutaminase 2 has been postulated to be involved in the pathogenesis of central nervous system neurodegenerative disorders. However, its role in neuronal cell death remains to be elucidated. Excitotoxicity is a common event underlying neurodegeneration. We aimed to evaluate the protein targets for transglutaminase 2 in cell response to NMDA-induced excitotoxic stress, using SH-SY5Y neuroblastoma cells which express high tranglutaminase 2 levels upon retinoic acid-driven differentiation toward neurons. NMDA-evoked calcium increase led to transglutaminase 2 activation that mediated cell survival, as at first suggested by the exacerbation of NMDA toxicity in the presence of R283, a synthetic competitive inhibitor of transglutaminase active site. Assays of R283-mediated transglutaminase inhibition showed the involvement of enzyme activity in NMDA-induced reduction in protein basal levels of pro-apoptotic caspase-3 and the stress protein Hsp20. However, this occurred in a way different from protein cross-linking, given that macromolecular assemblies were not observed in our experimental conditions for both proteins. Co-immunoprecipitation experiments provided evidence for the interaction, in basal conditions, between transglutaminase 2 and Hsp20, as well as between Hsp20 and Hsp27, a major anti-apoptotic protein promoting caspase-3 inactivation and degradation. NMDA treatment disrupted both these interactions that were restored upon transglutaminase 2 inhibition with R283. These results suggest that transglutaminase 2 might be protective against NMDA-evoked excitotoxic insult in neuronal-like SH-SY5Y cells in a way, independent from transamidation that likely involves its interaction with the complex Hsp20/Hsp27 playing a pro-survival role. © 2011 Springer-Verlag.

Transglutaminases:nature's biological glues

Transglutaminases (Tgases) are a widely distributed group of enzymes that catalyse the post-translational modification of proteins by the formation of isopeptide bonds. This occurs either through protein cross-linking via epsilon-(gamma-glutamyl)lysine bonds or through incorporation of primary amines at selected peptide-bound glutamine residues. The cross-linked products, often of high molecular mass, are highly resistant to mechanical challenge and proteolytic degradation, and their accumulation is found in a number of tissues and processes where such properties are important, including skin, hair, blood clotting and wound healing. However, deregulation of enzyme activity generally associated with major disruptions in cellular homoeostatic mechanisms has resulted in these enzymes contributing to a number of human diseases, including chronic neurodegeneration, neoplastic diseases, autoimmune diseases, diseases involving progressive tissue fibrosis and diseases related to the epidermis of the skin. In the present review we detail the structural and regulatory features important in mammalian Tgases, with particular focus on the ubiquitous type 2 tissue enzyme. Physiological roles and substrates are discussed with a view to increasing and understanding the pathogenesis of the diseases associated with transglutaminases. Moreover the ability of these enzymes to modify proteins and act as biological glues has not gone unnoticed by the commercial sector. As a consequence, we have included some of the present and future biotechnological applications of this increasingly important group of enzymes.

Novel sol–gel preparation of (P2O5)0.4–(CaO)0.25–(Na2O)X–(TiO2)(0.35−X) bioresorbable glasses (X = 0.05, 0.1, and 0.15)

Quaternary phosphate-based glasses in the P2O5–CaO–Na2O–TiO2 system with a fixed P2O5 and CaO content of 40 and 25 mol% respectively have been successfully synthesised via sol–gel method and bulk, transparent samples were obtained. The structure, elemental proportion, and thermal properties of stabilised sol–gel glasses have been characterised using X-ray diffraction (XRD), energy dispersive X-ray spectroscopy (EDX), 31P nuclear magnetic resonance (31P NMR), titanium K-edge X-ray absorption near-edge structure (XANES), fourier transform infrared (FTIR) spectroscopy, and differential thermal analysis (DTA). The XRD results confirmed the amorphous nature for all stabilized sol–gel derived glasses. The EDX result shows the relatively low loss of phosphorus during the sol–gel process and Ti K-edge XANES confirmed titanium in the glass structure is in mainly six-fold coordination environment. The 31P NMR and FTIR results revealed that the glass structure consist of mainly Q1 and Q2 phosphate units and the Ti4+ cation was acting as a cross-linking between phosphate units. In addition DTA results confirmed a decrease in the glass transition and crystallisation temperature with increasing Na2O content. Ion release studies also demonstrated a decrease in degradation rates with increasing TiO2 content therefore supporting the use of these glasses for biomedical applications that require a degree of control over glass degradation. These sol–gel glasses also offer the potential to incorporate proactive molecules for drug delivery application due to the low synthesis temperature employed.

Increases in renal ε-(γ-glutamyl)-lysine crosslinks result from compartment-specific changes in tissue transglutaminase in early experimental diabetic nephropathy:Pathologic implications

Diabetic nephropathy (DN) is characterized by an early, progressive expansion and sclerosis of the glomerular mesangium leading to glomerulosclerosis. This is associated with parallel fibrosis of the renal interstitium. In experimental renal scarring, the protein cross-linking enzyme, tissue transglutaminase (tTg), is up-regulated and externalized causing an increase in its crosslink product, e-(γ-glutamyl)-lysine, in the extracellular space. This potentially contributes to the extracellular matrix (ECM) accumulation central to tissue fibrosis by increasing deposition and inhibiting breakdown. We investigated if a similar mechanism may contribute to the ECM expansion characteristic of DN using the rat streptozotocin model over 120 days. Whole kidney e-(γ-glutamyl)-lysine (HPLC analysis) was significantly increased from Day 90 (+337%) and peaked at Day 120 (+650%) (p <0.05). Immunofluorescence showed this increase to be predominantly extracellular in the peritubular interstitial space, but also in individual glomeruli. Total kidney transglutaminase (Tg) was not elevated. However, using a Tg in situ activity assay, increased Tg was detected in both the extracellular interstitial space and glomeruli by Day 60, with a maximal 53% increase at Day 120 (p <0.05). Using a specific anti-tTg antibody, immunohistochemistry showed a similar increase in extracellular enzyme in the interstitium and glomeruli. To biochemically characterize glomerular changes, glomeruli were isolated by selective sieving. In line with whole kidney measurement, there was an increase in glomerular e-(γ-glutamyl) lysine (+ 361%); however, in the glomeruli this was associated with increases in Tg activity (+228%) and tTg antigen by Western blotting (+215%). Importantly, the ratio of glomerular e-(γ-glutamyl) lysine to hydroxyproline increased by 2.2-fold. In DN, changes in the kidney result in increased translocation of tTg to the extracellular environment where high Ca2+ and low GTP levels allow its activation. In the tubulointerstitium this is independent of increased tTg production, but dependent in the glomerulus. This leads to excessive ECM cross-linking, contributing to the renal fibrosis characteristic of progressive DN.

Transglutaminase 2 interacts with syndecan-4 and CD44 at the surface of human macrophages to promote removal of apoptotic cells

Tissue transglutaminase (TG2) is a multifunctional protein cross-linking enzyme that has been implicated in apoptotic cell clearance but is also important in many other cell functions including cell adhesion, migration and monocyte to macrophage differentiation. Cell surface-associated TG2 regulates cell adhesion and migration, via its association with receptors such as syndecan-4 and β1 and β3 integrins. Whilst defective apoptotic cell clearance has been described in TG2-deficient mice, the precise role of TG2 in apoptotic cell clearance remains ill-defined. Our work addresses the role of macrophage extracellular TG2 in apoptotic cell corpse clearance. Here we reveal TG2 expression and activity (cytosolic and cell surface) in human macrophages and demonstrate that inhibitors of protein crosslinking activity reduce macrophage clearance of dying cells. We show also that cell-impermeable TG2 inhibitors significantly inhibit the ability of macrophages to migrate and clear apoptotic cells through reduced macrophage recruitment to, and binding of, apoptotic cells. Association studies reveal TG2-syndecan-4 interaction through heparan sulphate side chains, and knockdown of syndecan-4 reduces cell surface TG2 activity and apoptotic cell clearance. Furthermore, inhibition of TG2 activity reduces crosslinking of CD44, reported to augment AC clearance. Thus our data define a role for TG2 activity at the surface of human macrophages in multiple stages of AC clearance and we propose that TG2, in association with heparan sulphates, may exert its effect on AC clearance via a mechanism involving the crosslinking of CD44.

Synthesis and characterisation of interfacial layers in organic solar cells

The quest for renewable energy sources has led to growing attention in the research of organic photovoltaics (OPVs), as a promising alternative to fossil fuels, since these devices have low manufacturing costs and attractive end-user qualities, such as ease of installation and maintenance. Wide application of OPVs is majorly limited by the devices lifetime. With the development of new encapsulation materials, some degradation factors, such as water and oxygen ingress, can almost be excluded, whereas the thermal degradation of the devices remains a major issue. Two aspects have to be addressed to solve the problem of thermal instability: bulk effects in the photoactive layer and interfacial effects at the photoactive layer/charge-transporting layers. In this work, the interface between photoactive layer and electron-transporting zinc oxide (ZnO) in devices with inverted architecture was engineered by introducing polymeric interlayers, based on zinc-binding ligands, such as 3,4-dihydroxybenzene and 8-hydroxyquinoline. Also, a cross-linkable layer of poly(3,4-dimethoxystyrene) and its fullerene derivative were studied. At first, controlled reversible addition-fragmentation chain transfer (RAFT) polymerisation was employed to achieve well-defined polymers in a range of molar masses, all bearing a chain-end functionality for further modifications. Resulting polymers have been fully characterised, including their thermal and optical properties, and introduced as interlayers to study their effect on the initial device performance and thermal stability. Poly(3,4-dihydroxystyrene) and its fullerene derivative were found unsuitable for application in devices as they increased the work function of ZnO and created a barrier for electron extraction. On the other hand, their parental polymer, poly(3,4-dimethoxystyrene), and its fullerene derivative, upon cross-linking, resulted in enhanced efficiency and stability of devices, if compared to control. Polymers based on 8-hydroxyquinoline ligand had a negative effect on the initial stability of the devices, but increased the lifetime of the cells under accelerated thermal stress. Comprehensive studies of the key mechanisms, determining efficiency, such as charge generation and extraction, were performed by using time-resolved electrical and spectroscopic techniques, in order to understand in detail the effect of the interlayers on the device performance. Obtained results allow deeper insight into mechanisms of degradation that limit the lifetime of devices and prompt the design of better materials for the interface stabilisation.

Comparative physical, mechanical and crystallographic properties of a series of gemfibrozil salts

Objectives Understanding the impact of the counterion on the properties of an acidic or basic drug may influence the choice of salt form, especially for less potent drugs with a high drug load per unit dose. The aim of this work was to determine the influence of the hydrogen bonding potential of the counterion on the crystal structure of salts of the poorly soluble, poorly compressible, acidic drug gemfibrozil and to correlate these with mechanical properties. Methods Compacts of the parent drug and the salts were used to determine Young's modulus of elasticity using beam bending tests. Crystal structures were determined previously from X-ray powder diffraction data. Key findings The free acid, tert-butylamine, 2-amino-2-methylpropan-1-ol and 2-amino-2-methylpropan-1, 3-diol salts had a common crystal packing motif of infinite hydrogen-bonded chains with cross-linking between pairs of adjacent chains. The tromethamine (trsi) salt, with different mechanical properties, had a two-dimensional sheet-like network of hydrogen bonds, with slip planes, forming a stiffer compact. Conclusions The type of counter ion is important in determining mechanical properties and could be selected to afford slip and plastic deformation. © 2010 Royal Pharmaceutical Society of Great Britain.

TG2: a credible therapeutic target in fibrotic diseasedue to its multifunctional roles

The role of TG2 in fibrosis is reported to be related to two important effects. The first in mediating the deposition and accumulation of the fibrotic extracellular matrix (ECM) via its cross-linking of proteins like fibronectin, collagen I and collagen III; and the second, the activation of latent matrix bound TGFβ1. We report here that the role of TG2 in fibrosis progression can be much more complex. We also report a new family of TG2-specific inhibitors that can not only inhibit protein cross-linking, but also regulate other functions of TG2, thus increasing their potency which can be demonstrated by their effectiveness in inhibiting fibrosis in two different fibrotic in vivo models.

Heterogeneously catalyzed lignin depolymerization

Biomass offers a unique resource for the sustainable production of bio-derived chemical and fuels as drop-in replacements for the current fossil fuel products. Lignin represents a major component of lignocellulosic biomass, but is particularly recalcitrant for valorization by existing chemical technologies due to its complex cross-linking polymeric network. Here, we highlight a range of catalytic approaches to lignin depolymerisation for the production of aromatic bio-oil and monomeric oxygenates.