Stabilizing Enzymes within Polymersomes by Coencapsulation of Trehalose.

Enzymes are essential biocatalysts and very attractive as therapeutics. However, their functionality is strictly related to their stability, which is significantly affected by the environmental changes occurring during their usage or long-term storage. Therefore, maintaining the activity of enzymes is essential when they are exposed to high temperature during usage or when they are stored for extended periods of time. Here, we stabilize and protect enzymes by coencapsulating them with trehalose into polymersomes. The anhydrobiotic disaccharide preserved up to about 81% of the enzyme's original activity when laccase/trehalose-loaded nanoreactors were kept desiccated for 2 months at room temperature and 75% of its activity when heated at 50 °C for 3 weeks. Moreover, the applicability of laccase/trehalose-loaded nanoreactors as catalysts for bleaching of the textile dyes orange G, toluidine blue O, and indigo was proven. Our results demonstrate the advantages of coencapsulating trehalose within polymersomes to stabilize enzymes in dehydrated state for extended periods of time, preserving their activity even when heated to elevated temperature.

Amino acid composition of nanofibrillar self-assembling peptide hydrogels affects responses of periodontal tissue cells in vitro.

BACKGROUND: The regeneration of tissue defects at the interface between soft and hard tissue, eg, in the periodontium, poses a challenge due to the divergent tissue requirements. A class of biomaterials that may support the regeneration at the soft-to-hard tissue interface are self-assembling peptides (SAPs), as their physicochemical and mechanical properties can be rationally designed to meet tissue requirements. MATERIALS AND METHODS: In this work, we investigated the effect of two single-component and two complementary ß-sheet forming SAP systems on their hydrogel properties such as nanofibrillar architecture, surface charge, and protein adsorption as well as their influence on cell adhesion, morphology, growth, and differentiation. RESULTS: We showed that these four 11-amino acid SAP (P11-SAP) hydrogels possessed physico-chemical characteristics dependent on their amino acid composition that allowed variabilities in nanofibrillar network architecture, surface charge, and protein adsorption (eg, the single-component systems demonstrated an ~30% higher porosity and an almost 2-fold higher protein adsorption compared with the complementary systems). Cytocompatibility studies revealed similar results for cells cultured on the four P11-SAP hydrogels compared with cells on standard cell culture surfaces. The single-component P11-SAP systems showed a 1.7-fold increase in cell adhesion and cellular growth compared with the complementary P11-SAP systems. Moreover, significantly enhanced osteogenic differentiation of human calvarial osteoblasts was detected for the single-component P11-SAP system hydrogels compared with standard cell cultures. CONCLUSION: Thus, single-component system P11-SAP hydrogels can be assessed as suitable scaffolds for periodontal regeneration therapy, as they provide adjustable, extracellular matrix-mimetic nanofibrillar architecture and favorable cellular interaction with periodontal cells.

Characterization and in ovo vascularization of a 3D-printed hydroxyapatite scaffold with different extracellular matrix coatings under perfusion culture.

For the fabrication of appropriate bone tissue-engineered constructs several prerequisites should be fulfilled. They should offer long-term stability, allow proper cell attachment and proliferation and furthermore be osteoinductive and easy to be vascularized. Having these requirements as background, we fabricated a novel porous 3D-printed hydroxyapatite (HA) scaffold and treated it with oxygen plasma (OPT). MG-63 pre-osteoblast-seeded bone constructs allowed good cell attachment and proliferation, which was even better when cultivated in a perfusion flow bioreactor. Moreover, the deposition of extracellular matrix (ECM) on the otherwise inorganic surface changed the mechanical properties in a favourable manner: elasticity increased from 42.95±1.09 to 91.9±5.1 MPa (assessed by nanoindentation). Compared to static conditions, osteogenic differentiation was enhanced in the bioreactor, with upregulation of ALP, collagen I and osteocalcin gene expression. In parallel experiments, primary human bone marrow mesenchymal stromal cells (hBMSCs) were used and findings under dynamic conditions were similar; with a higher commitment towards osteoblasts compared to static conditions. In addition, angiogenic markers CD31, eNOS and VEGF were upregulated, especially when osteogenic medium was used rather than proliferative medium. To compare differently fabricated ECMs in terms of vascularization, decellularized constructs were tested in the chorioallantoic membrane (CAM) assay with subsequent assessment of the functional perfusion capacity by MRI in the living chick embryo. Here, vascularization induced by ECM from osteogenic medium led to a vessel distribution more homogenous throughout the construct, while ECM from proliferative medium enhanced vessel density at the interface and, to a lower extent, at the middle and top. We conclude that dynamic cultivation of a novel porous OPT HA scaffold with hBMSCs in osteogenic medium and subsequent decellularization provides a promising off-the-shelf bone tissue-engineered construct.

Mechanical characteristics of beta sheet-forming peptide hydrogels are dependent on peptide sequence, concentration and buffer composition.

Self-assembling peptide hydrogels can be modified regarding their biodegradability, their chemical and mechanical properties and their nanofibrillar structure. Thus, self-assembling peptide hydrogels might be suitable scaffolds for regenerative therapies and tissue engineering. Owing to the use of various peptide concentrations and buffer compositions, the self-assembling peptide hydrogels might be influenced regarding their mechanical characteristics. Therefore, the mechanical properties and stability of a set of self-assembling peptide hydrogels, consisting of 11 amino acids, made from four beta sheet self-assembling peptides in various peptide concentrations and buffer compositions were studied. The formed self-assembling peptide hydrogels exhibited stiffnesses ranging from 0.6 to 205 kPa. The hydrogel stiffness was mostly affected by peptide sequence followed by peptide concentration and buffer composition. All self-assembling peptide hydrogels examined provided a nanofibrillar network formation. A maximum self-assembling peptide hydrogel dissolution of 20% was observed for different buffer solutions after 7 days. The stability regarding enzymatic and bacterial digestion showed less degradation in comparison to the self-assembling peptide hydrogel dissolution rate in buffer. The tested set of self-assembling peptide hydrogels were able to form stable scaffolds and provided a broad spectrum of tissue-specific stiffnesses that are suitable for a regenerative therapy.

Epithelial growth factor receptor expression influences 5-ALA induced glioblastoma fluorescence.

The extent of 5-aminolevulinic acid (5-ALA) guided tumor resection has a determining impact in high-grade glioma and glioblastoma surgery. Yet the intensity of the 5-ALA induced fluorescence may vary within the tumor. We aimed to correlate 5-ALA induced fluorescence with the expression of epithelial growth factor receptor (EGFR) and its constitutively active version EGFRvIII in different glioblastoma (GBM) cell lines. To elucidate the role of EGFR in the metabolism of 5-ALA in GBM cell lines with variable EGFR expression status, we analyzed the activation of EGFR by its primary ligand EGF, and its downstream effect on Heme oxygenase-1 (HO-1), a key enzyme regulating the metabolism of Protoporphyrin IX (PpIX), the fluorescent metabolite of 5-ALA. Effects of direct pharmacological inhibition by Tin(IV)-Protoporphyrin (SnPP) or gene knockdown by small interfering RNA (siRNA) on HO-1 enzyme were analyzed in respect to 5-ALA induced fluorescence. Furthermore, inhibition of EGFR by Gefitinib was tested. A significant difference in 5-ALA induced fluorescence was obtained in U87MG (low EGFR expression) and LN229EGFR cells (EGFR overexpression) compared to BS153 (EGFR overexpression/EGFRvIII+). Treatment of U87MG and LN229EGFR cells with EGF significantly reduced cellular fluorescence, by promoting HO-1 transcription and expression in a concentration-dependent manner. This effect could be reversed by EGFR-specific siRNA treatment, which reduced protein expression of about 80% in U87MG. Remarkably, inhibition of HO-1 activity by SnPP or reduction of HO-1 protein levels by siHO-1 treatment restored fluorescence in all cell lines, independently of EGFR quantitative and qualitative expression. Gefitinib treatment was able to restore fluorescence after EGF stimulation in U87MG cells but not in BS153 cells, overexpressing EGFR/EGFRvIII. In GBM cell lines, 5-ALA induced fluorescence is variable and influenced by EGF-induced downstream activation of HO-1. HO-1 protein expression was identified as a negative regulator of 5-ALA induced fluorescence in GBM cells. We further propose that co-expression of EGFRvIII but not quantitative EGFR expression influence HO-1 activity and therefore cellular fluorescence.

International Relations at Universities of Applied Sciences.

An overview of international relations at the Universities of Applied Sciences in Switzerland is presented.

Synthesis and Electrophoretic Properties of Novel Nanoparticles for Colored Electronic Ink and e-Paper Applications.

A new approach based on non-pigmented, stable colored nanoparticles able to migrate upon application of an electrical field (10-60 V) has been developed for the improvement of the color brightness of e-displays. The scientific challenges comprised the development of efficient syntheses of tri- and bifunctional dendrimers including branching points for further extension and individual decoration with dye (yellow, magenta, cyan). The covalent attachment of these scaffolds to silica nanoparticles was performed via hydrosilylation and final in situ charging generated attractive silica shells for the substractive CMY color space model.

International Relations at Universities of Applied Sciences.

An overview of international relations at the Universities of Applied Sciences in Switzerland is presented.

Self-Sealing and Puncture Resistant Breathable Membranes for Water-Evaporation Applications.

Breathable and waterproof membranes that self-seal damaged areas are prepared by modifying a poly(ether ester) membrane with an amphiphilic polymer co-network. The latter swells in water and the gel closes punctures. Damaged composite membranes remain water tight up to pressures of at least 1.6 bar. This material is useful for applications where water-vapor permeability, self-sealing properties, and waterproofness are desired, as demonstrated for a medical cooling device.

Novel microcalorimetric assay for antibacterial activity of implant coatings: The cases of silver-doped hydroxyapatite and calcium hydroxide.

Biomaterials with antimicrobial properties are now commonly used in different clinical specialties including orthopedics, endodontic, and traumatology. As a result, assessing the antimicrobial effect of coatings applied on implants is of critical importance. In this study, we demonstrate that isothermal microcalorimetry (IMC) can be used for monitoring bacterial growth and biofilm formation at the surface of such coatings and for determining their antimicrobial effects. The antibacterial effects of silver doped hydroxyapatite (HA) and calcium hydroxide coatings on Staphylococcus epidermidis were determined with a minimal workload. Using the Gompertz growth model we determined biofilm growth rates close to those values reported in the literature. Furthermore, we were able to estimate the reduction in the bacterial inocula originally applied at the surface of the coatings. Therefore, in addition to monitoring the antimicrobial effect of silver doped HA and calcium hydroxide coatings, we also demonstrate that IMC might be a valuable tool for assessing such antimicrobial properties of implant coatings at a minimal workload.

Surface chemistry at Swiss Universities of Applied Sciences.

In the Swiss Universities of Applied Sciences, a number of research groups are involved in surface science, with different methodological approaches and a broad range of sophisticated characterization techniques. A snapshot of the current research going on in different groups from the University of Applied Sciences and Arts Western Switzerland (HES-SO), the Zurich University of Applied Sciences (ZHAW) and the University of Applied Sciences and Arts Northwestern Switzerland (FHNW) is given.

Preparation and optimization of calcium fluoride particles for dental applications.

Fluorides are used in dental care due to their beneficial effect in tooth enamel de-/remineralization cycles. To achieve a desired constant supply of soluble fluorides in the oral cavity, different approaches have been followed. Here we present results on the preparation of CaF2 particles and their characterization with respect to a potential application as enamel associated fluoride releasing reservoirs. CaF2 particles were synthesized by precipitation from soluble NaF and CaCl2 salt solutions of defined concentrations and their morphology analyzed by scanning electron microscopy. CaF2 particles with defined sizes and shapes could be synthesized by adjusting the concentrations of the precursor salt solutions. Such particles interacted with enamel surfaces when applied at fluoride concentrations correlating to typical dental care products. Fluoride release from the synthesized CaF2 particles was observed to be largely influenced by the concentration of phosphate in the solution. Physiological solutions with phosphate concentration similar to saliva (3.5 mM) reduced the fluoride release from pure CaF2 particles by a factor of 10-20 × as compared to phosphate free buffer solutions. Fluoride release was even lower in human saliva. The fluoride release could be increased by the addition of phosphate in substoichiometric amounts during CaF2 particle synthesis. The presented results demonstrate that the morphology and fluoride release characteristics of CaF2 particles can be tuned and provide evidence of the suitability of synthetic CaF2 particles as enamel associated fluoride reservoirs.

An in vitro toxicity evaluation of gold-, PLLA- and PCL-coated silica nanoparticles in neuronal cells for nanoparticle-assisted laser-tissue soldering.

The uptake of silica (Si) and gold (Au) nanoparticles (NPs) engineered for laser-tissue soldering in the brain was investigated using microglial cells and undifferentiated and differentiated SH-SY5Y cells. It is not known what effects NPs elicit once entering the brain. Cellular uptake, cytotoxicity, apoptosis, and the potential induction of oxidative stress by means of depletion of glutathione levels were determined after NP exposure at concentrations of 10(3) and 10(9)NPs/ml. Au-, silica poly (ε-caprolactone) (Si-PCL-) and silica poly-L-lactide (Si-PLLA)-NPs were taken up by all cells investigated. Aggregates and single NPs were found in membrane-surrounded vacuoles and the cytoplasm, but not in the nucleus. Both NP concentrations investigated did not result in cytotoxicity or apoptosis, but reduced glutathione (GSH) levels predominantly at 6 and 24h, but not after 12 h of NP exposure in the microglial cells. NP exposure-induced GSH depletion was concentration-dependent in both cell lines. Si-PCL-NPs induced the strongest effect of GSH depletion followed by Si-PLLA-NPs and Au-NPs. NP size seems to be an important characteristic for this effect. Overall, Au-NPs are most promising for laser-assisted vascular soldering in the brain. Further studies are necessary to further evaluate possible effects of these NPs in neuronal cells.

Poly(N-vinylpyrrolidone)-poly(dimethylsiloxane)-based polymersome nanoreactors for laccase-catalyzed biotransformations.

Laccases (Lac) are oxidizing enzymes with a broad range of applications, for example, in soil remediation, as bleaching agent in the textile industry, and for cosmetics. Protecting the enzyme against degradation and inhibition is of great importance for many of these applications. Polymer vesicles (polymersomes) from poly(N-vinylpyrrolidone)-block-poly(dimethylsiloxane)-block-poly(N-vinylpyrrolidone) (PNVP-b-PDMS-b-PNVP) triblock copolymers were prepared and investigated as intrinsically semipermeable nanoreactors for Lac. The block copolymers allow oxygen to enter and reactive oxygen species (ROS) to leave the polymersomes. EPR spectroscopy proved that Lac can generate ROS. They could diffuse out of the polymersome and oxidize an aromatic substrate outside the vesicles. Michaelis-Menten constants Km between 60 and 143 µM and turn over numbers kcat of 0.11 to 0.18 s(-1) were determined for Lac in the nanoreactors. The molecular weight and the PDMS-to-PNVP ratio of the block copolymers influenced these apparent Michaelis-Menten parameters. Encapsulation of Lac in the polymersomes significantly protected the enzyme against enzymatic degradation and against small inhibitors: proteinase K caused 90% less degradation and the inhibitor sodium azide did not affect the enzyme's activity. Therefore, these polymer nanoreactors are an effective means to stabilize laccase.

Differentiation of human mesenchymal stem cells on plasma-treated polyetheretherketone.

Polyetheretherketone (PEEK) generally exhibits physical and chemical characteristics that prevent osseointegration. To activate the PEEK surface, we applied oxygen and ammonia plasma treatments. These treatments resulted in surface modifications, leading to changes in nanostructure, contact angle, electrochemical properties and protein adhesion in a plasma power and process gas dependent way. To evaluate the effect of the plasma-induced PEEK modifications on stem cell adhesion and differentiation, adipose tissue-derived mesenchymal stem cells (adMSC) were seeded on PEEK specimens. We demonstrated an increased adhesion, proliferation, and osteogenic differentiation of adMSC in contact to plasma-treated PEEK. In dependency on the process gas (oxygen or ammonia) and plasma power (between 10 and 200 W for 5 min), varying degrees of osteogenic differentiation were induced. When adMSC were grown on 10 and 50 W oxygen and ammonia plasma-treated PEEK substrates they exhibited a doubled mineralization degree relative to the original PEEK. Thus plasma treatment of PEEK specimens induced changes in surface chemistry and topography and supported osteogenic differentiation of adMSC in vitro. Therefore plasma treated PEEK holds perspective for contributing to osseointegration of dental and orthopedic load-bearing PEEK implants in vivo.

Photoreaction of a hydroxyalkyphenone with the membrane of polymersomes: a versatile method to generate semipermeable nanoreactors.

Block copolymer vesicles can be turned into nanoreactors when a catalyst is encapsulated in these hollow nanostructures. However the membranes of these polymersomes are most often impermeable to small organic molecules, while applications as nanoreactor, as artificial organelles, or as drug-delivery devices require an exchange of substances between the outside and the inside of polymersomes. Here, a simple and versatile method is presented to render polymersomes semipermeable. It does not require complex membrane proteins or pose requirements on the chemical nature of the polymers. Vesicles made from three different amphiphilic block copolymers (α,ω-hydroxy-end-capped poly(2-methyl-2-oxazoline)-block-poly(dimethylsiloxane)-block-poly(2-methyl-2-oxazoline) (PMOXA-b-PDMS-b-PMOXA), α,ω-acrylate-end-capped PMOXA-b-PDMS-b-PMOXA, and poly(ethylene oxide)-block-poly(butadiene) (PEO-b-PB)) were reacted with externally added 2-hydroxy-4'-2-(hydroxyethoxy)-2-methylpropiophenone under UV-irradiation. The photoreactive compound incorporated into the block copolymer membranes independently of their chemical nature or the presence of double bonds. This treatment of polymersomes resulted in substantial increase in permeability for organic compounds while not disturbing the size and the shape of the vesicles. Permeability was assessed by encapsulating horseradish peroxidase into vesicles and measuring the accessibility of substrates to the enzyme. The permeability of photoreacted polymersomes for ABTS, AEC, pyrogallol, and TMB was determined to be between 1.9 and 38.2 nm s(-1). It correlated with the hydrophobicity of the compounds. Moreover, fluorescent dyes were released at higher rates from permeabilized polymersomes compared to unmodified ones. The permeabilized nanoreactors retained their ability to protect encapsulated biocatalysts from degradation by proteases.

Nanoshell assisted laser soldering of vascular tissue.

BACKGROUND AND OBJECTIVES: Laser tissue soldering (LTS) is a promising technique for tissue fusion but is limited by the lack of reproducibility particularly when the amount of indocyanine green (ICG) applied as energy absorber cannot be controlled during the soldering procedure. Nanotechnology enables the control over the quantitative binding of the ICG. The aim of this study was to establish a highly reproducible and strong tissue fusion using ICG packed nanoshells. By including the chromophore in the soldering scaffold, dilution of the energy absorber during the soldering procedure is prevented. The feasibility of this novel nanoshell soldering technique was studied by assessing the local heating of the area and tensile strength of the resulting fused tissue. STUDY DESIGN/MATERIALS AND METHODS: Nanoshells with a diameter of 250-270 nm were loaded with ICG and included in a porous polycaprolactone (PCL) scaffold doped with albumin solder. The nanoshell scaffold was used in a flexible, semi-dry formulation suitable for surgical use. Heat development, tensile strength as well as tissue damage were assessed. RESULTS: Rabbit aortic arteries were successfully soldered using an ICG packed nanoshell scaffold. Tensile strengths of these nanoshell soldered anastomoses were found to be 734 ± 327 mN (median = 640 mN). Thermal damage was restricted to the adventitia at the irradiated area. In addition, absorber dilution was prevented during the soldering procedure resulting in significantly lower variance in maximum temperature (P = 0.03) compared to the classical liquid ICG soldering technique. CONCLUSION: Using nanoshells, controlled amounts of chromophore could successfully be bound into the polymer scaffold. Diode laser soldering of vascular tissue using ICG-nanoshell scaffolds leads to strong and reproducible tissue fusion. With optimally chosen settings of irradiation time, nanoshells coating and scaffold properties, our improved LTS procedure demonstrates the potential for a clinically applicable anastomosis technique.

Uptake and fate of surface modified silica nanoparticles in head and neck squamous cell carcinoma.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is currently the eighth leading cause of cancer death worldwide. The often severe side effects, functional impairments and unfavorable cosmetic outcome of conventional therapies for HNSCC have prompted the quest for novel treatment strategies, including the evaluation of nanotechnology to improve e.g. drug delivery and cancer imaging. Although silica nanoparticles hold great promise for biomedical applications, they have not yet been investigated in the context of HNSCC. In the present in-vitro study we thus analyzed the cytotoxicity, uptake and intracellular fate of 200-300 nm core-shell silica nanoparticles encapsulating fluorescent dye tris(bipyridine)ruthenium(II) dichloride with hydroxyl-, aminopropyl- or PEGylated surface modifications (Ru@SiO2-OH, Ru@SiO2-NH2, Ru@SiO2-PEG) in the human HNSCC cell line UMB-SCC 745. RESULTS: We found that at concentrations of 0.125 mg/ml, none of the nanoparticles used had a statistically significant effect on proliferation rates of UMB-SCC 745. Confocal and transmission electron microscopy showed an intracellular appearance of Ru@SiO2-OH and Ru@SiO2-NH2 within 30 min. They were internalized both as single nanoparticles (presumably via clathrin-coated pits) or in clusters and always localized to cytoplasmic membrane-bounded vesicles. Immunocytochemical co-localization studies indicated that only a fraction of these nanoparticles were transferred to early endosomes, while the majority accumulated in large organelles. Ru@SiO2-OH and Ru@SiO2-NH2 nanoparticles had never been observed to traffic to the lysosomal compartment and were rather propagated at cell division. Intracellular persistence of Ru@SiO2-OH and Ru@SiO2-NH2 was thus traceable over 5 cell passages, but did not result in apparent changes in cell morphology and vitality. In contrast to Ru@SiO2-OH and Ru@SiO2-NH2 uptake of Ru@SiO2-PEG was minimal even after 24 h. CONCLUSIONS: Our study is the first to provide evidence that silica-based nanoparticles may serve as useful tools for the development of novel treatment options in HNSCC. Their long intracellular persistence could be of advantage for e.g. chronic therapeutic modalities. However, their complex endocytotic pathways require further investigations.