High performance electrochromic devices based on anodized nanoporous Nb2O5

Despite the predictions, the true potential of Nb2O5 for electrochromic applications has yet to be fully realized. In this work, three-dimensional (3D) compact and well-ordered nanoporous Nb2O5 films are synthesized by the electrochemical anodization of niobium thin films. These films are formed using RF sputtering and then anodized in an electrolyte containing ethylene glycol, ammonium fluoride, and small water content (4%) at 50 °C which resulted in low embedded impurities within the structure. Characterization of the anodized films shows that a highly crystalline orthorhombic phase of Nb2O5 is obtained after annealing at 450 °C. The 3D structure provides a template consisting of a large concentration of active sites for ion intercalation, while also ensuring low scattering directional paths for electrons. These features enhance the coloration efficiency to 47.0 cm2 C?1 (at 550 nm) for a 500 nm thick film upon Li+ ion intercalation. Additionally, the Nb2O5 electrochromic device shows a high bleached state transparency and large optical modulation.

Ambient ionisation mass spectrometry for the characterisation of polymers and polymer additives : A review

The purpose of this review is to showcase the present capabilities of ambient sampling and ionisation technologies for the analysis of polymers and polymer additives by mass spectrometry (MS) while simultaneously highlighting their advantages and limitations in a critical fashion. To qualify as an ambient ionisation technique, the method must be able to probe the surface of solid or liquid samples while operating in an open environment, allowing a variety of sample sizes, shapes, and substrate materials to be analysed. The main sections of this review will be guided by the underlying principle governing the desorption/extraction step of the analysis; liquid extraction, laser ablation, or thermal desorption, and the major component investigated, either the polymer itself or exogenous compounds (additives and contaminants) present within or on the polymer substrate. The review will conclude by summarising some of the challenges these technologies still face and possible directions that would further enhance the utility of ambient ionisation mass spectrometry as a tool for polymer analysis. (C) 2013 Elsevier B. V. All rights reserved.

Poly(2-oxazoline) hydrogels as next generation three dimensional cell supports

Synthetic hydrogels selectively decorated with cell adhesion motifs are rapidly emerging as promising substrates for 3D cell culture. When cells are grown in 3D they experience potentially more physiologically relevant cell-cell interactions and physical cues compared with traditional 2D cell culture on stiff surfaces. A newly developed polymer based on poly(2-oxazoline)s has been used for the first time to control attachment of fibroblast cells and is discussed here for its potential use in 3D cell culture with particular focus on cancer cells towards the ultimate aim of high throughput screening of anti-cancer therapies. Advantages and limitations of using poly(2-oxazoline) hydrogels are discussed and compared with more established polymers, especially polyethylene glycol (PEG).

Stability of endoglucanases from mesophilic fungus and thermophilic bacterium in acidified polyols

Recent developments in chemical pretreatments of lignocellulosic biomass using polyols as co-solvents (e.g., glycerol and ethylene glycol) at temperatures less than 100 °C may allow the effective use of thermostable and non-thermostable cellulases in situ during the saccharification process. The potential of biomass saccharifying enzymes, endoglucanases (EG) from a thermophilic bacterium (Thermotoga maritima) and a mesophilic fungus (Trichoderma longibrachiatum), to retain their activity in aqueous buffer, acidified glycerol, and acidified ethylene glycol used as co-solvents at pretreatment temperatures at or below 100 °C were examined. The results show that despite its origin, T. longibrachiatum EG (Tl-EG) retained 75% of its activity after exposure to 100 °C for 5 min in aqueous buffer while T. maritima EG (Tm-EG) retained only 5% activity. However, at 90 °C both enzymes retained over 87% of their activity. In acidified (0.1% (w/w) H2SO4) glycerol, Tl-EG retained similar activity (80%) to that obtained in glycerol alone, while Tm-EG retained only 35%. With acidified ethylene glycol under these conditions, both Tl-EG and Tm-EG retained 36% of their activity. The results therefore show that Tl-EG is more stable in both acidified glycerol and ethylene glycol than Tm-EG. A preliminary kinetic study showed that pure glycerol improved the thermal stability of Tl-EG but destabilized Tm-EG, relative to the buffer solution. The half-lives of both Tl-EG and Tm-EG are 4.5 min in acidified glycerol, indicating that the effectiveness of these enzymes under typical pretreatment times of greater than 15 min will be considerably diminished. Attempts have been made to explain the differences in the results obtained between the two enzymes.

Doxycycline-inducible expression of SPARC/Osteonectin/BM40 in MDA-MB-231 human breast cancer cells results in growth inhibition

SPARC (secreted protein acidic and rich in cysteine)/BM40/Osteonectin is a matricellular protein with multiple effects on cell behaviour. In vitro, its major known functions are anti-adhesive and anti-proliferative, and it is associated with tissue remodelling and cancer in vivo. SPARC is overexpressed in many cancers, including breast cancer, and the effects of SPARC seem to be cell type-specific. To study the effects of SPARC on breast cancer, we transfected SPARC into the MDA-MB-231 BAG, human breast cancer cell line using the Tet-On inducible system. By western analysis, we found low background levels in the MDA-MB-231 BAG and clone X parental cells, and prominent induction of SPARC protein expression after doxycycline treatment in SPARC transfected clones X5, X21, X24 and X75. Induction of SPARC expression did not affect cell morphology or adhesiveness to collagens type I and IV, but it slowed the rate of proliferation in adherent cultures. Cell cycle analysis showed that SPARC slowed the progression to S phase. Doxycycline induction of SPARC also slowed the rate of monolayer wound closure in the cultured wound healing assay. Thymidine inhibition of proliferation abrogated this effect, confirming that it was due to anti-proliferation rather than inhibition of migration. Consistent with this, we were unable to detect any differences in migration and Matrigel outgrowth analysis of doxycycline-stimulated cells. We conclude that SPARC is inhibitory to human breast cancer cell proliferation, and does not stimulate migration, in contrast to its stimulatory effects reported for melanoma (proliferation and migration) and glioma (migration) cells. Similar growth repression by SPARC has been reported for ovarian cancer cells, and this may be a common feature among carcinomas.

Paracrine interactions between LNCaP prostate cancer cells and bioengineered bone in 3D in vitro culture reflect molecular changes during bone metastasis

As microenvironmental factors such as three-dimensionality and cell–matrix interactions are increasingly being acknowledged by cancer biologists, more complex 3D in vitro models are being developed to study tumorigenesis and cancer progression. To better understand the pathophysiology of bone metastasis, we have established and validated a 3D indirect co-culture model to investigate the paracrine interactions between prostate cancer (PCa) cells and human osteoblasts. Co-culture of the human PCa, LNCaP cells embedded within polyethylene glycol hydrogels with human osteoblasts in the form of a tissue engineered bone construct (TEB), resulted in reduced proliferation of LNCaP cells. LNCaP cells in both monoculture and co-culture were responsive to the androgen analog, R1881, as indicated by an increase in the expression (mRNA and/or protein induction) of androgen-regulated genes including prostate specific antigen and fatty acid synthase. Microarray gene expression analysis further revealed an up-regulation of bone markers and other genes associated with skeletal and vasculature development and a significant activation of transforming growth factor β1 downstream genes in LNCaP cells after co-culture with TEB. LNCaP cells co-cultured with TEB also unexpectedly showed similar changes in classical androgen-responsive genes under androgen-deprived conditions not seen in LNCaP monocultures. The molecular changes of LNCaP cells after co-culturing with TEBs suggest that osteoblasts exert a paracrine effect that may promote osteomimicry and modulate the expression of androgen-responsive genes in LNCaP cells. Taken together, we have presented a novel 3D in vitro model that allows the study of cellular and molecular changes occurring in PCa cells and osteoblasts that are relevant to metastatic colonization of bone. This unique in vitro model could also facilitate cancer biologists to dissect specific biological hypotheses via extensive genomic or proteomic assessments to further our understanding of the PCa-bone crosstalk.

Sertoli cells as paracrine modulators of DNA synthesis in rat peritubular myoid cells in culture

To determine whether Sertoli cells influence DNA synthesis by rat peritubular myoid cells in vitro, the effects of Sertoli cells on [3H]thymidine incorporation by peritubular myoid cells in a coculture situation were examined. Incubation of testicular peritubular myoid cells with Sertoli cells in coculture induced a significant increase in [3H]thymidine incorporation by peritubular myoid cells. This indicates a cell-cell cooperation between Sertoli and peritubular myoid cells in the testis in terms of DNA synthesis. Secreted factors from Sertoli cells, as tested in a parabiotic culture situation, also increased [3H]thymidine incorporation by peritubular myoid cells. Moreover, in terms of total cellular protein, cocultures of Sertoli cells and peritubular myoid cells resulted in a significant increase when compared with the monocultures, and this coculture effect substituted for the stimulatory response of serum on peritubular myoid cell monoculture. This study investigated the cooperative role of Sertoli cells and peritubular myoid cells in paracrine regulation of testicular functions.

Production of levulinic acid and other chemicals from sugarcane fibre

Lignocellulosics represent a renewable resource for producing fuels and chemicals as an alternative to fossil resources. This study utilised an organic acid catalyst and a co-solvent to develop an environmentally friendly processing technology for the production of levulinic acid and furfural from a waste material, sugarcane fibre.

Use of the biolistic particle delivery system to transform fungal genomes

Biolistic delivery of transforming DNA into fungal genomes, especially when performed on uninucleate haploid conidia, has proven successful in bypassing the time-consuming repetitive purification of protoplasts used for the widely applied polyethylene glycol-mediated method. Biolistic transformation is also relatively quick compared to other available methods and provides a high percentage of stable transformants.

Development of a polymer theranostic for prostate cancer

Theranostics offers an improved treatment strategy for prostate cancer by facilitating simultaneous targeting of tumour cells with subsequent drug delivery and imaging. In this report we describe the synthesis of hyperbranched polymers that are biocompatible, can specifically target and be internalised by prostate cancer cells (through targeting of prostate-specific membrane antigen – PSMA) and ultimately facilitate controlled delivery of a model drug. The theranostic also incorporates a far-red fluorescent dye that allows tracking of the polymer via optical imaging. Controlled synthesis of the polymer is achieved via reversible addition fragmentation chain transfer polymerisation of polyethylene glycol monomethyl methacrylate, with ethylene glycol dimethacrylate as the branching agent. Incorporation of 20 mol% of an hydrazide-methacrylate monomer allows post-ligation of a model drug, fluorene-2-carboxaldehyde, through a hydrolytically-degradable hydrazone linkage. The rate of degradation of this particular linker was enhanced at endosomal pH (pH = 5.5) where [similar]95% of the model drug was released in 4 hours compared to less than 5% released over the same period at physiological pH. The theranostic showed high uptake into prostate cancer cells expressing prostate-specific membrane antigen, while minimal uptake was observed in PC3 cells negative for PSMA, highlighting the enhanced efficacy of the targeting ligand.

Chondrocyte redifferentiation and construct mechanical property development in single-component photocrosslinkable hydrogels

Hydrogels are promising materials for cartilage repair, but the properties required for optimal functional outcomes are not yet known. In this study, we functionalized four materials that are commonly used in cartilage tissue engineering and evaluated them using in vitro cultures. Gelatin, hyaluronic acid, polyethylene glycol, and alginate were functionalized with methacrylic anhydride to make them photocrosslinkable. We found that the responses of encapsulated human chondrocytes were highly dependent on hydrogel type. Gelatin hydrogels supported cell proliferation and the deposition of a glycosaminoglycan rich matrix with significant mechanical functionality. However, cells had a dedifferentiated phenotype, with high expression of collagen type I. Chondrocytes showed the best redifferentiation in hyaluronic acid hydrogels, but the newly formed matrix was highly localized to the pericellular regions, and these gels degraded rapidly. Polyethylene glycol hydrogels, as a bioinert control, did not promote any strong responses. Alginate hydrogels did not support the deposition of new matrix, and the stiffness decreased during culture. The markedly different response of chondrocytes to these four photocrosslinkable hydrogels demonstrates the importance of material properties for chondrogenesis and extracellular matrix production, which are critical for effective cartilage repair.

The suitability of DEAE-Cl active groups on customized poly(GMA-co-EDMA) continuous stationary phase for fast enzyme-free isolation of plasmid DNA

The creation of a commercially viable and a large-scale purification process for plasmid DNA (pDNA) production requires a whole-systems continuous or semi-continuous purification strategy employing optimised stationary adsorption phase(s) without the use of expensive and toxic chemicals, avian/bovine-derived enzymes and several built-in unit processes, thus affecting overall plasmid recovery, processing time and economics. Continuous stationary phases are known to offer fast separation due to their large pore diameter making large molecule pDNA easily accessible with limited mass transfer resistance even at high flow rates. A monolithic stationary sorbent was synthesised via free radical liquid porogenic polymerisation of ethylene glycol dimethacrylate (EDMA) and glycidyl methacrylate (GMA) with surface and pore characteristics tailored specifically for plasmid binding, retention and elution. The polymer was functionalised with an amine active group for anion-exchange purification of pDNA from cleared lysate obtained from E. coli DH5α-pUC19 pellets in RNase/protease-free process. Characterization of the resin showed a unique porous material with 70% of the pores sizes above 300 nm. The final product isolated from anion-exchange purification in only 5 min was pure and homogenous supercoiled pDNA with no gDNA, RNA and protein contamination as confirmed with DNA electrophoresis, restriction analysis and SDS page. The resin showed a maximum binding capacity of 15.2 mg/mL and this capacity persisted after several applications of the resin. This technique is cGMP compatible and commercially viable for rapid isolation of pDNA.

Towards the design of a scalable and commercially viable technique for plasmid purification using a methacrylate monolithic stationary phase

A monolithic stationary phase was prepared via free radical co-polymerization of ethylene glycol dimethacrylate (EDMA) and glycidyl methacrylate (GMA) with pore diameter tailored specifically for plasmid binding, retention and elution. The polymer was functionalized. with 2-chloro-N,N-diethylethylamine hydrochloride (DEAE-Cl) for anion-exchange purification of plasmid DNA (pDNA) from clarified lysate obtained from E. coli DH5α-pUC19 culture in a ribonuclease/ protease-free environment. Characterization of the monolithic resin showed a porous material, with 68% of the pores existing in the matrix having diameters above 300 nm. The final product isolated from a single-stage 5 min anion-exchange purification was a pure and homogeneous supercoiled (SC) pDNA with no gDNA, RNA and protein contamination as confirmed by ethidium bromide agarose gel electrophoresis (EtBr-AGE), enzyme restriction analysis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This non-toxic technique is cGMP compatible and highly scalable for production of pDNA on a commercial level.

Three dimensional in-vitro models for studying cancer angiogenesis

Introduction Hydrogels prepared from star-shaped poly(ethylene glycol) (PEG) and maleimide-functionalized heparin provide a potential matrix for use in developing three dimensional (3D) models. We have previously demonstrated that these hydrogels support the cultivation of human umbilical vein endothelial cells (HUVECs). We extend this body of work to study the ability to create an extracellular matrix (ECM)-like model to study breast and prostate cancer cell growth in 3D. Also, we investigate the ability to produce a tri-culture mimicking tumour angiogenesis with cancer spheroids, HUVECs and mesenchymal stem cells (MSCs). Materials and Methods The breast cancer cell lines, MCF-7 and MDA-MB-231, and prostate cancer cell lines, LNCaP and PC3, were seeded into starPEG-heparin hydrogels and grown for 14 Days to analyze the effects of varying hydrogel stiffness on spheroid development. Resulting hydrogel constructs were analyzed via proliferation assays, light microscopy, and immunostaining. Cancer cell lines were then seeded into starPEG-heparin hydrogels functionalized with growth factors as spheroids with HUVECs and MSCs and grown as a tri-culture. Cultures were analyzed via immunostaining and observed using confocal microscopy. Results Cultures prepared in MMP-cleavable starPEG-heparin hydrogels display spheroid formation in contrast to adherent growth on tissue culture plastic. Small differences were visualized in cancer spheroid growth between different gel stiffness across the range of cell lines. Cancer cell lines were able to be co-cultivated with HUVECs and MSC. Interaction was visualized between tumours and HUVECs via confocal microscopy. Further studies intend to further optimize and mimic the ECM environment of in-situ tumour angiogenesis. Discussion Our results confirm the suitability of hydrogels constructed from starPEG-heparin for HUVEC and MSC co-cultivation with cancer cell lines to study cell-cell and cell-matrix interactions in a 3D environment. This represents a step forward in the development of 3D culture models to study the pathomechanisms of breast and prostate cancer.

Developing a three-dimensional in-vitro cell culture model for studying breast and prostate cancer using starPEG-heparin hydrogels

Introduction Hydrogels prepared from poly(ethylene glycol) (PEG) and maleimide-functionalized heparin provide a potential matrix for use in developing three dimensional (3D) models. We have previously demonstrated that these hydrogels support the cultivation of human umbilical vein endothelial cells (HUVECs) (1). We extend this body of work to study the ability to create an extracellular matrix (ECM)-like model to study breast and prostate cancer cell growth in 3D. Also, we investigate the ability to produce a tri-culture mimicking tumour angiogenesis with cancer spheroids, HUVECs and mesenchymal stem cells (MSC). Materials and Methods The breast cancer cell lines, MCF-7 and MDA-MB-231, and prostate cancer cell lines, LNCaP and PC3, were seeded into starPEG-heparin hydrogels and grown for 14 Days to analyse the effects of varying hydrogel stiffness on spheroid development. Resulting hydrogel constructs were analyzed via Alamar Blue assays, light microscopy, and immunofluorescence staining for cytokeratin 8/18, Ki67 and E-Cadherin. Cancer cell lines were then pre-grown in hydrogels for 5-7 days and then re-seeded into starPEG-heparin hydrogels functionalised with RGD, SDF-1, bFGF and VEGF as spheroids with HUVECs and MSC and grown for 14 days as a tri-culture in Endothelial Cell Growth Medium (ECGM; Promocell). Cell lines were also seeded as a single cell suspension into the functionalised tri-culture system. Cultures were fixed in 4% paraformaldehyde and analysed via immunostaining for Von Willebrand Factor and CD31, as well as the above mentioned markers, and observed using confocal microscopy. Results Cultures prepared in MMP-cleavable starPEG-heparin hydrogels display spheroid formation in contrast to adherent growth on tissue culture plastic. Small differences were visualised in cancer spheroid growth between different gel stiffness across the range of cell lines. Cancer cell lines were able to be co-cultivated with HUVECs and MSC. HUVEC tube formation and cancer line spheroid formation occured after 3-4 days. Interaction was visualised between tumours and HUVECs via confocal microscopy. Slightly increased interaction was seen between cancer tumours and micro-vascular tubes when seeded as single cells compared with the pre-formed spheroid approach. Further studies intend to utilise cytokine gradients to further optimise the ECM environment of in situ tumour angiogenesis. Discussion and Conclusions Our results confirm the suitability of hydrogels constructed from starPEG-heparin for HUVECs and MSC co-cultivation with cancer cell lines to study cell-cell and cell-matrix interactions in a 3D environment. This represents a step forward in the development of 3D culture models to study the pathomechanisms of breast and prostate cancer. References 1. Tsurkan MV, Chwalek K, Prokoph S, Zieris A, Levental KR, Freudenberg U, Werner C. Advanced Materials. 25, 2606-10, 2013. Disclosures The authors declare no conflicts of interest