Research Report - FHNW

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Fachhochschule NordwestschweizHochschule für Life SciencesGründenstrasse 40CH-4132 Muttenz

T +41 61 467 42 42F +41 61 467 47 01info.lifesciences @ fhnw.chwww.fhnw.ch /lifesciences

The FHNW incorporates nine faculties:

– School of Applied Psychology– School of Architecture, Civil Engineering and Geomatics– Academy of Art and Design–SchoolofLifeSciences– Academy of Music– School of Teacher Education– School of Social Work– School of Engineering– School of Business

Editorial 3

Molecular Technologies (MT) 7

Rapid Development of Stable Cell Lines with Multiple Transgenes Using Variants of Flp Recombinases and Specific Target Sites 8Understanding Biomineralization and its Implication for the Environmental Chemistry of Selenium 10Hormonal Activity of Chemicals to which Humans Are Frequently Exposed: Are there Potential Risks? 12Hormonal Activity of the UV-Filter Benzophenone-4 Determined by Target Gene Expression Profile in Zebrafish Embryos 14Activation of the JAK-STAT Pathway as Potential Molecular Basis for Diagnosis and Therapy of Macular Degeneration 16Understanding the Degradation of Hydroquinone, a Key Metabolite in Xenobiotic Degradation 18How to Detect Genetically Modified FP967 Flax by Event-Specific Polymerase Chain Reaction 20Molecular Identification of a Bacterial Production Strain Used in the Pharmaceutical Industry 22MangaCat: Flame Spray Pyrolysis Synthesis of Manganese Oxide Nanocomposite Particles for Catalysis 24 Engineered Supramolecular Surfaces for High-Throughput Polymorphism Screening of APIs 26

Therapeutic Technologies (TT) 31

LANCE: LAccase-Nanoparticle Conjugates for the Elimination of Micropollutants from Wastewater in Bioreactors 32Mistletoe-Preparations (Iscador®) Encapsulated in Silica Nanoparticles as a Therapy for Breast Cancer 34Porous Shape-Memory-Scaffolds for Bone Implants 36Best Environmental Practices in the Healthcare Sector 38INOFEA, Innovative Nanomaterials for Environmental Applications, an Ecopreneurship Venture 40Absorption Mechanisms of Poorly Water-Soluble Drugs from Self-Emulsifying Formulations in the Caco-2 Cell Model 42New Technologies for In Vitro Testing of Lipid-Based Drug Formulations 44NAPTIS: Nano- and Micro-Scaled Porous Surfaces of Titanium Implants Produced by Spark-Assisted Anodizing 46Development of a Functional Model of a Hollow Rotor Axial Flow Pump for Cardiac Assistance 48

Environmental Management (EM) 53

Waterless Mobile Toilet for Hospital Use 54 Recovering Phosphorus and Closing the Nutrient Cycle 56Water Loss Reduction – A Focus on Pressure Management 58Adapting a Membrane Bioreactor to Industrial Wastewater Treatment 60

Appendix 64

Publications 66Project Portfolio 75 Research Seminars 76 Topics and Competences 78Contact 80

Technologies for the development of innovative healthcare products and therapy solutions as well as methods and procedures for environmental sustain ability are at the center of our research interests.

Here we present the second research report of our school. Apart from training of qualified, practice-oriented Life Sciences experts we have a strong commitment to high quality research and innovation. This is achieved by projects with partners from industry and other institutions or via outsourcing of promising business ideas into spin-offs. The report illustrates projects from our three areas of research: “Mole-cular Technologies” (MT), “Therapeutic Technologies” (TT) and “Environmental Ma-nagement” (EM). The thematically focused institutes co-operate often with each other within a given project but – more importantly – are also closely interconnected with partners from within the region of Northwestern Switzerland as well as beyond.

Humans as patients, innovative therapy solutions and environmental technologies are located at the center of our work. Questions are addressed in the fields of disease diagnostics, effective therapeutic products and solutions (medicines, bio medical products) as well as procedures for sustainable industrial production. In environ-mental research we are concerned with “Clean Technologies” such as environmental biotechnology, water purification and with concepts for the use of bio wastes (Green chemicals and bio energy). Use and risks of technologies and products are critically evaluated by means of ecotoxicology, analytics and economic assessments. The quali-ty of the projects receives national attention, for example the acknowledgment shown by the inclusion of a diagnostics project at the exhibition “Future Expo” within the Swiss Innovation Forum 2010.

Researchers from our School are broadly engaged in Switzerland and abroad. Apart from cooperation in established consortia such as Biotechnet they carry out rele-vant foundation work for new consortia like the CTI network for Sustainable Engi-neering. They also organize technical and scientific workshops for exchanging prac-tice-relevant results. Scientific findings from projects are internationally presented, for example the waste water treatment technologies presented at the World Expo in Shanghai in spring 2010.

Finally we are engaged in the establishment of start-up enterprises. As a condition for the promotion of spin-offs an incubator was launched in 2009 in cooperation with the university of Basel, the canton Basel-Stadt and the EVA (the Basel life sciences start up agency). In this environment researchers from both universities can transfer ideas into young enterprises under professional coaching. A spin-off of our School took advantage of the possibility to prepare for the foundation of their company in this stimulating environment. The business idea of the INOFEA team is based on a new technology for removal of unwanted, harmful particles in water.

The spectrum of our research activities is rich and the passion and motivation of the research staff substantial – they shape the profile of our school!

Gerda HuberDirector, School of Life Sciences FHNW

Editorial

2 | 3

6 | 7

Molecular Technologies (MT)

Technologies for the synthesis and analysis of active compounds and biological systems

IntroductionThe development of cell based assays is very time consuming in general. Therefore, many laboratories apply specially designed molecular biology tools such as the FlpIn system to accelerate the generation of stable cell lines. This system is based on homo-logous recombination, using a naturally occurring recombinase (Flp) from yeast and a specifically recognised FLP recombinase target (FRT) site. In most cases the FRT site is artificially inte-grated into the genome of a mammalian cell line. Subsequently, vector constructs which harbour an identical FRT site and a gene of interest can be inserted into the genome by site directed recombination through transient expression of the Flp recom-binase. Using a fixed chromosomal locus for transgene integra-tion will most likely lead to similar expression levels of various cDNAs when the same targeting vector is used. Therefore it is assumed that variability due to potential differences in the flan-king DNA can be excluded. However, the default system only lends itself to the integration of a single transgene and is thus not suitable for the heterologous expression of multi-subunit protein complexes. To address this problem, we designed plas-mid vector constructs for the development of FlpIn host cells with two different FRT sites, which can be targeted in a selective manner by specific Flp recombinases. The system was further optimised by the use of target constructs with bidirectional and inducible promotors for the regulated expression of up to four transgenes in one cell line.

Results

Accelerated generation of FlpIn host cells with a tagged FRT siteThe commercially available FlpIn system is designed to ensure that only cells with a precisely integrated target vector into the chromosomal FRT site will survive subsequent antibiotic se-lection (e.g. hygromycin B). Nevertheless, experience has shown that false positive cells occasionally emerge after selection and in addition, host cells can become heterogeneous after continu-ed cultivation. To address these problems we have designed a construct where the chromosomal integration of the FRT site is linked to functional expression of a fusion protein consisting of the fluorescent protein AcGFP1 and a zeocin resistance mar-ker (ZeoR-AcGFP1) (Fig. 1). This modification allowed continued quality control of selected host cell clones with standard flu-orescence microscopy. In addition, this offers the potential to dramatically accelerate the selection of zeocin resistant host cells with fluorescence activated cell sorting (FACS). Upon inte-gration of a construct into the FRT site by homologous recom-bination, the expression of the respective fluorescent protein was abolished together with the zeocin resistance marker. The

novel modified FlpIn system yielded cells which could also be sorted for loss of fluorescence after selection for the newly integrated hygromycin B resistance (HygR).

Stable integration and validation of a second FRT siteThe functional expression of drug targets in mammalian cell lines often requires coordinated expression of several trans-genes. To facilitate these kinds of tasks, we successfully de-veloped a FlpIn host cell with two independently addressable FRT sites for homologous recombination. For this purpose we applied a mutated recombinase in combination with a mutated and highly specific FRT site as described by others in earlier reports [1]. For our purposes we selected a recombinase which showed high frequency of recombination with a mutated FRT site and low frequency with the wild type FRT site. Further-more, the wild type recombinase showed low frequency of re-combination with the chosen mutated FRT site. Based on this knowledge, we created a construct for stable integration of the selected FRT site into mammalian cell lines. The construct was designed to link the integration of the FRT site with functio-nal expression of DsRed and a puromycin antibiotic resistance marker (DsRed-IRES-PuroR).

Finally, a Chinese hamster ovary (CHO) cell line was generated with two independently addressable FRT sites linked to ZeoR-AcGFP1 and DsRed-IRES-PuroR, respectively (Fig. 2). Integra-tion experiments were carried out with cotransfections of a plasmid construct driving expression of the wt Flp recombi-nase and a target vector carrying a wt FRT site linked to the expression of a hygromycin B resistance marker. The desired specific integration events led to newly acquired hygromycin B resistance without affecting the puromycine resistance. Correct integration into the wt FRT site was monitored by the abolishing of green fluorescence without effects on the red fluorescence. In contrast, transfection of the mutated FRT construct together with the mutated recombinase and selection for zeocin and hygromycin B resistance, led to cells with transgene integration exclusively into the mutated FRT site. Correct integration was also confirmed by the disappearance of red fluorescence.

Conclusion and OutlookWe report the successful construction of a modified FlpIn sys-tem, which allows the fast generation of host cells with two independently addressable FRT target sites. Preliminary vali-dation experiments confirmed specific integration of target vec-tors into the wt FRT and a mutated FRT site, in combination with the wt recombinase and a mutated recombinase, respec-tively. In our laboratory we routinely apply a FlpIn compati-ble target vector with a bidirectional and inducible promoter,

School of Life Sciences

which allows concurrent integration and controlled expression of two genes. Fitting this construct with the mutated FRT site will enable expression of up to four proteins simultaneously after integration of two constructs into host cells, which carry two FRT sites. In addition, the number of expressed transgenes could theoretically be increased up to eight through the use of internal ribosome entry sites (IRES). In future we also plan to modify the system for independent activation of integrated transcripts. For this purpose cells will be fitted with compatibi-lity for tetracycline and ponasterone induced expression. This will allow integration of constructs with tetracycline inducible promoters in one FRT site and constructs with ponasterone inducible promoters in the second FRT site.

In general, the newly adapted FlpIn system for accelerated deve-lopment of cellular assays will be most suitable for laboratories working in the field of pharmaceutical drug development. Re-levant applications will be for High-Throughput Screening and subsequent compound profiling during the lead optimisation process. In addition, potential uses are possible for recombi-nant protein expression and development of cell-based sensor systems for diagnostics and ecotoxicology.

References:[1] Voziyanov Y, Konieczka JH, Francis Stewart A, Jayaram M. Stepwise Mani-

pulation of DNA Specificity in Flp Recombinase: Progressively Adapting Flp to

Individual and Combinatorial Mutations in its Target Site. J. Mol. Biol.

2003;326 (1):65–76

[2] Invitrogen. System Manual: Flp-In System. For Generating Stable Mamma-

lian Expression Cell Lines by Flp Recombinase-mediated Integration. Availa-

ble under: http://biochem.dental.upenn.edu/GATEWAY/Vector_manual/flpin-

system_man.pdf

Research Focus Area: Molecular Technologies (MT)

Project Team: René F. Prétôt, Hugo Albrecht (Institute for Chemistry and Bioanalytics, School of Life Sciences FHNW)Eleonora Chiri, master student at Università Degli Studi Di Palermo, Faculty of Science

Partner: Università Degli Studi Di Palermo

Funding: Erasmus Program (European Region Action Scheme for the Mobility of University Students)

Economic efficiency and benefit to society: The modified FlpIn system will speed up the construction of cell based assays in general and facilitate the heterologous expression of multi-subunit protein complexes in mammali-an cells, thereby reducing development costs.

Rapid Development of Stable Cell Lines with Multiple Transgenes Using Variants of Flp Recombinases and Specific Target Sites

A FlpIn host cell with two independently addressable Flp recombinase target sites (FRT sites) has been generated for site directed integration of DNA target vectors. This cell line has the potential to dramatically accelerate the development of highly sophisticated cell-based assay systems through controlled expression of multiple transgenes.

René F. Prétôt, Eleonora Chiri, Hugo AlbrechtSchool of Life Sciences FHNW

Keywords: Flp recombinase, site directed recombination, cell based assays, High-Throughput Screening (HTS)

8 | 9

expression of ZeoR-AcGFP1 fusion protein from the integrated FRT site

Figure 1

plasmid to be integrated into the chromosomal FRT site

ATG PSV40 Amp pUC ori

ZeoR-AcGFP1

- B PCMV

Amp pUC ori

Amp pUC ori

expression of hygromycinB resistance gene

expression of gene of interest

no promoter no start codon

Flp recombinase

FRT

GOI FRT HygBR

ATG PSV40

- B FRT HygBR PCMV

GOI ZeoR-AcGFP1 FRT

Figure 2

A B CHO TetOn FRTZeoAcGFP/FRT*PuroIRESDsRed CHO wt

Figure 1: Integration of a FRT harbouring plasmid via homologous recombination During the recombination process, the entire plasmid containing the gene of interest is integrated into the chromosomal FRT site. This integration event deprives the coding sequence for the zeocin resistance-AcGFP1 fusion protein of its promoter as well as its start codon.

Figure 2: Analysis of cells with two intact FRT sites by FACS X-axes indicate green fluorescence, Y-axes indicate red fluorescence with each dot representing a single analysed cell. A) CHO wt cells show matched green and red fluorescence B) CHO cells with integrated ZeoR-AcGFP1 and DsRed-IRES-PuroR elements show increased green and red fluorescence.

Figure 1: Transmission microscopy image of a hCMEC/D3 cell monolayer (left panel). Confocal microscopy analysis of hCMEC/D3 cells demonstrating claudin-5 immunostaining (right panel) [4].

School of Life Sciences 10 | 11

IndroductionSelenium has been referred to as “the essential toxin” due to its ambivalent character as essential yet highly toxic trace element in human and animal health. Selenium deficiency is a problem of worldwide concern, affecting an estimated 0.5 to 1 billion people [1], whereas selenium toxicity has been ob-served on large scale, e.g. in the San Joaquin valley (California, USA) [2], representing one of the most productive agricultural areas in the USA. Selenium is heterogeneously distributed in the earth’s crust resulting in certain countries suffering from both selenium deficiency and toxicity effects, although affected regions are sometimes separated by a distance of only a few kilometres. Thus, environmental problems related to selenium are pro-blems of improper distribution. Within Europe, for example, Central-Eastern England shows a highly heterogenous sele-nium soil distribution, with deficient (<0.1 mg/kg) and sele-niferous (>0.5 mg Se/kg) soils directly adjacent. The global selenium cycle is influenced crucially by anthro-pogenic activities, mainly by combustion of fossil fuels, non-ferrous metal melting and agricultural fertilization. When se-lenium enters the aquatic environment, trace concentrations of selenium (>5 µg/L) can lead to disastrous toxicity effects on water birds and fish due to its tendency to bioaccumulate. The toxicity of selenium is strongly dependent on its specia-tion, i.e. the specific chemical form it is present in. Compared to water soluble oxyanions (i.e. selenite, selenate), elemental selenium is considered less toxic. The ability of microorga-nisms to reduce selenium oxyanions to elemental selenium is widespread in the environment. Specialized dissimilatory reducers can “respire” selenium oxyanions to produce energy for growth, whereas other microbial groups can reduce sele-nium oxyanions to elemental selenium, yet do not gain energy by the reduction. Therefore, various remediation approaches try to use such microorganisms for the biotreatment of sele-nium contaminated waters [3]. It has been observed that ele-mental selenium formed by these microorganisms does not crystallize to larger particles, yet consists of almost perfectly spherical nanoparticles (mostly around 300–500 nm in dia-meter) (Fig. 1), whereas chemical synthesis results in preci-pitates of dissimilar morphology. In the aquatic environment, such bionanominerals do not settle from solution, thus they are subject to transport processes within the water bodies, re-oxidation, uptake and assimilation by biota. The probable reason for the tendency of biogenic selenium to remain in solution suspended as nanoparticles is an organic polymer layer modifying the surface, preventing crystallization and conferring the selenium core with physico-chemical proper-ties different from particles without such a layer (Fig. 2).

Results Until recently, it was not known which molecules form the organic polymer layer around the nanoparticles. We therefo-re investigated the presence of proteins within the organic polymer layer by means of Capillary Liquid Chromatography-Electro Spray Ionization-tandem Mass Spectrometry (LC-ESI-MS/MS). We studied two distinct dissimilatory selenate reducers, i.e. Bacillus selenatarsenatis and Sulfurospirillum barnesii, and one non-dissimilatory selenite reducing species, Rhodospirillum rubrum. In a first step, selenium nanoparti-cles potentially bearing a protein layer were isolated from residual biomass by low speed density based centrifugation. Using centrifugation in polytungstate solution with a density

Understanding Biomineralization and its Implication for the Environmental Chemistry of Selenium

Selenium is a trace element that is essential to humans, yet toxic at elevated concentrations. Water soluble, toxic forms of selenium can be converted to non-toxic, elemental selenium by different microorganisms. This so called “bio-mineralized” selenium is particular regarding its behavior in the environment, in contrast to conventionally produced selenium.

Markus Lenz, Philippe F.X. Corvini School of Life Sciences FHNW

Keywords: Trace element cycling, bioremediation, biomineralization, bionanomineral

of 3 g/cm³, selenium particles were pelleted (density of 4.8 g/cm³), whereas biomass can be removed from the superna-tant (Fig. 3). Subsequently, proteins were subjected to sodi-um dodecyl sulfate (SDS) polyacrylamid gel electrophoresis, followed by LC-ESI-MS/MS for protein identification. For the first time we were able to qualitatively characterize the protein fraction associated with selenium bionanominerals of different microbial origin. For all microorganisms used, a number of proteins with diverse cellular functions were iden-tified. Consequently, one can postulate that the association of proteins is a general feature of biogenic selenium particles. Furthermore, for the first time, we were able to identify pro-teins with an anticipated direct functional role in selenium reduction in spatial association with the bionanominerals formed. Such proteins include specialized oxyanion reduc-tases and proteins involved in electron transport during mi-crobial respiration. It has to be stressed that the proteins found associated with the selenium particles are inseparable by numerous centrifugation and washing steps and are thus also expected to be associated under environmental condi-tions. Such surface modification with proteins will strongly influence sedimentation/transport processes and eventually the environmental fate of selenium. Currently, experiments conducted at the FHNW aim to quantify further organic po-lymers associated with the selenium particles and the extent of altered environmental behavior of biogenic selenium in contrast to chemically synthesized selenium.

Conclusion and Outlook This study showed that microbially formed selenium parti-cles are modified by a series of proteins associated with them, independent from the physiology of the microorganisms that produce them (dissimilatory and non dissimilatory selenium reducing, strict/facultative anaerobes, gram positive/negati-ve) and their conditions of formation (salt, selenium source, phototrophic/dark conditions, etc.). These proteins form high affinity complexes that do not dissociate upon repeated cen-trifugation/washing. Future studies will aim to investigate whether the protein layer on selenium particles is responsible for the particular environmental fate observed for biogenic selenium. In nature, biodegradation of such protein modifi-cation might occur – altering the environmental fate in turn – hence opening a fascinating yet challenging future field of

interdisciplinary biogeochemical research. It is a future goal to control selenium biomineralization for (bio)nanotechnolo-gy purposes. For instance, if selenium can be produced in an easily recoverable form, i.e. in the form of large, pure particles, operational costs of remediative systems can be reduced in turn, since selenium is a valuable product. Such recovered se-lenium could then be re-used to increase selenium nutritional levels in deficient areas, eventually closing the selenium cycle.

References: [1] Haug A, Graham RD, Christophersen OA, Lyons GH. How to use the world's

scarce selenium resources efficiently to increase the selenium concentration in

food. Microbial Ecology in Health and Disease 2007;19 (4):209–28.

[2] Presser TS, Luoma SN. Forecasting selenium discharges to the San Francisco

Bay-delta estuary: Ecological effects of a proposed San Luis Drain extension-

Available at http://pubs.usgs.gov/pp/p1646/pdf/pp1646.pdf 2007.

[3] Lenz M, Lens PNL. The essential toxin: the changing perception of selenium

in environmental sciences. Science of the total Environment 2009;407 (12):3620–33.

[4] Chen T, Wong Y-S, Zheng W, Bai Y, Huang L. Selenium nanoparticles fabricated

in Undaria pinnatifida polysaccharide solutions induce mitochondria-mediated

apoptosis in A375 human melanoma cells. Colloids and Surfaces B: Biointerfaces

2008;67 (1):26–31.

Research Focus Area:Molecular Technologies (MT)

Project Team: Markus Lenz, Philippe F.X. Corvini (Institute for Ecopreneur-ship, School of Life Sciences FHNW)

Partner: Suzette Moes (Biozentrum, University of Basel)

Funding: Swiss National Science Foundation (SNSF, 200021–126899)

Economic efficiency and benefit to society:Minerals that are produced by living organisms – so called biominerals – often bear particular physico-chemical cha-racteristics when compared to chemically synthesized ana-logues. Recently efforts have been made to economically explore biomineralization for the production of valuable materials (e.g. nanoselenium as anti-cancer agent [4]). Since biological processes are mostly catalysed at physiological conditions, exploring biomineralization might represent an alternative to energy consuming chemical synthesis of na-nomaterials.

Figure 1: Scanning Electron Microscopy picture of selenium particles for-med by Bacillus selenatarsenatis

Figure 2: Core-organic polymer layer model of biogenically formed selenium precipitates

Figure 3: Purification of biogenic selenium particles by a density density based centrifugation in polytungstate: selenium sinks to the bottom (right), whereas biomass swims up (selenium free control, left)


IndroductionFlame retardants, antimicrobial agents and phthalates have nu-merous applications in various products. They finally may end up in the environment by leaching from products and from the product’s use. Analytical chemical measurements documented that residues occur in human tissues and the environment due to high usage, relative persistence and also because of their high lipophilicity. Brominated flame retardants are particularly rele-vant. They are used in construction materials, furniture, plastics, electronic equipment, textile, and other products. Until recently, polybrominated diphenyl ethers (PBDEs) accounted for a lar-ge proportion of flame retardants used in polyurethane foam and electronic applications. But penta-, octa-, and deca-BDE have been banned or voluntarily phased out in Europe because of their persistence and bioaccumulation. Other flame retar-dants currently used are tetrabromobisphenol A (TBBPA) and hexabromocyclodecane (HBCD). The antimicrobial compounds triclosan (TCS) and triclocarban (TCC) are frequently detected in the environment. They are extensively used in consumer pro-ducts, including household and personal care products, soaps and textiles. Phthalates have a wide spectrum of industrial and commercial applications, including use as plasticizers, solvents and in flexible plastics (food and beverage packaging), and may occur in some medicines. Phthalates are not covalently bound to plastic products and therefore may leak out to contaminate food products. Some phthalates are developmental and reproductive toxicants in laboratory animals showing adverse effects on the male reproductive system [1].

Previous studies in humans and animals have shown that some of these compounds negatively interfere with the hormonal sys-tem and are therefore called endocrine disrupters. Exposure to endocrine-disrupting chemicals may result in adverse effects on reproduction, foetal/child development, tumor development and other physiological processes. Potential risks of such con-taminants to human reproductive and developmental health are supported by reports on the decrease in anogenital distance among male infants with prenatal phthalate exposure [2], effects on germ cells in male rodent gonads by phthalates, and effects posed by brominated flame retardants [3].

An important mechanism of endocrine disruption is direct in-teraction with hormone receptors as agonists or antagonists. For instance, anti-androgenic compounds can disrupt the action of steroidal androgens in the foetus, with irreversible conse-quences (incomplete masculinization and malformations of the reproductive organs) [4].

The aim of this study was to analyse selected flame retardants, antimicrobials and phthalates including:

– tetrabromobisphenol A (TBBPA), – hexabromocyclodecane (HBCD)– penta-bromodiphenylether (BDE) (BDE-100) and hexa-BDE

(BDE-155)– the antimicrobial compounds TCS and TCC – eight phthalates

for their androgenic and anti-androgenic activity in vitro in the MDA-kb2 cell line. This widely applied human mammary carcinoma cell line expresses endogenous androgen receptor (hAR) and an androgen-responsive luciferase reporter plasmid driven by the mouse mammary tumor virus promoter (MMTV) [5]. The androgen receptor can act through the MMTV promoter. Therefore compounds that act through the androgen receptor activate the MMTV luciferase reporter. After incubation of the MDA-kb2 cells with dehydrotestosterone or a test compound (androgenic activity) or the co-exposure of the cells with dehy-drotestosterone and a test compound (anti-androgenic activity) the amount of expressed luciferase is measured. One advantage of this cell system is the high sensitivity and the high repro-ducibility of results because each cell is derived from a single clone and the expression of the luciferase reporter is stable over an extended time and number of passages. The intra-assay and inter-assay variability is low. Additionally, the cells are easy to cultivate and assays rapidly performed in 96-well plates [5]. Although humans are exposed to these contaminants, the toxi-cological risks are unclear. By using this in vitro test system, we provide data that may help in the risk assessment of these chemicals.

ResultsNo or only weak androgenic activity was observed in all te-sted compounds. TBBPA showed weak anti-androgenic activity, which is demonstrated for the first time. The flame retardants HBCD, BDE-100 and BDE-155 enhanced the dehydrotestostero-ne-dependent activation of androgen receptor-responsive gene expression but exhibited little or no agonistic activity (Fig. 1). The enhancement of the dehydrotestosterone response reached 150%, which was similar to the antimicrobials (TCS up to 180% and TCC up to 130%) (Fig. 2). This enhancement of androgenic activity represents a novel mode of action of the endocrine ac-tivity of flame retardants. In contrast, most phthalates showed anti-androgenic activity. Butylbenzyl phthalate (BBP), dibutyl phthalate (DBP) and diethyl phthalate (DEP) showed strong an-tiandrogenicity (Fig. 3). An up to 80% inhibition of the dehydro-testosterone response was detected at the highest concentra-

Hormonal Activity of Chemicals to which Humans Are Frequently Exposed: Are there Potential Risks?

Frequently-used chemicals in materials, household products and personal care products are analysed for hormonal activity in cell culture. Some flame retardants and the antimicrobials triclosan and triclocarban are found to increase androgenic activity of dehydrotestosterone, whereas phthalates have anti-androgenic activity.

Karl Fent, Verena ChristenSchool of Life Sciences FHNW

Keywords: Flame retardants, biocides, phthalates, hormonal activity, androgenic activity, anti-androgenic activity, cell systems

tions of these three phthalates. The anti-androgenic activity of diethylhexyl phthalate (DEHP), dipentyl phthalate (DPP), dime-thyl phthalate (DMP), and the DEHP metabolite monoethylhexyl phthalate (MEHP) was lower. Di-octyl-phthalate (DOP) did not show any anti-androgenic activity. The data are published in detail by Christen et al. [6].

Conclusion and OutlookOur in vitro cell culture study demonstrates for the first time a weak anti-androgenic activity of TBBPA and an enhancement of the androgenic activity of dehydrotestosterone by HBCD, BDE-100 and BDE-155, which represents a novel mechanism of hormonal activity of flame retardants. Currently, we are inves-tigating the activity of compound mixtures, since human and

environmental exposure occurs not by single compounds, but as mixture. We will analyse the mixture activity of phthalates by using the concentration addition model. With this model, mixtures can be analysed with regard to their synergistic or antagonistic activity. We will start with binary mixtures follo-wed by mixtures of three and four phthalates. Further studies should show whether the activities found in our in vitro study also occur in vivo, and whether there are effects on reproduc-tion. These data will help in the toxicological and environmen-tal risk assessment of these chemicals.

References:[1] Sharpe RM. Phthalate exposure during pregnancy and lower anogenital index

in boys: wider implications for the general population? Environ Health Persp.

2005;113 (8): A504–505.

[2] Swan SH, Main KM, Liu F, Stewart SL, Kruse RL, Calafat AM, Mao CS, Redmon

JB, Ternand CL, Sullivan S, Teague JL. Decrease in anogenital distance among

male infants with prenatal phthalate exposure. Environ Health Persp. 2005;113

(8):1056–106

[3] Legler J. New insights into the endocrine disrupting effects of brominated

flame retardants. Chemosphere 2008;73 (2): 216–222.

[4] Wilson VS, Blystone CR, Hotchkiss, AK, Rider CV, Gray LE. Diverse mechanis-

ms of anti-androgen action: impact on male rat reproductive tract development.

Int J Androl. 2008;31 (2): 178–187

[5] Wilson VS, Bobseine K, Lambright CR, Gray LE, Jr. A novel cell line, MDA-kb2,

that stably expresses an androgen- and glucocorticoid-responsive reporter for

the detection of hormone receptor agonists and antagonists. Toxicol Sci.2002;66

(1): 69–81.

[6] Christen C, Crettaz P, Oberli-Schrämmli A, Fent K. Some flame retardants and

the antimicrobials triclosan and triclocarban enhance the androgenic activity in

vitro. Chemosphere 2010; 81 (10): 1245–1252.


Project Team: Karl Fent, Verena Christen (Institute for Ecopreneurship, School of Life Sciences FHNW)

Partner: Pierre Crettaz, Aurelia Oberli-Schrämmli (Federal Office of Public Health, FOPH)

Funding: Federal Office of Public Health (FOPH)

Economic efficiency and benefit to society:The use of chemicals exerting hormonal activity represents a potential risk to the health of humans and the environment. Knowledge of such compounds and their activity is impor-tant for risk assessment and for the safe handling of these compounds.

Figure 1: Flame retardants HBCD, BDE-100 and BDE-155 enhance androgen activity

Figure 2: Biocides triclosan and triclocarban enhance androgen activity.

Figure 3: Phthalates show antiandrogenic activity


IntroductionToxicological and ecotoxicological sciences at FHNW are concerned with the study of potential impacts of chemicals on human health and the environment. These data will ulti-mately help develop better risk assessment of compounds and products in which the chemicals occur. In our group we focus on chemicals to which humans and aquatic organisms are exposed and whose potential effects are not fully known. The research aims to elucidate the potential toxicity and me-chanisms of chemicals in personal care products, pharma-ceuticals and other materials.Chemicals that absorb UV-irradiation are called UV-filters and are added to consumer products including sunscreens and cosmetics (creams, lipsticks, lotions, fragrances, skin lotions, hair sprays and shampoos). In addition, they find application in the UV-protection of numerous materials and products. Consequently, they ultimately enter the aquatic environment directly or indirectly via wastewater, where re-sidues of several UV-filters have been detected. Among UV-filters detected in Swiss river waters benzophenone-4 (BP-4) was found to be most prevalent [1]. BP-4 is water-soluble and occurred up to 3000 ng/L and 1480 ng/L in lakes and waste-water in Spain, respectively [2]. In addition, more lipophilic UV-filters are accumulated in aquatic animals including fish and cormorants [1].To date, potential adverse effects of BP-4 exposure in humans and in aquatic organisms remain elusive. Our previous in vi-tro studies showed multiple hormonal activities including estrogenic, antiestrogenic and antiandrogenic activities [3]. This indicated that hormonal activities of BP-4 remain to be further investigated in vivo for assessing their potential risk to aquatic organisms. In the light of the potential endocrine-disrupting activity of BP-4, we evaluate the effects at mole-cular level on the expression of genes involved in hormonal pathways in early developmental stages of zebrafish (Danio rerio). The aim was to elucidate mechanisms of action of BP-4 and its potential effects on hormone signalling as well as on steroidogenesis in fish embryos. Embryos were exposed up to 3 days after hatching to con-centrations of 30 and 3000 µg/L BP-4 in the laboratory. Each treatment of 30 µg/L and 3000 µg/L of BP-4, as well as the control, consisted of six replicates. The embryos were con-tinuously incubated at 27±1 °C for approximately 5 days throughout gastrulation, organogenesis and early larval development using a static-water renewal procedure by re-placing the appropriate BP-4 concentrations in new beakers every 48 h. At the end of the exposure (120 hpf) eleuthero-embryos were anaesthetized and a total of 15 eleuthero-embryos per replicate were pooled. Total RNA was extracted

and used for qRT-PCR analysis. To analyse for effects of BP-4 we followed a targeted gene concept by focusing on mRNA expression levels determined by quantitative reverse tran-scription PCR (qRT-PCR). We selected oestrogen-related genes (vitellogenin 1, vitellogenin 3), sex-steroid receptors, (oestrogen receptor alpha, oestrogen receptor beta 1, andro-gen receptor) and genes involved in formation of steroid hor-mones (hydroxysteroid 17-ß dehydrogenase-3, P450aromA, P450aromB) after exposure during embryogenesis. By app-lying this approach we aimed to elucidate the toxicological profile for use in environmental risk assessment.

ResultsIn exposed free-swimming eleuthero-embryos we found the transcripts of vtg1 (Fig. 1), vtg3, esr1, esr2b (Fig. 2), hsd17ß3, cyp19b (Fig. 3), cyp19a, hhex and pax8 induced at 3000 µg/L BP-4. The gene vtg1 encodes vitellogenin, a precursor for the yolk sac protein, which is expressed under the regulation of estrogens. Vitellogenin is a known and well established bio-marker for estrogenic activity. Among the altered genes, the estrogen receptor esr2b transcript (Fig. 2) was significantly induced at 30 µg/L BP-4. The transcripts of the ar remained unaffected. BP-4 led to a significant induction of aromatase cyp19b (Fig. 3) and cyp19a transcripts at 3000 µg/L, which points to an effect on steroidogenesis. The aromatases con-vert testosterone to estradiol. The magnitude of mRNA induc-tion for all the investigated transcripts was below 2.5-fold. This indicates a low estrogenic activity of BP-4. The induction of hhex and pax8 suggests an interference with early thyroid development in addition to the estrogenic activity.

Hormonal Activity of the UV-Filter Benzophenone-4 Deter-mined by Target Gene Expression Profile in Zebrafish Embryos

UV-absorbing chemicals such as benzophenone-4 (BP-4) are frequently used in cosmetics, personal care products and in the protection of materials. They enter the aquatic environment, but the potential toxicological mode of action and the adverse effects are unknown. In this study we analysed molecular effects of BP-4 in zebrafish embryos. The data will be used to improve environmental risk assessment.

Karl Fent, Sara Zucchi, Nancy BlüthgenSchool of Life Sciences FHNW Keywords: Personal care products, benzophenone-4, zebrafish, molecular effects, hormonal activity

Conclusion and OutlookIn conclusion, the transcription profile reveal that BP-4 in-terferes with the expression of genes involved in hormonal pathways and steroidogenesis [4]. The effects of BP-4 obser-ved at high concentrations point to an estrogenic activity in embryos. The fact that BP-4 interferes with the sex hormone system of embryos may have implications for the environ-mental risk assessment of this UV-filter. At the same time the data suggest that a better risk characterization of this com-pound should be performed with the focus on human health.

References:[1] Fent K, Zenker A, Rapp M. Widespread occurrence of estrogenic UV-filters

in aquatic ecosystems in Switzerland. Environmental Pollution 2010;158

(5):1817–24.

[2] Rodil R, Quintana JB, López-Mahía P, Muniategui-Lorenzo S, Prada-Rodrí-

guez D. Multi-residue analytical method for the determination of emerging pol-

lutants in water by solid-phase extraction and liquid chromatography-tandem

mass spectrometry. Journal of Chromatography A 2009;1216 (14):2958–69.

[3] Kunz PY, Fent K. Multiple hormonal activities of UV filters and comparison

of in vivo and in vitro estrogenic activity of ethyl-4-aminobenzoate in fish.

Aquat. Toxicol. 2006;79 (4):305–24.

[4] Zucchi S, Nancy Blüthgen N, Ieronimo A, Fent K. The UV-absorber benzo-

phenone-4 alters transcripts of genes involved in hormonal pathways in zebra-

fish (Danio rerio) eleuthero-embryos and adult males. Toxicol. Appl. Pharmacol.

2011;250 (2):137–46.


Project Team: Karl Fent, Sara Zucchi, Nancy Blüthgen, Andrea Ieronimo (Institute for Ecopreneurship, School of Life Sciences FHNW)

Partner: none

Funding: Funded by Swiss National Science Foundation (SNSF, 31003A-121829) and Federal Office for the Environment (FOEN)

Economic efficiency and benefit to society:The use of chemicals which exert hormonal activity poses a potential risk for human health and the environment. This is particularly important for chemicals in personal care products. Knowledge of such compounds and their activity allows the estimation of potential risks and can guide safe handling of these compounds.

Figure 1: Induction of vitellogenin 1 (vtg1) transcript by BP-4 exposure in zebrafish embryos

Figure 2: Induction of estrogen receptor (esr2b) transcript by BP-4 exposure in zebrafish embryos

Figure 3: Induction of P450aromB (cyp19b) transcript by BP-4 exposure in zebrafish embryos


IntroductionAge-related macular degeneration (AMD) is a degenerative dis-ease of the macula leading to irreversible visual impairment and blindness, affecting nearly 50 million people worldwide [1]. Clinically and histologically, AMD can be classified into two major subtypes: dry and wet AMD. Dry AMD is characte-rized by macular changes consisting of abnormalities of the retinal pigment epithelium (RPE) and drusen, photoreceptor dysfunction and degeneration. The key feature of wet AMD is choroidal neovascularization (CNV), described as the growth of new blood vessels from the choroid into the region under-lying the RPE. The etiology of AMD is unclear but includes hereditary components. The earlier stage of AMD treatment is limited to risk factor management. Major changes during AMD disease development occur in the outer retina, affecting the photoreceptors, the RPE and the Bruch’s membrane.Recent studies have shown that immune mechanisms play an important role in the development of AMD. RPE cells consti-tute a specialized phagocytic system similar to that of macro-phages. In addition, the RPE can modulate immune response through pro-inflammatory cytokine production, including IL-1β, IL-6 and tumor necrosis factor alpha (TNF-α). RPE cells also produce pigment epithelial growth factor, which decre-ases and increases IL-12 and IL-10 production, respectively. RPE plays a critical role in photoreceptor renewal, since de-fects lead to photoreceptor death and retinal degeneration. Although it is known that dysfunctions of RPE cells are the major cause of degenerative diseases including AMD, the ex-act molecular mechanisms of these processes remain to be elucidated.

The JAK-STAT pathway has been implicated in the control of cell progression, cell survival and cell death. STATs are a class of transcription factors activated upon tyrosine phosphory-lation. The cytoplasmic protein family functions by signaling and transcription factor regulation and by participating in normal cellular responses to cytokines and growth factors [2].The JAK-STAT pathway can lead to angiogenesis by triggering angiogenic factor production including VEGF and MMP. Our recent research has shown abnormal STAT3 activation in choroidal neovascular membranes of AMD patients [3]. Therefore we chose to focus on the expression and regulati-on of the JAK-STAT pathway using the ARPE-19 cell line, a signaling cascade not extensively studied in the context of AMD pathogenesis. Our results demonstrate STAT3 specific up-regulation by interferon-α (IFN-α) and IL-6. We also found a strong activation of STAT1 induced by interferon-γ (IFN−γ). Furthermore, in AMD where multiple growth factor pathways are involved, STAT proteins, in particular STAT3 because of

its central regulatory role, represent an attractive develop-ment target for potentially effective AMD therapies.

MethodsElectromobility shift assay, immunofluorescence staining and flow cytometry were used to evaluate the JAK-STAT path-way in the ARPE-19 cell line.

Results

Effect of IFN-α and IFN-γ on HLA modulationWe first tested the effects of IFN-α and IFN-γ on HLA-ex-pression on ARPE-19 cells by flow cytometry. We found that untreated ARPE-19 cells express high levels of MHC class I (Fig. 1). When the cells were treated with 500 U/ml of IFN-α or with 500 U/ml of IFN-γ for 48 hours, we observed the up-regu-lation of MHC class I expression (Fig. 1). Moreover, 48 hours' treatment with 500 U/ml of IFN-γ induced the expression of MHC class II molecule HLA-DR (Fig. 1), whereas IL-4 and IL-6 treatments did not have any effect on the expression of MHC molecules on ARPE-19 cells (data are not shown).

Intracellular signalingWe further studied the potential effects of IFN-α, IFN-γ, IL-6 and IL-4 on the activation of STAT proteins in ARPE-19 cells. To this end, the electromobility shift assay (EMSA) was car-ried out on nuclear extracts and probed with four oligonu-cleotides O15, M67, βCas and Cε, which recognize all known STAT proteins. Untreated cells were used as negative controls and for positive controls Daudi, HepG2, THP-1 and Colo 205 cell lines were treated with the appropriate cytokines. Stimu-lation with IFN-α activated a STAT1-STAT2-p48 complex that binds to the O15 probe. All positive nuclear extracts of sti-mulated cells were verified in SuperShift experiments using specific anti-STAT antibodies.

Activation of the JAK-STAT Pathway as Potential Molecular Basis for Diagnosis and Therapy of Macular Degeneration

Age-related macular degeneration (AMD) is a degenerative disease of the macula leading to irreversible visual impairment and blindness. Retinal pigment epithelial cells play a pivotal role in the development of AMD. Understanding the underlying molecular mechanisms is a prerequisite for developing therapeutic strategies for the treatment of this disease.

Elizaveta Fasler-Kan, Daniel GygaxSchool of Life Sciences FHNW

Keywords: JAK-STAT pathway, ARPE-19, age-related macular degeneration, MHC expression

When tested on βCas probe, all nuclear extracts stimulated with human IL-4 showed activated STAT6. A very strong acti-vation of STAT1:1 homodimer shift was observed after IFN-γ and IFN-α stimulation on M67 probe. Furthermore, IL-6 acti-vated STAT3 in the ARPE-19 cells. The STAT1:STAT3 complex after stimulation with IFN-α appeared as a band of interme-diate intensity migrating slightly slower than STAT1:1 homo-dimers. Both STAT1:1 homodimers and STAT1:3 heterodimers could be SuperShifted with antibodies specific for STAT1. STAT3:3 homodimers and STAT1:3 heterodimers could be SuperShifted with antibodies specific for STAT3; whereas STAT2-specific antisera had no effect (data are not shown). Untreated cells revealed no activation of STAT proteins. To sum up, our EMSA data showed that the intracellular STAT pathways are activated in ARPE-10 cells in response to cytokine treatment, with IFN-γ activating STAT1 and IFN-α activating STAT1, STAT2 and STA3. Similarly, Il-6 Activated STAT3 and Il-4 activated STAT6.

Immunofluorescence stainingIn the next set of experiments we confirmed activated STAT protein nuclear localization by immunofluorescence confocal laser microscopy. The immunofluorescence assay confirmed the data obtained from the SuperShift experiments. Repre-sentative results of the nuclear expression of STAT1 proteins are shown in Fig. 2. ARPE-19 cells were left untreated (left panel) or were treated with IFN-γ (500 U/ml) for 20 minutes (right panel). Strong nuclear localization of STAT1 was obser-ved in the cells after treatment with IFN-γ (Fig. 2, red color). The cytoplasm was stained with anti-cytokeratin 8 antibo-dies (green color). Strong translocation of STAT6 was obser-ved after incubation with Il-4 and IFN-α induced the nuclear translocation of both STAT1 and STAT3 (data are not shown).

Conclusion and OutlookIn the present study, JAK-STAT pathway activation in ARPE-19 cells was demonstrated with various cytokines using the techniques of EMSA, flow cytometry and immunofluorescence. Our current data show that multiple STAT proteins (STAT1, STAT2, STAT3 and STAT6) are activated in ARPE-19 cells upon stimulation with cytokines.The individual STAT activation patterns were obviously cy-tokine-specific. We foresee the ARPE-19 cell line as a model for screening of novel drugs through interference with the JAK-STAT signaling pathway and for future research using primary RPE culture.

References:[1] Klein R, Klein BP, Linton KLP. Prevalence of age-related maculopathy. The

Beaver Dam Eye Study. Ophthalmology. 1992, 99(6):933–943

[2] Darnell JE Jr, Kerr IM and Stark GR. Jak-STAT pathways and transcripti-

onal activation in response to IFNs and other extracellular signalling proteins.

Science. 1994; 264(5164):1415–1421

[3] Fasler-Kan E, Wunderlich K, Hildebrand P, Flammer J, Meyer P. Activated

STAT3 in choroidal neovascular membranes of patients with age-related ma-

cular degeneration. Ophthalmologica. 2005; 219(4):214–221


Project Team: Elizaveta Fasler-Kan and Daniel Gygax (Institute for Chemi-stry and Bioanalytics, School of Life Sciences FHNW)

Partner:Natasha Barteneva (Immune Diseases Institute and Pro - gram in Cellular and Molecular Biology, Children Hospital of Boston, Department of Pathology, Harvard Medical School) and Peter Meyer (Institute of Pathology, University Hospital Basel)

Funding: Internal Funding

Economic efficiency and benefit to society:Our data will be used for further studies in diagnostics and therapy of age-related macular degeneration. The full work is published in International Journal of Interferon, Cytokine and Modulator Research, 2010, 2010(2):127–136.

Figure 1: Up-regulation of MHC expression. Black histogram: control; grey: MHC expression; white: MHC expression + INF-α treatment. Axis Y- rel. cell counts, X- rel. intensity

Figure 2: Nuclear localization of STAT1 observed by confocal laser microsco-py after IFN-γ treatment: STAT1 red and Cytokeratin green color


IntroductionHydroquinone (HQ) is a key compound occurring in the degra-dation of diverse xenobiotics such as building blocks in poly-carbonate production (bisphenol A, BPA) [1], flame retardants (tetrabromobisphenol A) [2], intermediates of pharmaceuticals, ammunition and dye production (4-nitrophenol) [3] and de-gradation products of industrial detergents (branched nonyl-phenols) [1]. HQ degradation can either proceed via direct ring cleavage of the benzene ring or ring cleavage of 1,2,4-trihydro-xybenzene. The two classes of enzymes that can be involved in these two pathways, extradiol and intradiol dioxygenases respectively, represent two evolutionary distinct classes of en-zymes. Extradiol dioxygenases are more versatile, as they usu-ally cleave a wider variety of substrates and are also involved in a wider variety of metabolic pathways, including those de-grading non-aromatic compounds [4]. Intradiol dioxygenases, which cleave HQ subsequent to ring hydroxylation, have been quite well characterized and even reaction mechanisms have been proposed. For HQ extradiol dioxygenases, numerous genes have been found which were attributed to HQ dioxygenase activity, but data on the biochemical properties of these en-zymes are scarce, as only one member of this family has been characterized.Quinonoide compounds derived from HQs are agents of oxi-dative stress and have a high toxic potential. A further rapid metabolization of this intermediate is necessary to minimize exposure time and thus to avoid damage to the cell. Therefore the elucidation of HQ degradation can lead to a better under-standing of the mechanisms that prevent oxidative stress.Sphingomonas sp. strain TTNP3 is a bacterium isolated from sewage sludge. It is known to degrade endocrine disrupting chemicals such as bisphenol A and several branched nonylphe-nol isomers [1]. It belongs to the genus Sphingomonas (sensu latu), which comprises strictly aerobic heterotrophic, gram-ne-gative bacteria. Its members are frequently isolated from envi-ronmental samples and studied as degraders of (substituted) polyaromatic hydrocarbons, furan, dibenzo-p-dioxin, carbazol, estradiol and numerous other compounds [5].

Results

Biochemical characteristics of HQ dioxygenase from 'Sphingomonas' sp. strain TTNP3HQ dioxygenase from strain TTNP3 could be purified to homo-geneity by sequential chromatographic purification steps. To identify enzymatically active fractions, HQ ring cleavage acti-vity was routinely measured by following the formation of the reaction product 4-hydroxymuconic semialdehyde at 320 nm at pH 7.0. The purification was not straightforward however, as

the enzyme readily lost activity when handled in the absence of 4-hydroxybenzoic acid, a competitive inhibitor of the enzyme. Moreover, it was found to be relatively unstable in the presence of oxygen but could be stabilized under an argon atmosphere. The enzyme activity on a broad range of substrates was tested by means of an oxygen-sensitive Clarke-type electrode (Table 1).Analysis of the purified enzyme by inductively coupled plasma mass spectrometry revealed it to contain iron. Based on a de-termination of its molecular mass by size exclusion chromato-graphy, it was calculated to contain 1.4 moles of iron per mole enzyme. Moreover, inactivation studies with chelators of ferrous iron and hydrogen peroxide, which oxidizes ferrous iron to fer-ric iron, strongly indicated that the enzyme contained ferrous iron in its catalytic center. Further analysis by SDS-PAGE produ-ced two bands corresponding to molecular masses of 18.5 and 38 kDa, which indicates them to be the large and small subunit respectively, of an α2β2 heterotetramer.

Genetic background on HQ dioxygenase from Sphingomonas sp. strain TTNP3The respective bands of the enzyme subunits were cut out of the SDS-PAGE gel and after extraction and subsequent tryptic di-gestion, analysed via QqTOF. From the mass fingerprints of the small and the large subunits of the enzyme, four and six oligo-peptides respectively could be generated by de novo sequencing. These peptides could be perfectly matched to two neighbouring open reading frames found in the genome sequence of strain TTNP3 (designated hqdA and hqdB). Interestingly, the amino acid sequences derived from these open reading frames did not show any resemblance to sequences of HQ dioxygenases that have been identified in Sphingomonads until now. Rather, they showed significant similarities to the small and large subunit of a HQ dioxygenase that has been identified and purified in

Understanding the Degradation of Hydroquinone, a Key Metabolite in Xenobiotic Degradation

Hydroquinone is a metabolite occurring in a number of degradation pathways of environmentally relevant xenobi-otics. In this study, a novel type of hydroquinone dioxygenase could be purified and characterized. A gene cluster containing the gene encoding for this dioxygenase and the enzymes presumably involved in the further degradation of hydroquinone metabolites were also identified.

Boris A. Kolvenbach, Hyazinth Dobrowinski, Markus Lenz, Philippe F.X. CorviniSchool of Life Sciences FHNW

Keywords: dioxygenases, aromatic ring cleavage

Pseudomonas fluorescens strain ACB (47% and 62% amino acid identities of the small and the large subunits, respectively). The greatest similarities however were to two open reading fra-mes coding for putative proteins from Photorhabdus lumine-scens subsp. laumondii TTO1, a bacterium that can be found in the gut of entomopathogenic nematodes (51% and 63% amino acid identity respectively).

More genes related to the degradation of HQIn the vicinity of the open reading frames attributed to the HQ dioxygenase subunits of strain TTNP3, further open reading fra-mes were identified, whose derived amino acid sequences bear similarities to enzymes probably involved in the further degra-dation of 4-hydroxymuconic semialdehyde. They revealed 68%, 64%, 56% and 32% amino acid identity to a 4-hydroxymuconic semialdehyde dehydrogenase (hqdC), a maleylacetate reducta-se (hqdD), a 1,2,4-trihydroxybenzene dioxygenase (hqdE), and a putative ferredoxin (hqdF) respectively. The maleylacetate reductase could already be shown to be enzymatically active, as an E. coli strain which was transformed with the sequence contained in an expression vector was shown by GC-MS analy-sis to degrade maleylacetate to 3-oxoadipic acid, while another strain bearing the same vector with a control insert did not. Hence, it is probable that the other gene products of these open reading frames are also involved in the degradation of HQ in strain TTNP3 (Fig. 1).

Conclusion and OutlookHQ dioxygenase is a novel member of the class of HQ ring-cleaving enzymes. It was shown to degrade a broad range of substituted HQs, such as halogenated and alkylated derivatives.The HQ degradation pathway in strain TTNP3 is remarkable regarding both the enzymes and their organization. Sequence similarities of hqdE indicate that 1,2,4-trihydroxybenzene may play a role in HQ degradation, yet in contrast to the other me-tabolites involved in this pathway, its presence could not be confirmed in degradation experiments. The enzymes of the HQ pathway were rather related to bacteria other than Sphingomo-nads (such as Photorhabdus, Burkholderia, Pseudomonas and Ralstonia). Of the six enzymes, only the maleylacetate reduc-tase and the 1,2,4-trihydroxybenzene dioxygenase appeared to have similar homologs in Sphingomonads. This was an unex-pected result, as enzymes attributed to xenobiotic degradation in Sphingomonads are usually very similar within the genus (often more than 90% amino acid sequence identity). Moreover, Sphingomonads are known to possess a relatively complex or-ganization for degradative genes, as these are often scattered throughout the genome rather than clustered as in this case [5].

References:[1] Kolvenbach B, Schlaich N, Raoui Z, Prell J, Zuhlke S, Schaffer A, Guengerich

FP, Corvini PFX. Degradation Pathway of Bisphenol A: Does ipso Substitution

Apply to Phenols Containing a Quaternary {alpha}-Carbon Structure in the

para Position? Appl. Environ. Microbiol. 2007;73 (15):4776–84.

[2] Moonen MJH, Kamerbeek NM, Westphal AH, Boeren SA, Janssen DB,

Fraaije MW, van Berkel WJH. Elucidation of the 4-hydroxyacetophenone cata-

bolic pathway in Pseudomonas fluorescens ACB. Journal of Bacteriology

2008;190 (15):5190–8.

[3] Spain JC, Gibson DT. Pathway for biodegradation of para-nitrophenol in a

Moraxella sp. Applied and Environmental Microbiology 1991;57 (3):812–9.

[4] Vaillancourt FH, Bolin JT, Eltis LD. The ins and outs of ring-cleaving dio-

xygenases. Critical Reviews in Biochemistry and Molecular Biology 2006;41

(4):241–67.

[5] Stolz A. Molecular characteristics of xenobiotic-degrading sphingomo-

nads. Applied Microbiology and Biotechnology 2009;81 (5):793–811.


Project Team: Boris A. Kolvenbach, Hyazinth Dobrowinski, Markus Lenz, Philippe F.X. Corvini (Institute for Ecopreneurship, School of Life Sciences FHNW)

Partner: Dirk Benndorf, Erdmann Rapp (Otto von Guericke University, Magdeburg, Germany) Jan Fousek, Cestmir Vlcek (Institute of Molecular Genetics, Academy of Sciences of the Czech Repu-blic, Centre for Applied Genomics, Prague, Czech Republic)Frédéric L.P. Gabriel (Institut für Klinische Chemie und La-boratoriumsmedizin, Rostock, Germany) Hans-Peter E. Koh-ler (EAWAG, Zürich, Switzerland) Andreas Schäffer (RWTH, Aachen, Germany)

Funding: Swiss National Science Foundation (SNSF, 200021-120574 1) and Czech Ministry for Education

Economic efficiency and benefit to society:The understanding of hydroquinone degradation in bacteria helps identifying the reasons for persistence of micropol-lutants in the environment. It constitutes information for the development of efficient bioremediation methods.

Table 1: Substrate specifity of HQ dioxygenase (relative enzymatic activity compared to hydroquinone)

Figure 1: Proposed degradation pathway for hydroquinone in strain TTNP3 (gene coding for enzymes whose functionality is proven is boxed)

Substrate(200M) Activity(%) SD

Hydroquinone 100 12.8

Chlorohydroquinone 29 0.8

2-Methoxyhydroquinon 59 6.7

2-Methylhydroquinone 139 9.3

2-Ethylhydroquinone 83 4.3

2-Propylhydroquinone 23 2.6

2-t-Butylhydroquinone 5 0.6

2-Pentylhydroquinone 19 1.1

2-Hexylhydroquinone <2 1.1

2-(1-methyl-1-octyl)-hydroquinone <2 0.5

IntroductionLinum usitatissimum L., commonly known as flax, is an im-portant oilseed crop that provides diversity in crop rotations in Canada. Because some sulfonylurea herbicides are very persistent in soil, the choice of crops for use in rotations is limited. To address this problem, Mc Hughen developed a synthase (ALS) gene cloned from Arabidopsis thaliana using Agrobacterium-based transformation. The coding sequence of the gene is modified by a single cytosine-to-thymine substitu-tion at the 589th nucleotide, which results in reduced affinity for sulfonylurea herbicides. The inserted construct contains additional to the ALS gene a neomycin phosphotransferase II gene (nptII), a spectinomycin/streptomycin resistance gene (Spec), a beta-lactamase gene (bla), and the nopaline syntha-se gene (NOS). The transgenic line was designated CDC Triffid (FP967).After safety assessment by the Canadian Government, the CDC Triffid line was authorized for food and feed use in Canada in 1996. In 1998 the line was approved in the United States. No GM linseed has been approved in any other country so far.In September 2009, Germany posted a notification on an inter-nal EU system (Rapid Alert System for Food and Feed – RASFF) that several linseed products originating from Canada were found to contain genetically modified (GM) DNA, which was attributed to the GM linseed (Linum usitatissimum) event FP967 (“CDC-Triffid”). In order to prevent further imports of unauthorised GM lin-seed form Canada, the European Commission adopted a sam-pling and testing protocol for Canadian flaxseed exported to the EU [Sampling and Testing Protocol for the Canadian Flax-seed Exported to the EU]. For detection of the genetic modifi-cation in linseed products the protocol refers to a real-time PCR method, which is targeting a construct of two elements, which are specifically present in event FP967 [Ref. 1) and Ref. 2)]. This “NOST-Spec” construct-specific real-time PCR method has been extensively validated for its sensitivity and specifi-city at the methods developer’s laboratory [1] and in a verifica-tion study done by the European Union Reference Laboratory for GM Food and Feed (EURL-GMFF) [2].For specificity reasons however, it is preferable to apply an event-specific detection method to confirm the presence of a defined genetically modified plant. For all the approved varie-ties in the EU event-specific methods have been developed and made publically available [1].

ResultsThe polymerase chain reaction has revolutionized DNA ana-lytics in general. It is also the basis for numerous attempts to isolate and identify unknown genomic regions adjacent to a known region, a process that is referred to as “genome wal-king”. The PCR based methods are much faster and less labour intensive than the traditional genome walking method that re-quires the construction of a genomic library with subsequent screening for the correct clone. The PCR based approaches can generally be classified into 3 groups that differ in their methods of “surrounding” the unknown DNA sequence with known sequences. (1) In the inverse PCR method, the known region with part of the unknown regions upstream and down-stream is generated using specific restriction enzymes. Ligati-on of the fragment results in a circular piece of DNA in which the unknown DNA sequence is now flanked by the known DNA sequence and can thus be identified. (2) In the ligation medi-ated PCR, a small synthetic piece of DNA is attached to the unknown flanking region and again, this leads to the unknown sequence being flanked by known sequences. (3) In the ran-dom primed PCR method, a primer with a randomly chosen sequence then binds at an unknown place within the unknown region. The rational is that as long as the primer binds within a relatively short distance of the known region, a PCR product can be generated and analysed.


Several attempts using all the approaches have been tried out in the laboratory and we succeeded in identifying the flan-king region of the FP967 integration event by applying the random primed PCR method. Using the sequence information obtained, we were able to establish an event-specific TaqMan Real Time PCR system. The difficulty was to find a TaqMan probe that binds the target sequence with high specificity and high sensitivity. This was accomplished by using the “locked-nucleic-acid” technology where some nucleotides within the probe are chemically changed in such a way that the binding to the target molecule is stronger and more specific. In Figures 1a) and b), the target molecule, i.e. the genome of the FP967 flax, has been serially diluted and measured using the newly established assay system. The results show that the assay is efficient and reproducible. Further measurements using either wild-type line seed DNA of other transgenic plant DNA as tar-get molecules showed a 100% specificity.

Often transgenic plant seeds are mixed with wild-type plant seeds. It is therefore of paramount importance that an assay to detect transgenic plants has a sensitivity that allows for the quantification of even small amounts of transgenic sample within a wild-type sample. We assessed the sensitivity of the quantification by determining the absolute and relative Limit of Detection (LOD) and Limit of Quantification (LOQ).LOD and LOQ refer to the lowest quantity of target that can be reliably detected and quantified with a probability higher than 95%. The absolute LOD and LOQ is the lowest number of initial haploid genomic copies that can be detected and quan-tified. Relative LOD and LOQ refer to the lowest percentage of GMO that can be detected and quantified in a fixed total amount of wild type DNA.In order to determine the absolute LOD and LOQ of the quanti-fication assay, a serial dilution containing 1400 to 5 FP967 ha-ploid genomic copies was analyzed in 6 parallel real-time PCRs. The ability to detect FP967 decreased as the copy numbers decreased. We were able to detect FP967 in all six parallel re-actions down to 20 haploid copies (Table 1). From these results we fixed the absolute LOD and LOQ at about 20 haploid geno-mic copies of FP967 flax (Table 1).The absolute LOD and LOQ were estimated in pure GM flax. In real food and feed samples, the presence of a large back-ground of non-target DNA – e.g. wild-type DNA – may modify these estimations. In order to evaluate the performance of our detection method under more realistic conditions, the relative

LOD and LOQ were estimated using a series of dilutions from 1 to 0.01% FP967 DNA in the presence of wild-type flax DNA (Table 2).We fixed the LOD and LOQ relative to our quantification test at about 0.06% of GMO content in a total amount of 100 ng/ reaction of flax DNA (Table 2). This value is about ten times less than the threshold set by the EU and Swiss authorities for non-authorised GM products in foods.

Conclusion and OutlookThe establishment of the first event-specific assay now allows for the detection and quantification of the defined FP967 flax. This had not previously been possible as the existing test only identifies the inserted DNA but not the inserted DNA at the defined locus within the genome. The assay will be published and could be chosen to undergo extensive validation by the European Union Reference Laboratory.

References:[1] European Commission, Joint Research Centre. Report on the Verification of

the Performance of a Construct-Specific Assay for the Detection of Flax CDC

Triffid Event FP967 Using Real-Time PCR. 2009. Availabe under: http://gmo-crl.

jrc.ec.europa.eu/doc/Flax_FP967_verification_report.pdf

[2] Nakamura K, Akiyama H, Yamada C, Satoh R, Makiyama D, Sakata K,

Kawakami H, Mano J, Kitta K, Teshima R. Novel method to detect a construct-

specific sequence of the acetolactate synthase gene in genetically-modified flax

CDC Triffid (FP967). Biol Pharm Bull. 2010;33 (3): 532–534

Research Focus Area: Molecular Technologies (MT) Project Team: Eric Kübler, Rosario Vanella, Anna Weston (Institute for Chemistry and Bioanalytics, School of Life Sciences FHNW)

Partner: Peter Brodman (Kantonales Laboratorium Basel-Stadt)

Funding: Master thesis project

Economic efficiency and benefit to society: Genetically modified flax can now be specifically detected and quantified.

How to Detect Genetically Modified FP967 Flax by Event-Specific Polymerase Chain Reaction

In this work the unknown DNA sequence of the plant Linum usitatissimum adjacent to the known FP967 inserted sequences has been identified by applying random primed site PCR. Using the sequence generated with this method, an event-specific real-time PCR process has been developed and tested successfully.

Eric Kübler, Rosario Vanella, Peter Brodmann, Anna WestonSchool of Life Sciences FHNW

Keywords: Transgenic plant, event-specific assay, FP967 flax

20 | 21

Figure 1: a) Amplification plot generated using primers FP967 right3, FP967 left1 and the LNA probe with serial dilutions of FP967 DNA containing 1400, 350, 87, 21 initial haploid genomic copies. b) Representative standard curve generated from the amplification data given in a).

Tables 1 and 2: Table 1 shows the sensitivity of the assay in terms of the genome copy number that can be detected. Table 2 shows the sensitivity of the assay in terms of the relative amount of GMO within 100 ng of total DNA.

Table 1. Amplification data used to determine absolute LOD and LOQ

DNAamount Haploidgenomic No.ofpositive MeanCt SDofobserved (ng) copies signals values Ctvalues

100 1400 6/6 28.97 0.12

25 350 6/6 30.64 0.36

6.25 87.5 6/6 32.68 0.16

1.56 21.8 6/6 36.09 0.43

0.4 5.4 0/6 – –

Table 2. Amplification data used to determine relative LOD and LOQ

GMO%(100ngtotalDNA) No.ofpositive MeanCtvalues SDofobserved signals Ctvalues

1 6/6 29.29 0.27

0.25 6/6 31.61 0.11

0.0625 6/6 34.25 0.17

0.01 0/6 – –


IntroductionThe company Laves-Arzneimittel produces Colibiogen, a bio-logical drug for patients suffering from inflammatory gut mu-cosa, e.g. irritable bowel syndome (IBS), infammatory bowel diseases and radiation colitis. Colibiogen stabilizes the inte-stinal flora, reduces inflammation and induces relaxation ef-fects in the gut. For the production of Colibiogen, Escherichia coli strain L1000 (DSM 13792), originally isolated from a heal-thy human subject by J. Vorschütz in 1931, is used. As Esche-richia coli bacteria are ubiquitous in the human environment, there is a realistic contamination risk for biotechnological systems. Hence, it is important to develop and validate highly discriminating diagnostic methods for strain identification in strain maintanance, inoculum generation and fermentation process control. In this study a PCR-based method was deve-loped for identifying E. coli L1000 and reliably distinguishing it from other strains of this genus.

ResultsThe challenge of the research project consisted in the un-ambiguous identification of E. coli and the discrimination of Escherichia coli strain L1000 adjacent to other, very similar Escherichia coli strains. It was therefore necessary to iden-tify molecular markers which are specific for Escherichia coli L1000 and markers which are universal to E. coli.As a universal E. coli marker we selected two genes, namely uidA, which codes for a β-D-glucuronidase and lacY, which codes for lactose permease. These markers were selected ba-sed on a literature survey [1, 2].The identification of markers which are highly specific for E. coli L1000 was more complicated. For this purpose the geno-mic information on about 30 E. coli strains available at public databases was used. These sequences amounted to about 150 Megabase. Together with the analysis of a partially complete L1000 genome sequence it was possible to identify five geno-mic regions which are present in E. coli L1000 but absent in the other E. coli strains. For the two universal markers and the five specific markers, primer pairs were designed [3, 4]. The primers were subsequently used in polymerase chain re-action (PCR) assays and optimized for efficiency and specifi-city in PCR. In all these reactions, the sequences framed by the primer pairs were very specifically amplified, resulting in DNA fragments of modest size which could then easily be de-tected in agarose gel electrophoresis and fluorescence stain-ing (Fig. 1).To analyze the performance of the seven molecular markers, we chose the strain L1000, five strains which are descendants of L1000 and 24 E. coli strains unrelated to E. coli L1000. The two universal markers should be present in all strains

whereas the L1000 specific marker should only be detected in L1000 and the five descendant strains. The universal mar-ker uidA (β-D-glucuronidase) was present in all E. coli strains tested but the second universal marker lacY (lactose perme-ase) could not be detected in one strain of E. coli. Concerning the L1000 specific markers, we observed that the five mar-kers were present in L1000 and its derivatives. In addition, the markers could be detected in one to two of the unrelated control strains. In order to increase the reliability of the test we therefore decided to use two universal makers and two specific markers for the discrimination between L1000 and the control strains.In view of an efficient workflow we combined the single PCR reactions in a multiplex PCR reaction. A multiplex PCR reac-tion uses several primer pairs and therefore generates sever-al PCR products which are then simultaneously analyzed by gel electrophoresis. For multiplex PCR, the primer pairs and the PCR product lengths were optimized in order to ensure efficient and reliable amplification as well as clear distinc-tion of the different amplified fragments (Fig. 1). In the final assay we used the universal marker uidA and lacY, which produced fragments of 592 and 508 basepairs respectively, and the specific markers Microcin B17 and one unique mar-ker sequence of contig 48, resulting in the fragments 462 and 281 base pairs respectively. The specific Microcin B17 related marker originates from the operon for the biosynthesis of the microbial toxin/antibioticum Microcin B17. The selected mo-lecular marker of contig48 derived from an intergenic region of unknown function.The assay developed allows easy identification of the E. coli strain L1000. A small aliquot of the culture is lysed and then subjected to the multiplex PCR with four primer pairs. After the multiplex PCR, the PCR fragments are separated by gel electrophoresis and visualized by staining. The whole pro-cedure can be accomplished in three hours by a technician. The interpretation of the results is also straightforward. If all four PCR fragments are detected, E. coli L1000 is identified unambiguously. If only two large fragments derived from the universal markers are present, the test culture/colony clone did not contain L1000 but another E. coli strain. If no molecu-lar marker is detected then either the assay failed technically or a non E. coli strain grew in the culture.

Molecular Identification of a Bacterial Production Strain Used in the Pharmaceutical Industry

The company Laves-Arzneimittel GmbH uses the Eschrichia coli strain L1000 for the production of the product Colibio-gen. During quality control it is necessary to identify repeatedly that only this strain is used in the production.

Jacqueline Büttiker, Dieter Grimmecke, Georg LippsSchool of Life Sciences FHNW

Keywords: Escherichia coli, Laves-Arzneimittel, PCR, identification, quality control

Conclusion and OutlookThe techniques developed in this project now allow the technically simple, cost-effective and highly-specific identi-fication of the strain E. coli L1000. The reliable detection of production strains is important in order to ensure that the product – in this case a pharmaceutical – is of constant qua-lity. The work was carried out in cooperation with Laves Arz-neimittel GmbH. Jacqueline Büttiker accomplished the work with expertise and accuracy and consequently received an award for the successful thesis. It also underscores the power of PCR based detection techniques in microbiology and biotechnology.


Project Team:Georg Lipps, Jacqueline Büttiker (Institute for Chemistry and Bioanalytics, School of Life Sciences FHNW)

Partner:Hans-Dieter Grimmecke (Laves-Arzneimittel GmbH)

Funding:Laves-Arzneimittel GmbH

Economic efficiency and benefit to society:The method developed enables cost-effective, simple and re-liable identification of an industrial E. coli production strain.

Figure 1: Multiplex PCR detection of E. coli L1000 A: The culture is either analyzed by four separate PCR reactions (lane 2-5) or a single multiplex PCR (lane 6). Four different sized fragments are genera-ted and separated by gel-electrophoresis. B: Analysis of twenty E. coli strains by multiplex PCR. Only in the case of E. coli L1000 all four DNA fragments could be detected.

A

References:[1] Horáková K, Mlejnková H, Mlejnek P. Specific detection of Escherichia coli

isolated from water samples using polymerase chain reaction targeting four

genes: cytochrome bd complex, lactose permease, β-d-glucuronidase, and β-d-

galactosidase. Journal of Applied Microbiology 2008;105 (4):970–6.

[2] Horáková K, Mlejnková H, Mlejnek P. Direct detection of bacterial faecal in-

dicators in water samples using PCR. Water Sci Technol. 2006; 54(3):135–40.

[3] Kalendar R, Lee D, Schulman AH Invited Review: FastPCR Software for PCR

Primer and Probe Design and Repeat Search. Genes, Genomes and Genomics

2009; 3(1).

[4] Kämpke T, The Reference Point Method in Primer Design. PCR Primer Design.

Edited by Anton Yuryev. Humana Press 2001, 75–93.

B

Substrates Conversion(%) Crudeyield(%) NMRanalysis 1 44 62 educt+product 2 76 74 educt+product 3 52 Quantitative educt+product 4 12 verylow manyside products 5 35 Quantitative educt+product 6 35 Quantitative educt+product

Substrates Conversion(%) Crudeyield(%) NMRanalysis 1 49 11 educt+product 3 45 44 educt+product 5 45 Quantitative educt+product

IntroductionFlame Spray Pyrolysis (FSP) is a versatile technology to produce inorganic nanoparticulate composite materials in a combusti-on process. The technology has been known since the 1950s but recently experienced a renaissance due to the development of new composite additives used in polymer formulations. A prominent example is the silver-silica composite polymer ad-ditive produced by HeiQ Materials, the industrial partner of this project. Based on their know how and technology, FSP has been expanded to the production of the new class of silica-supported catalysts allowing environmentally benign chemical transformation in chemical production. The process allows the synthesis of multifunctional catalytic composite materials.

ResultsThis project is designed to build upon existing expertise in the synthesis and scale-up to industrial production of nano- and micro-dimensioned composite particles using flame spray py-rolysis. The target is the preparation of manganese oxide com-posite nanoparticles by FSP, either as pure phases or supported on a silica matrix, as well as their evaluation as environmentally benign oxidants. The method developed involved a metal salt or complex dissolved in a flammable liquid. The solution was sprayed with a high flow of gas through a pilot flame and small droplets like a fog were formed. Metal species in solution were then oxidized. The size of the particles can be controlled by the size of the flame, the flame temperature and the concentration of the precursor liquid. The process of the FSP synthesis was significantly improved by modifying the concentration of the precursor solution and diminishing the liquid flow. Furthermo-re an improved nozzle geometry and gas feed were developed and implemented in the FSP apparatus. This optimisation affor-ded a considerably higher nanomaterial production of 1.5 g per day. The concept was to use the manganese oxide nanoparticles as oxidation equivalent transfer species utilising hypochlorite, hydrogen peroxide, ozone or oxygen as primary oxidant. Diffe-rent batches of pure manganese oxide nanoparticles were pre-pared and subjected to classical oxidation conditions with a 5-fold excess of oxidant in TBME at room temperature for up to 10 days (Table 1). The nanoparticles easily formed a suspension during the reaction and the work up was simplified. Filtration was performed by means of a small disposable syringe filter. The same filter was easily clogged during the work up of oxidations carried out with commercial manganese dioxide. Six substrates suitable for controlled and selective oxidation with manganese dioxide MnO2 were selected to test the reactivity of the nano-composite particles prepared (Fig. 3). Oxidation of benzylic al-cohols and allylic alcohols by commercial manganese dioxide or freshly prepared activated reagent is widely described in the

literature [1, 2]. The first experiments performed aimed to define the exact reaction conditions required for a complete transfor-mation of alcohol to aldehyde using either commercial man-ganese (IV) oxide activated from Sigma-Aldrich (21,764-6, 5um,

~85%) or MnO2 prepared according to the procedure described by J. Attenburrow [3]. Identical reaction conditions were used

for the six substrates and involved the use of 3-fold excess of the oxidant in tert-butylmethyl ether (TBME) or toluene for the benzyl alcohols (1–4) and allylic alcohols (5–6), depending on the solubility of the substrates. Compounds 2, 5 and 6 are colorless oils easily solubilized in apolar solvents; other substrates re-quired sonication or to be warmed up before addition of the oxi-dant. Reactions were strongly stirred at room temperature for 16 hours. The conversion of the reactions (percentage of educt transformed to aldehyde) was calculated from 1H NMR spectra (Bruker Avance 400 MHz) by integration of significant peaks of the starting material and product. Powder XRD analysis of the particles obtained by FSP revealed that the product was not the expected MnO2, but a lower oxide Mn3O4, which was not ideal as oxidant catalyst. A transfer step from Mn3O4 to the higher oxidation state of Manganese in MnO2 was therefore required. Based on the simple observation that nanoparticles on silica (loading 10%) activated overnight by concentrated sulfuric acid generated a new compound on Tlc in presence of trans-2-methyl-3-phenyl-2-propen-1-ol, we investigated experimen-tal conditions including an acidic treatment step (H2SO4) and subsequent neutralization of the composite material prior to the oxidation reaction. A proposed mechanism of the conversion [4] is shown in the formula scheme.

Mn3O4 + 4H+ à MnO2 + 2Mn2+ + 2H2O.

The activation procedure leads to uniform nanoneedle struc-tures (Fig. 2) which showed very good oxidation activities. We


determined that a neutralization step with sodium hydrogen carbonate after activation with concentrated sulfuric acid was necessary to avoid over-oxidization, potential decomposition or side reactions in the substrates. The combined activation and neutralization step led to a significant improvement in reactivity of the catalyst and yield. The reactions were stopped after ten days and worked up. It was demonstrated in the case of the reaction with trans-2-methyl-3-phenyl-2-propene-1-ol that longer reaction times gave higher conversions (Table 1, Fig. 5). The slow reactivity of the activated nanoparticles re-presents the limiting factor in the demonstrated properties of the nanocomposite particles. The handling of the reagent was satisfactory, the work up procedure was facilitated and the recovery of organic compounds was significantly improved. Comparable results were obtained upon treating manganese oxide nanoparticles on silica (50% loading) with concentrated sulfuric acid followed by the neutralization and isolation step. After 5 days and using a 5-fold excess of the manganese oxide, the conversion obtained was nearly 50%. The first results with nanoparticles on silica with a loading of 50% are described in Table 2. It is interesting to note that nanoparticles on silica with a loading of 10% did not lead to the oxidation products even after activation and neutralization.

Conclusion and OutlookFSP is a very versatile technology allowing the synthesis of a broad variety of composite materials, which can be used in a many fields of applications. In particular the synthesis of mixed oxides and solid supported catalysts can be produced, which is otherwise impossible or at least very difficult to achieve. The synthesis of manganese oxide via this route is a versatile al-ternative to the precipitation procedure commonly used, which suffers from bad reproducibility and difficult handling. Reac-tions carried out with MnO2 produced by FSP are far easier to work up and therefore give better yields compared to commer-cially available materials. Furthermore, current FSP technology will be expanded towards new materials exhibiting multifunc-tionality by combining various catalytic active materials in one composite.

References:[1] A.J. Fatiadi AJ. Active Manganese Dioxide Oxidation in Organic Chemistry –

Part II and Part II. Synthesis 1976 (2):65-104 and Synthesis 1976 (3):133–167

[2] Hirano M, Yakabe S, Chikamori H, H. Clark J, Morimoto T. Oxidation by Che-

mical Manganese Dioxide. Part 3.1 Oxidation of Benzylic and Allylic Alcohols,

Hydroxyarenes and Aminoarenes. Journal of Chemical Research, Synopses 1998

(12):770–1.

[3] Attenburrow J, Cameron AFB, Chapman JH, Evans RM, Hems BA, Jansen ABA,

Walker T. Synthesis of vitamin A from cyclohexanone. J. Chem. Soc. 1952 (Copy-

right (C) 2010 American Chemical Society (ACS). All Rights Reserved.):1094–111.

[4] Askar M, Abbas H. Chemically activated manganese dioxide for dry batteries.

Journal of Power Sources 1994;51 (3):319–30


Project Team: Uwe Pieles, Christelle Jablonski, Gerhard Grundler, Christian Siebenhaar, Marcus Waser (Institute for Chemistry and Bio-analytics, School of Life Sciences FHNW)Edwin Constable (University of Basel)

Partner: Murray Height (HeiQ Materials Ltd)

Funding: Swiss Nanoscience Institute (SNI)

Economic efficiency and benefit to society:Manganese oxide catalysts and other catalytic composite ma-terials produced by FSP allow an environmentally benign large scale oxidation of compounds in the chemical industry or can be used for oxidative waste water treatment.

MangaCat: Flame Spray Pyrolysis Synthesis of Manganese Oxide Nanocomposite Particles for Catalysis

Versatile Flame Spray pyrolysis technology has been further developed to produce a new class of silica supported catalytically active nanoparticulate materials. Among other things, composite manganese oxide has been produced, which allows the environmentally benign oxidative transformation of a variety of precursors for pharmaceutical drug synthesis.

Uwe Pielesa, Christelle Jablonskia, Gerhard Grundlera, Edwin Constableb

a School of Life Sciences FHNW, bUniversity of Basel

Keywords: Flame-Spray-Pyrolysis, Catalysis, Manganese oxide, Nanocomposite, Nanoparticles, oxidation

24 | 25

Figure 2: MnO2 nanoneedles obtained after treatment of Mn3O4 with sulfuric acid.

Figure 1: Structures of Model compounds

Table 1: Oxidation reaction performed with 5 eq of nanoparticles (activated) for 5 days

Table 2: Oxidation reactions performed with 5 eq of nanoparticles (activa-ted) on silica in TBME for 5 days

IntroductionCrystal polymorphism is defined as the ability of a solid sub-stance to exist as more than a single unique crystalline phase. These different polymorphs have different physical characteri-stics, both spectroscopically, which aids in their identification, and physico-chemically, such as their rate of dissolution and bio-availability. A fixed polymorph composition is therefore le-gally required for active pharmaceutical ingredients (APIs) to be allowed as a drug [1].In this project a new technological approach has been deve-loped, based on finely controlled surface modifications at the nanometer scale in order to create the appropriate template effect, [2] which will allow the creation of tailor-made solutions for high-throughput screening of polymorphic forms of APIs.Our partner in this project is the recently established compa-ny RPD Tool AG, who are developing automation solutions for API crystal screening for pharmaceutical industry. This scree-ning is traditionally done in small disposable glass vials and centres on varying the parameters: solvent, temperature and degree of over-saturation. The vials are used only once, because minute remnants of some crystalline phases are known to di-rect subsequent crystallisations [3].The project aims to provide synthetically modified surfaces to direct crystallisation, achieved by calix[4]arene based am-phiphiles, which are synthesised, characterised and appropri-ately derivatised to be suitable for their designed purpose. In the first instance, the influence of these amphiphiles is assessed when supplied as a monolayer at the solution/air interface.

ResultsA series of calix[4]arene amphiphiles was synthesised, follow-ing known literature procedures, starting from tert-butylphenol. In the first instance, this gave para-tert-butyl-calix[4]arene 1, which served as a precursor for all further modified calix[4]arenes [4]. The amphiphile with the most promising results car-ried four carboxylate groups on the para-positions and dode cyl chains at the phenolic rim. It was prepared from the precursor calix[4]arene 1 by aromatic dealkylation, followed by O-alkyla-tion with an n-dodecyl chain, bromination of the ring, to allow a copper(I) mediated nitrile exchange, and finally alkaline hy-drolysis of the nitrile to give the tetra-carboxy calix[4]arene 2 [5]. Further derivatives of this material 3a–c were prepared to allow further probing of crystallisation behaviour.

The properties of the tetra-carboxylate derivative, 2, when spread at the air-water interface, were tested by the Langmuir balance technique, which confirmed their amphiphilic beha-viour. Its interaction with dissolved APIs was assessed by the same method, which suggested favourable interactions with

both aspirin and paracetamol. The behaviour of the amino-phenol derivatives 3a–c was then studied, which suggested that there is a much stronger interaction between the 4-ami-nophenol derivative 3c and the paracetamol solution when comparing the 3 derivatives 3a–c.Having found these interactions, the effect of 2 on actual solu-tion phase crystallization of paracetamol from a concentrated aqueous solution was investigated by a series of experiments. For this purpose, a warm, concentrated solution of the API was prepared and distributed in an array of glass vials, and the tet ra-carboxylate 2 added to the air/solution interface. The amount of amphiphile was varied across the array of experi-ments, in order to learn what effect the density of calix[4]arene has on the crystallization. The density is of particular inte rest, since the packing of the molecules at the interface depends on the amount of area available to each molecule, and is therefo-re expected to have an impact on a potential templating effect. After addition of the amphiphile, the array was cooled to room temperature to cause supersaturation in the vials and favor crystallization. The amount of amphiphile was shown to have a significant effect on crystallization. Within the timeframe of the experiment a modestly dense layer was statistically much more likely to produce crystals, all of which formed at the in-terface, an example of which is shown in Figure 2, whereas a dense layer or the blank reference vials with no layer showed no crystal formation.


With the knowledge of the density favorable to crystallization, a Langmuir-Blogett film was deposited on glass slides made partly hydrophobic by silanisation. A transfer ratio close to 1 on the hydrophobic part at constant surface pressure suggested a good film deposition of the tetra-carboxylate 2. As subphase, a concentrated solution of paracetamol was used, so that the glass slide was dropped into a container submerged in the sub-phase. This procedure was deemed necessary, as it is known that any further crossing of the air/solution interface has a detri-mental effect on the integrity of Langmuir-Blogett films, which are not chemically bonded but held in place by hydrophobic interactions alone. Due to the limitations that this procedure involves, only a small number of experiments were performed, also tuned to probe the effect of layer density on crystallization. Figure 3 shows an example of one such experiment with a clear distinction in crystallization behavior between the lower area of the slide, which carries the deposited film and the upper area, which is not modified. Raman analysis of the crystals obtained under the different conditions only showed evidence of the for-mation of the thermodynamically more stable form I, which is expected to be obtained from crystallizations from water.

Conclusion and OutlookThe effect of the calix[4]arene amphiphile monolayer in the model crystallization of paracetamol has been demonstrated. The focus of the project is now on extending the crystallization tests to other APIs, as well as on creating analogous templating materials which are chemically bonded to glass surfaces. This will allow testing of the full range of crystallization parame-ters to be used, most importantly a range of commonly used organic solvents, which are liable to dissolve the free calix[4]arene amphiphiles, and could therefore not be a part of the mo-del testing. Once a suitable method for the immobilization has been developed, the glass vials used by our project partner, RPD TOOL, may be modified and tested in the live screening system.

References:[1] a) FDA Center for Drug Evaluation and Research, Guidance for Industry –

ANDAs: Pharmaceutical Solid Polymorphism – Chemistry, Manufacturing, and

Controls Information, 2007 b) http://www.ich.org, Q6: Specifications for New Drug

Substances and Products

[2] Toworfe GK, Compostoa RJ, Shapiroa IM, Ducheyne P. Nucleation and growth

of calcium phosphate on amine-, carboxyl- and hydroxyl-silane self-assembled

monolayers. Biomaterials. 2006;27 (4): 631–642

[3] Müller M, Meier U, Wieckhusen D, Beck R, Pfeffer-Hennig S, Schneeberger R.

Process Development Strategy to Ascertain Reproducible API Polymorph Manu-

facture. Crystal Growth & Design. 2006;6 (4): 946–954

[4] Gutsche CD. Calixarenes. Cambridge: Royal Society of Chemistry. 1989.

[5] Gutsche CD, Pagoria PF. Calixarenes 16. Functionalized calixarenes: the direct

substitution route. J. Org. Chem. 1985;50 (26): 5795–5802


Project Team: Dirk Elend, Negar Moridi, Patrick Shahgaldian (Institute for Chemistry and Bioanalytics, School of Life Sciences FHNW)

Partner: RPD TOOL AG

Funding: Confederation’s Innovation Promotion Agency (CTI)

Economic efficiency and benefit to society:The control of the polymorphism of active pharmaceutical in-gredients is a major issue in the development of new pharma-ceutically active ingredients from the early research stage to the final formulation steps. It is expected that new tailor-made solutions for high-throughput screening of polymorphic forms of APIs will be of great interest to the pharmaceutical industry and have a relevant market value. Additionally, the originality of the approach developed in the frame of the current project is expected to allow a better understanding of interfacial crystal-lization phenomena and thus to contribute to the advancement of knowledge in this particular field.

Engineered Supramolecular Surfaces for High-Throughput Polymorphism Screening of APIs

Polymorphism control of crystalline active pharmaceutical ingredients (APIs) is of ongoing interest in the pharma-ceutical industry. The aim of the project is to use the template effect of a chemically modified interface with a highly regular, quasi crystalline surface to direct crystallization and thereby control polymorph growth.

Dirk Elend, Negar Moridi, Patrick Shahgaldian School of Life Sciences FHNW

Keywords: amphiphilic macrocycle, calixarene, monolayer, polymorphism control

26 | 27

Figure 1: R=C12H25 a) CH2O, NaOH, Ph2O b) PhOH, AlCl3, toluene c) R-Br, NaH, DMF d) NBS, MEK e) CuCN, NMP f) KOH, EtOH g) C2O2Cl2, DCM h) aminophenol, pyridine

Figure 2: paracetamol growing at amphiphile interface

Figure 3: paracetamol crystals on LB film

30 | 31

Therapeutic Technologies (TT)

Technologies for the development and production of pharmaceutical and biomedical products taking into consideration sustainable manufacturing

IntroductionEndocrine disrupting compounds (EDCs) have been in the focus of environmental and health institutions of many countries for a few decades. They are defined as “exogenous substances or mixtures that alter function of the endocrine system and conse-quently cause adverse health effects in an intact organism, or its progeny, or (sub) populations” [1]. Bisphenol A is a building block for the production of flame-retardants, polycarbonate plastics and epoxy resins and belongs to EDCs. For a few years, enzyme-catalyzed polymerization (mainly by peroxidases) and precipi-tation processes have been explored as methods for the removal of phenolic contaminants such as BPA [2]. Several researchers proposed that instead of peroxidases, laccases may be useful for removing phenolic contaminants from water or wastewater. Laccases utilize molecular oxygen as a substrate for the oxida-tion of a variety of phenols, including BPA [3].While enzyme-based biocatalytic processes can easily be im-plemented in batch systems, the situation is quite different in continuous systems like waste water treatment plants (WWTPs). Enzyme immobilization on solid supports represents a strategy to prevent wash-out in the systems where they are added. Fur-thermore, several studies have described stabilization effects of enzymatic activities through the immobilization onto carrier material, which constitutes a protective micro-environment [4]. Prerequisites for the efficient biocatalytic activity of the immobi-lized enzyme encompass various aspects. For instance, the solid supports must be biocompatible, while the immobilized en-zyme should remain accessible to its cofactors and substrates. In the past decades, porous inorganic materials, especially me-soporous silica, have been widely used as an enzyme carrier material due to their large specific surface area, negligible swelling, high stability and low price [5].

Aims of this studyOur final goal is to demonstrate at pilot-scale, at the ARA Birs wastewater treatment plant, the efficiency of laccase-NP conju-gates for the removal of micropollutants. The tests are perfor-med in a fixed-bed reactor coupled to a membrane bioreactor developed by our industrial partners Aquaren AG and MMS AG, respectively (Fig. 3). The coupling of the enzyme to the carrier material was the first crucial step of this project. We achieved the efficient binding of laccase produced by the fungus Corio-lopsis polyzona (supplied by our partner Wetlands Incubator S.P.R.L, Belgium) to the surface of aggregates of nanoparticles (NPs) produced by means of a flame spray pyrolysis process (fsNP). Various coupling procedures were tested to obtain the highest loads of laccase activity on the porous nanostructured material. A method allowing for the production of NP/Laccase conjugates at kilogram-scale was optimized.

Results

Conjugate development of nano structured material and laccaseLaccase-modified NPs were obtained using surface modifica-tion, i.e. silanization, followed by a glutaraldehyde cross-linking procedure. Both, the protein load on fsNP and the binding effici-ency (up to 98%) of the laccase activity were maximized (2.9 U/mg fsNP; 11.3 U/mg protein) with the developed and optimized coupling method. The final products were characterized by both Scanning Electron Microscopy (SEM) (Fig. 1) and Brunauer-Em-mett-Teller (BET) surface area measurements. Both analyses showed changes of the surface characteristics. For instance the BET-analysis showed that the initial surface of fsNP (320 m2/g fsNP) decreased step by step during the immobilization procedure to a value of 109 m2/g fsNP. In addition, enzyme ac-tivity and stability were assessed in waste water to evaluate their applicability for BPA degradation in advanced waste water treatment systems.

The stability of the enzymatic activity of laccase- fsNP conju-gates (according to the optimized coupling method) and of free and sorbed laccase was assessed under application-relevant conditions in waste water (pH=8.2; value of treated effluent of ARA Birs) over one month. The conjugates were stable as 78% of the initial laccase activity remained after the test period. On the other hand, the activity of both the sorbed and free laccase decreased to 10% and 2.5% respectively during one month of incubation (Fig. 2).

Pilot-scale experimentsThe pilot plant was designed and constructed (Fig. 3) with the support of our industrial partners AQUAREN AG and MMS AG. The fixed-bed-membrane reactor system is being operated in the Birs WWTP in Basel, Switzerland. It consists of a fixed bed cascade bioreactor coupled to an ultrafiltration (UF) unit. The function of the fixed-bed reactor is to remove organic macropo-llutants (organic carbon, nitrogen, phosphorous, etc.) while the UF membrane unit is designed to retain the laccase-NP conju-gates as well as biomass from the fixed bed in the system and produce permeate free of particles.


Conclusion and OutlookLaccase of the white-rot fungi Coriolopsis polyzona was suc-cessfully immobilized on fsNP using a novel method. Experi-ments showed that immobilization to fumed silica is a highly ef-ficient procedure to stabilize laccase. In waste water, 78% of the laccase activity remained on the NP over one month, whereas the activity of free laccase dropped to 2.5%. This drastic increase in stability demonstrates the promising potential of immobi-lized laccase for waste water treatment process applications. To demonstrate the feasibility of the LANCE concept a pilot plant was designed and operated at the Birs WWTP (Basel) in order to perform degradation experiments under relevant conditions.

References:[1] Greim HA. The Endocrine and Reproductive System: Adverse Effects of Hor-

monally Active Substances? Pediatrics 2004;113 (4):1070–5.

[2] Tsutsumi Y, Haneda T, Nishida T. Removal of estrogenic activities of bisphe-

nol A and nonylphenol by oxidative enzymes from lignin-degrading basidiomyce-

tes. Chemosphere 2001;42 (3):271–6.

[3] Tanaka T, Tonosaki T, Nose M, Tomidokoro N, Kadomura N, Fujii T, Taniguchi

M. Treatment of model soils contaminated with phenolic endocrine-disrupting

chemicals with laccase from Trametes sp. in a rotating reactor. Journal of Biosci-

ence and Bioengineering 2001;92 (4):312–6.

[4] Stanescu M, Fogorasi M, Shaskolskiy B, Gavrilas S, Lozinsky V. New Potential

Bio catalysts by Laccase Immobilization in PVA Cryogel Type Carrier. Applied Bio-

chemistry and Biotechnology 2009;160 (7):1947–54.

[5] Rekuc A, Bryjak J, Szymanska K, Jarzebski AB. Very stable silica-gel-bound

laccase biocatalysts for the selective oxidation in continuous systems. Bioresource

Technology 2010;101 (7):2076–83.

Research Focus Area: Therapeutic Technologies (TT)

Project Team: Philippe Corvini, Thomas Wintgens, Gregor Hommes, Liang Yu, Jan Svojitka, Xue Feng Tang, Patrick Shahgaldian (Institute for Ecopreneurship and Institute for Chemistry and Bioanalytics, School of Life Sciences FHNW)

Partner: European partners are SINTEF (Norway), UCL-GEBI (Belgium) and WETLANDS INCUBATOR S.P.R.L. (Belgium); National part-ners are University of Basel, HeiQ Materials Ltd, Aquaren AG, MMS AG, Amt für industrielle Betriebe and Balewa AG.

Funding: Confederation’s Innovation Promotion Agency (CTI) in the fra-me of the Materanet programme initiated by the European Uni-on and the Federal Office for the Environment (FOEN).

Economic efficiency and benefit to society:Chemical pollution of natural waters has become a major pu-blic concern in almost all parts of the world. The LANCE con-cept is based on the use of biological catalysts to develop an eco-friendly “de-pollution” technology for the advanced treat-ment of contaminated water.

LANCE: LAccase-Nanoparticle Conjugates for the Elimination of Micropollutants from Wastewater in BioreactorsRecent advances in nanomaterial sciences have opened new perspectives for environmental biotechnology appli-cations. The LANCE-project proposes an alternative to classical physico-chemical treatment for the elimination of micropollutants, which can pass waste water treatment plants almost unchanged and enter the water cycle through receiving waters. Robust enzymes immobilized to nanomaterials represent an eco-efficient depollution technology.

Gregor Hommes, Yannick Zimmermann, Liang Yu, Xue Feng Tang, Jan Svojitka, Patrick Shahgaldian, Thomas Wintgens, Philippe CorviniSchool of Life Sciences FHNW

Keywords: Nanoparticles; Laccase immobilization; Endocrine disrupters; Fixed-bed; Membrane bioreactor

32 | 33

Raw domestic waste water is pumped into the cascade of 6 fixed bed chambers, which was kindly supplied by AQUAREN AG. The carrier material is made of polyethylene and has a surface area of 150 m2/m3. We could demonstrate that by passing succes-sively through the six chambers of the fixed-bed reactors, large amounts of organic carbon, ammonia nitrogen and particles are removed from the wastewater by the biofilm that developed on the fixed-bed carrier. The fixed-bed is followed by a settling tank to retain biomass detached from the biomass carrier. The fixed bed cascade reaches full nitrification, high removal of biologi-cal oxygen demand and suspended solids (average effluent con-centrations <10 mg/L). After this treatment, the effluent quality meets the regulation standards.The effluent from the fixed bed is directly pumped into a UF membrane reactor which was constructed by the project partner MMS AG in Switzerland. The UF-unit is a single reactor contai-ning a submerged flat-sheet membrane module BIO-CEL® from Microdyn-Nadir with a total membrane area of 10 m2 and a pore size of ca. 0.04 µm. To prevent membrane clogging and to ensure a stable filtration process, the membrane is periodically back-washed with filtrate. The complete filtration process is automa-ted and controlled by computer software. After the on-going stabilization phase of the pilot system, the laccase-NP conjuga-tes will be dosed in this UF unit to react with the BPA which is not degraded in the preceding fixed-bed treatment.

Figure 1: SEM picture of fsNP (left), fsNP after silanization (middle), fsNP after modification with laccase and glutaraldehyde according to the opti-mized protocol (right). Samples were taken from a suspension in phosphate-buffer (pH=7), 150’000× magnification, 20 kV.

Figure 2: Stability assay of laccase-fsNP conjugates, laccase sorbed onto fsNP, and free laccase as a reference in wastewater (pH=8.2) over one month.

Figure 3: The fixed-bed-membrane reactor system

IntroductionBreast cancer ranks as the most frequently diagnosed form of malignant disease and the second most relevant cause of cancer-related death in women living in Europe and North America. In spite of recent improvements in hormonal thera-pies and in the use of adjuvant cytotoxic therapies, the reduc-tion in the overall mortality rate has been rather modest and approximately 40% of breast cancer patients will eventually succumb to their disease [1]. Given that the conventional the-rapies often lead to only partial success, complementary treat-ment to limit or prevent the symptoms associated with cancer is becoming increasingly important [2,3]. More frequently and often at the patient’s request, alternative medicines are in-cluded in the therapy plan of cancer, particularly mistletoe therapy. Of the complementary medicines, European mistletoe extracts (Viscum Album Peparations; VAP i.e. Iscador®) are the most commonly used in Europe, especially in Germany. Mistletoe (Viscum Album) is a photosynthetic plant that lives on different types of trees including the oak. The lectins pu-rified from the mistletoe extracts have been shown to exert cytotoxic effects on carcinoma cells and have been shown to possess immunomodulatory and anti-angiogenic properties. In vitro experiments with cell lines have shown that the various VAPs can be cytotoxic to a variety of carcinoma cells, either through the activation of the apoptotic cascade, or by leading to necrosis. Furthermore the extracts possess concentration-dependent cytotoxic properties whose extent varied with the host tree, but did not always correlate with the corresponding mistletoe lectin content. The cytotoxic effect of VAPs is likely to be at least partially caused by mistletoe lectin I, but the in vitro toxicity of VAPs does not always correlate with their lectin con-tent, suggesting that other cytotoxic components present in mistletoe extracts might also play a role. Therefore, more study is needed to know about mistletoe’s effects, the mechanism of action which is still unclear, and to shed light on the discre-pancy between the extraordinary cytotoxic properties of the mistletoe-extracts and their relative modest clinical success.

Aim of the project and strategy The scope of this project is to develop a core-shell silica nano-particle (SNP) with a dual capability simultaneously enabling imaging and delivery of mistletoe extracts to tumor cells. These bimodal SNPs represent a good alternative that on one hand allows enhance the therapeutic efficacy of mistletoe ex-tracts and on the other hand insight into shed light on their mechanism by tracking the loaded extracts optically using an infrared marker. Because of the high flexibility of their synthetic approach, SNPs are good candidates to design a system that combines diagnostic and therapeutic modalities.

It is conceivable that using such nanoparticle conjugates we might learn more about the secrets behind the high cytotoxic activity of mistletoe extracts and their mode of action. To the best of our knowledge, the concept of combining anticancer mistletoe extracts (Iscador® M, P, Qu) with an infrared marker within a single nanoparticulate system for the treatment of breast cancer is novel. However, a series of new strategies using nanoparticles to treat cancer patients have emerged over the past years and served as a basis. As a carrier for delivery, silica offers many advantages as a multifunctional drug carrier. In addition to being able to encapsulate organic or inorganic materials, a variety of functional molecules can be attached to the surface via silane linkers and serve as anchoring sites for potential targeting moieties. SNPs have been explored for a multitude of delivery applications and a number of studies have consistently shown that encapsula-ting Cis-platin or coupling it to nanoparticles improved the therapeutic index of this drug in murine and human cancer cell lines and a mouse model [4,5].

Preliminary resultsIn this work, we demonstrate the successful embedding of three different mistletoe extracts (Iscador® M, P, Qu) in a core-shell SNP using a modified Stöber sol-gel method (Fig. 1). The synthetic approach is highly modular and allows appropriate encapsulation of individual components of VAPs in the silica shell around a core labeled with an infrared fluorochrome. Although very small components of mistletoe extracts may be able to escape from the mesoporous framework of nano-particles and diffuse into cells, the focus here was to deliver not only one or two but all the components of the extracts equally to the targeted cells and to study their synergetic cytotoxic effects.

In vitro experiments were studied on MCF-7 and MFM-223 breast cancer cells and the cytotoxic effect of Iscador®-nanoparticle conjugates was characterized by using MTT (3(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bro-mide)-colorimetric assays. To better compare the efficacy of


the different Iscador®-nanoparticles, the data from the MTT-assays were used to determine the concentrations which re-sult in a 50% inhibition of cell growth (IC50, see Fig. 2). This also allows a comparison with the IC50-values previously published for the different mistletoe extracts on the same cell lines. To assess the therapeutic index of the different mistle-toe-nanoparticles, Iscador® M, P or Qu extracts were tested side-by-side. The emphasis here was put on the comparison of the time course of cell death between the nanoparticle for-mulations and the naked Iscador extracts.

The data depicted in Figure 2 reveal in all cases a significant shift of the curves to the right, suggesting that encapsulation of the Iscador-preparations in silica nanoparticles (curves in red) have increased their cytotoxic effect (curves in black) on the breast cancer cells tested. This increase can be seen even at low concentrations of Iscador nanoparticles. The most interesting outcome of this work is that the increased cytoto-xic effect was obtained on Iscador nanoparticles loaded with only 15% of the drug as compared to the standard dose (5 mg/ml) in the case of “naked” Iscador extracts which is indica-tive of a wider therapeutic window in the case of Iscador®-nanoparticle conjugates.

Conclusion and OutlookIn the frame of this study, three different mistletoe extracts (Iscador® M, P, and Qu) were successfully encapsulated into silica core shell nanoparticles. The effect of the mistletoe na-noparticle conjugates (Iscador@SNP) on the in-vitro growth of breast cancer cell lines (MCF-7 and MFM-223) was examined and characterized using MTT colorimetric assays. The results suggest that encapsulation of Iscador-preparations in nano-particles has increased their cytotoxic effect on the breast cancer cells tested. Interestingly, this increase can be seen even at low concentrations on Iscador® nanoparticle conju-gates loaded with only 15% of the drug which is indicative of their wider therapeutic window. Future investigations will focus on the use of confocal fluorescence microscopy to fol-low the internalization of Iscador® nanoparticles by cells, to characterize the nuclear morphology of the breast carcinoma cells upon treatment and to assess the cell damage index as compared to the conventional mistletoe extracts.

References:[1] Prosnitz RR, Iglehart, JD, Winer, EP. Breast Cancer. In: Rubin P. editor. clinical

oncology, a multidisciplinary approach for physicians and students. New York,

2001 pp. 267–99.

[2] Ganz P, Desmond K, Leedham B, Rowland JH, Meyerowitz BE, Belin TR. Qua-

lity of life in long-term, disease-free survivors of breast cancer: a follow-up study.

J Nat Cancer Inst 2002;94 (1):39–49.

[3] Molassiotis A, Fernadez-Ortega P, Pud D, Ozden G, Scott JA, Panteli V, Mar-

gulies A, Browall M, Magri M, Selvekerova S, Madsen E, Milovics L, Bruyns I, Gud-

mundsdottir G, Hummerston S, Ahmad AM, Platin N, Kearney N, Patiraki E. Use of

complementary and alternative medicine in cancer patients: a European survey,

Annals of Oncology 2005;16 (4): 655–663.

[4] Zhao Y, Trewyn BG, Slowing II, Lin VS-Y. Mesoporous Silica Nanoparticle-

Based Double Drug Delivery System for Glucose-Responsive Controlled Release

of Insulin and Cyclic AMP. J Am Chem Soc 2009;131 (24): 8398–8400.

[5] Burger KNJ, Staffhorst RWHM, de Vijlder HC, Velinova MJ, Bomans PH, Frede-

rik PM, de Kruijff B. Nanocapsules: lipid-coated aggregates of cisplatin with high

cytotoxicity. Nat Med 2002;8 (1):81–4.


Project Team: Amina Wirth, Uwe Pieles (Institute for Chemistry and Bio-analytics, School of Life Sciences FHNW)

Partner: A. Paula Simões-Wüst (Paracelsus Hospital, Research Depart-ment, CH-8805 Richterswil)

Funding: Internal Funding (School of Life Sciences FHNW) and Partners

Economic efficiency and benefit to society: Based on this finding, it is conceivable that VAP-nanoparticle formulations will open new possibilities to explore the full potential of Viscum album in breast and in bladder cancer therapy.

Mistletoe-Preparations (Iscador®) Encapsulated in Silica Nanoparticles as a Therapy for Breast CancerThe project aims to design a novel core shell nanoparticle encapsulating mistletoe extracts, one of the most commonly used forms of complementary cancer therapy in Europe. Mistletoe extracts nanoparticles conjugates have been tested against two different tumor cell lines to assess their therapeutic index and to clarify the in-vitro growth inhibitory effects versus the respective tumor cell type.

Amina Wirtha and A. Paula Simões-Wüstb

aSchool of Life Sciences FHNW, bResearch Department, Paracelsus Hospital, Richterswil

Keywords: Silica core shell nanoparticles, breast cancer, mistletoe extracts, Iscador®, complementary therapy, MTT colori-metric assays.

34 | 35

Figure 1: Synthetic strategy for the encapsulation of mistletoe preparations (Iscador® M, P, Qu). (a) Scanning electron micrograph of Iscador® M silica nanoparticles conjugates (magnification 125K), scale bar: 200 nm

Figure 2: Effects of VAPs on breast carcinoma cell growth. MCF-7, and MFM-223 breast carcinoma cells were treated with different concentrations of Iscador® Qu, M or P during 48 hours. Thereafter cell growth was determined using the standard MTT-colorimetric assays.

IndroductionSelective laser melting (SLM) is an Additive Manufacturing method that allows the production of complex-shaped metal-lic structures based on a virtual Computer-Aided Design (CAD) or a clinical dataset e.g. for a bone scaffold. In the SLM Rea-lizer 100 (MTT Technologies, Lübeck , Germany) a continuous wave Ytterbium fibre laser (wavelength: 1068–1095 nm) is used to solidify dedicated areas in a powder bed layer by layer until the final rapid prototyped structure of desired shape is built. This so-called Additive Manufacturing has become a profici-ent technology for efficiently producing small quantities of high quality, complex shaped parts within a short time. The laser-based SLM process is a promising technology to produce individualized bone implants, e.g. for patient-specific ortho-pedic treatment, as an alternative to conventional machining like turning or milling.

In this project we use nickel-titanium (NiTi) powder, with par-ticle size of around 50 µm provided by SAES MEMRY GmbH, as a starting material. These nearly equiatomic binary compo-sed nickel titanium alloys are successfully used in biomedi-cal applications [1] because of their biocompatibility [2]. NiTi is FDA-approved for permanent implants including stents and has outstanding properties as it can change its shape as the result of thermo-mechanical stimuli. Thus, NiTi exhibits pseudo-elastic behaviour. These shape memory phenomena originate from the reversible phase transformation between martensite and austenite. This diffusionless transformation between the two crystallographic structures (see Fig. 1) takes place on the nanoscopic length scale.

So far, the generatively produced NiTi-microstructures have been investigated by means of metallographic analysis (inclu-

ding careful sample preparation by grinding, electropolishing, etching and inspection of the specimen), differential scanning calorimetry (DSC) of both NiTi-powder and the SLM-micro-structures (DSC 30 from Mettler-Toledo, measurement range between -100°C and 125°C) and high-resolution micro compu-ted tomography (µCT, SkyScan 1172TM, SkyScan, Kontich, Bel-gium, 100 kV, 100 µA, 32.3 µm pixel size, 360° scan, Al/Cu filter, NRecon v1.6.1.2 software) equipped with a heating stage as well as advanced micro computed tomography (nanotomTM, GE).

ResultsThe metallographic analysis of the prepared solids shows an oriented microstructure with elongated grains in building di-rection, see arrow in Figure 2 left (process parameters: laser energy density: 84 J/mm3, laser power: 76 W and laser speed: 150 mm/s). Such epitaxial solidification was previously found in other titanium alloys [3]. Figure 2 right illustrates the globu-lar microstructure in the support structure. The pore density in the material depended on the laser parameters. DSC mea-surements served for the determination of the transition tem-peratures. The starting powder, as well as the SLM parts pro-duced, exhibited the expected behaviour: Upon cooling down the austenite from 125°C to the martensite region, the phase transformation starts at the martensite starting temperature Ms, maximizes the heat flow at the exothermic peak position Mp and is finally completed at Mf, resulting in pure martensite phase. Subsequently, during heating up the martensite from -100°C a diffusionless thermally reversible martensite to auste-nite phase change characterized by a change in crystal struc-ture starts at As, reaches its minimum of the heat flow at the endothermic peak position Ap and is completed at Af, resulting in pure austenite phase. Dependence of the transformation temperatures, e.g. of austenite and martensite peak tempera-


tures Ap and Mp, on the energy density applied during the pro-duction process was published [5]. Interestingly, the transition temperatures can be adjusted to the physiological range.

The one-way shape memory effect (explanation see Fig. 1) of the SLM structures was verified by mechanical expansion of a spiral spring, followed by heating and measuring the change in sample shape, see Figure 3. The pseudo-plastic behaviour of the SLM NiTi materials was clearly demonstrated during this one-way shape memory effect. Furthermore, these pseu do-plastic deformations were quantified using µCT to calculate the strain field of the entire specimen on the basis of three-di-mensional non-rigid registration of the tomographic datasets in successive steps [4]. For this procedure, a dedicated heating stage was integrated into the µCT-system to scan the spiral spring in-situ at various temperatures in the martensite and austenite states. Because larger and thicker SLM structures are highly X-ray absorbent, they were scanned with accelera-ting voltages as high as 180 kV by means of the nanotomTM to uncover the pore density, pore sizes and pore shapes [4].

Conclusion and OutlookIn collaboration, the University of Applied Sciences Northwe-stern Switzerland, the University and the University Hospi-tal of Basel, SAES Memry GmbH and Thommen Medical have applied selective laser melting to successfully fabricate NiTi shape-memory alloy microstructures that exhibit the expected shape-memory properties. DSC measurements demonstrated that thermally activated rapid prototyped microstructures re-versibly switch between martensite and austenite states. The related local deformations were uncovered by means of µCT. The fabrication process will be further optimized to create complex shaped implants to be tested for cyto-compatibility within a dedicated compression bioreactor system after bone cell seeding. The physico-chemical properties will be further investigated by means of X-ray diffraction and inert gas fusi-on test method. Additionally, intense thermo-mechanical cha-racterization including training for intrinsic two-way shape memory effect will be performed.

References:[1] Es-Souni M, Fischer-Brandies H. Assessing the biocompatibility of NiTi sha-

pe memory alloys used for medical applications. Anal Bioanal Chem 2005;381

(3):557–567.

[2] ASTM Standard. Standard Specification for Wrought Nickel-Titanium Shape

Memory Alloys for Medical Devices and Surgical Implants. ASTM Standard

F2063 2005.

[3] Thijs L, Verhaeghe F, Craeghs T, Van Humbeeck J, Kruth JP. A study of the

microstructural evolution during selective laser melting of Ti–6Al–4V. Acta

Mater 2010;58 (9):3303-3312.

[4] Bormann T, Friess S, de Wild M, Schumacher R, Schulz G, Müller B. Determi-

nation of strain fields in porous shape memory alloys using micro computed

tomography. Proc. of SPIE 2010;7804 78041M.

[5] Bormann T, Schumacher R, Müller B, Mertmann M, Pieles U, de Wild M. Pro-

perties of NiTi-structures fabricated by selective laser melting. Europ Cells and

Materials 2010;20 (Suppl 1): 13.


Project Team: Therese Bormann, Conradin Döbeli, Waldemar Hoffmann, Uwe Pieles, Erik Schkommodau, Ralf Schumacher, Michael de Wild (Institute for Medical and Analytical Technologies and Institute for Chemistry and Bioanalytics, School of Life Sciences FHNW)

Partner:Matthias Mertmann (SAES Memry GmbH), Falko Schlottig (Thommen Medical), Bert Müller (Biomaterials Science Cen-ter, University of Basel), Ivan Martin (Institute for Surgical Research and Hospital Management, University Hospital Basel).

Funding:Swiss National Science Foundation (SNSF, 406240 126123/1) and Confederation’s Innovation Promotion Agency (CTI) within the National Research Programme NFP 62 “Smart Materials”.

Economic efficiency and benefit to society:The unique behaviour of the porous microstructures opens perspectives and adaptations concerning biofunctionality for the benefit of patients, especially in dentistry. The collabora-tion between the University of Applied Sciences Northwestern Switzerland, the University of Basel and the University Hos-pital of Basel is leading to symbiotic exchange of knowledge and technology in the field of biomedical engineering.

Porous Shape-Memory-Scaffolds for Bone Implants

In this project we created porous and highly complex microstructures of nickel-titanium (NiTi) shape-memory alloys (SMA) using selective laser melting and tested them in-vitro. It is our goal to identify optimized structures and architectures in order to specifically enhance osteoprogenitor cell function. The aim of this project is the preparation of artificial 3D microstructures as future implantable biomaterial with improved bone-tissue performance.

Michael de Wild, Uwe Pieles, Therese Bormann, Conradin Döbeli, Waldemar Hoffmann, Uwe Pieles, Erik Schkommodau, Ralf Schumacher.School of Life Sciences FHNW

Keywords: NiTi shape memory alloy, implants, selective laser melting, metallography, differential scanning calorimetry, micro computed tomography

36 | 37

Figure 1: The schematic sketch shows the martensite-austenite phase transition for the one-way and two-way shape memory effects and pseudoelasticity on macroscopic level (spring) and atomic level as known for NiTi (Fig. from [5]).

Figure 2: Metallographic investigations revealed the microstructure in SLM fabricated samples. Arrows indicate building direction.

Figure 3: One-way shape memory effect in SLM fabricated helix: The original structure a) is mechanically deformed b) and returns to the original shape c) upon heating above the austenite finish temperature Af [5].

IntroductionWhile environmental management in the healthcare sector in industrialised countries is common practice due to legisla-tion and standards, it is not the case in developing and tran-sition countries, where hospitals especially often still have a relevant regional impact on the environment that is neg-lected or even ignored. Additionally, the costs for energy, wa-ter and the disposal of waste can be quite substantial. These issues therefore offer potential for improvement in efficien-cy, safety and quality of services provided by adopting Best Environmental Practices (BEP) in the healthcare sector. In a recent paper, the World Health Organisation [1] pointed in the same direction: “It is clear that the health sector can also play a leadership role in mitigating climate change – that is reducing its magnitude and consequences […]. By doing so the health sector will create a series of health, economic and social co-benefits that improve the health of the population in addition to the traditional role of the health sector in the delivery of quality health care”. Since 2008 we have applied extensive experience and know-how to the field of cleaner production as well as to the de-velopment of cooperation in the healthcare sector in deve-loping countries. Cleaner production pursues an integrated strategy, aiming at reducing environmental impacts in a pre-ventive and cost-effective manner, compared to increasingly expensive end-of-pipe solutions. For the “Best Environmental Practices in the Healthcare Sector” project, the first step was to assess the potential of cleaner production methodology in healthcare facilities. In a second step, a hands-on guide for implementation of this me-thodology as “Best Environmental Practice” in the healthcare sector was developed especially for developing and transiti-on countries.

Applied ResearchIntensive literature research revealed several fields in the healthcare sector, with significant potential for the imple-mentation of BEP. Figure 1 represents a way to understand healthcare facilities from a BEP perspective in an initial stage. This systemic approach represents the main hospital divisions with related inputs and outputs. This approach functions as a basis to select relevant issues and divisions, based on economic, environmental and safety criteria. In a second stage, it allows focusing on a more detailed assess-ment and implementation of measures. For example, hospi-tals consume large amounts of energy and water. Most of the energy consumption can be accounted for by heating, ven-tilation and air conditioning. Relevant quantities are also used in the laundry and food services divisions as well as in

administration due to its large consumption of electricity for office equipment and lighting. Cafeteria and food services, laundry and gardening are significant contributors to the consumption of water.

Another important topic regarding the environmental impact of hospitals, as well as the question of human health, is the issue of hazardous waste and toxic materials. In patient care, pharmacies and laboratories large amounts of waste are pro-duced every day. Some of these are hazardous for the envi-ronment and are often also a threat to human health if not handled and treated adequately. There are also various other toxic materials which need special handling to prevent risks to employees, patients and the environment. For example, the use of mercury is still an issue in many developing countries. Fortunately, in recent years large international campaigns have been launched to eliminate this potent neurotoxin in hospitals all over the world [3]. As part of the project, a one week study tour was organised for a delegation of Jordanian experts by the healthcare sec-tor in Switzerland. Several field visits were carried out to learn more about best environmental practice in Swiss hos-pitals. In addition, numerous presentations of experts from Switzerland and Germany about energy efficiency, waste ma-nagement and other subjects were given. All activities con-ducted during this week were valuable for both sides, the visiting experts, with potential implementation of lessons learned in Jordan and as input for the compilation of the guide. Following the study tour, two environmental audits were conducted in Jordanian hospitals. One was carried out in Prince Hamzah public hospital (Fig. 2) and the second in


Luzmilla private hospital (Fig. 3), both in Amman. These au-dits provided an overview of the relevant issues in Jordan and allowed testing of the auditing instruments. The audits revealed the potential to improve the safety of stakeholders and the environmental footprint while also generating finan-cial savings.

Simultaneously, two master theses were conducted both in Jordan and in Switzerland. The thesis evaluating cleaner pro-duction potential in Swiss healthcare facilities showed that the standards of environmental management are generally high and revealed fairly low potential for improvements. On the other hand, the thesis on cleaner production conducted in three selected Jordanian hospitals revealed high potential for improvement of environmental practices in the fields of electricity, water consumption and waste management.

ResultsOne of the main results was the development of the “Best En-vironmental Practices in the Healthcare Sector” guide. [2] The aim of this guide is first of all to raise awareness of the ne-cessity and the benefits of implementing BEP. The guide pro-vides a methodology to overview, understand and characte-rize the different hospital activity processes. Furthermore it allows the benchmarking of a hospital's status with similar hospitals as well as the monitoring and evaluation of imple-mentation progress. Following the introduction to the hospi-tal divisions` specialities and challenges concerning cleaner production, several options for environmental performance improvement have been suggested according to the different issues. The guide also includes both BEP case studies carried out in public and private hospitals in Jordan. In the appen-

dix of the guide several instruments and tools for an initial environmental audit, such as questionnaires, checklists, etc. are provided. The guide can be downloaded from the web un-der the following URL: http://www.fhnw.ch/lifesciences/iec/forschungsfelder-und-projekte/download-projekte/projekte/best-environmental-practices-for-the-healthcare-sector.

Conclusion and OutlookThe research conducted here revealed a substantial poten-tial for implementing BEP in healthcare in developing and transition countries. The guide developed offers a tool for assessing the situation. However, successful implementation of BEP still remains a challenge. While the guide provides tips and incentives to overcome typical obstacles and shows how to implement BEP in hospitals successfully, concrete approaches and solutions are not yet covered. Such concrete approaches could be “clean technologies”, which are broadly suggested in the guide but not properly assessed regarding their applicability under specific local and organisational circumstances. Further studies and practical implementati-on cases are needed to prove the economic feasibility, real ecological benefits and the technical and organisational via-bility of proposed measures and technologies.

References:[1] World Health Organisation, Health Care Without Harm. Healthy Hospitals

Healthy Planet Healthy People. Addressing climate change in health care set-

tings, 2008.

[2] IEC, SBA, RSS. Best Environmental Practices in the Healthcare Sector. A guide

to improve your environmental performance. 2010 (not published).

[3] Zimmer C, McKinley D. New approaches to pollution prevention in the health-

care industry. Journal of Cleaner Production (2008)16: 734–742

Research Focus Area: Therapeutic Technologies (TT);

Project Team:Emmanuel Oertlé, Dieter Mutz, Olga Steiger (Institute for Ecopreneurship, School of Life Sciences FHNW)

Partner: Sustainable Business Associates (SBA) Lausanne, Royal Sci-entific Society (RSS) Jordan

Funding: Swiss Secretariat for Economic Affairs (SECO)

Economic efficiency and benefit to societyThe project showed that BEP in the healthcare sector are beneficial not only for the environment but often go hand in hand with economic savings and increased safety for pati-ents and employees.

Best Environmental Practices in the Healthcare Sector

The environmental impact of the healthcare sector in developing and transition countries is often neglected or not addressed at all. We are now applying our long-lasting competence in Cleaner Production to the healthcare sector by developing tools to improve the environmental performance of hospital facilities.

Emmanuel Oertlé, Olga SteigerSchool of Life Sciences FHNW

Keywords: Best Environmental Practices (BEP), healthcare, sustainable development, cleaner production, environmental audit

38 | 39

Figure 1: The concept of BEP in hospitals

Figure 2: Prince Hamzah hospital in Amman, Jordan

Figure 3: Conduction of an environmental audit in Luzmilla hospital in Amman, Jordan

IntroductionResearch carried out into innovative nanomaterials at the School of Life Sciences FHNW has recently led to the idea of creating a spin-off company dedicated to the commercial valorization of these materials. One of our first products is a novel type of environmentally friendly nanomaterial with remarkable virus recognition properties, for which a patent has been filed this year. It is based on a molecular imprinting technology and it uses an innovative process for assembling organo-silica building blocks around a specific virus (the tar-get template). Once the template is removed, the final product consists of a spherical nanomaterial (400 nm-diameter) on the surface of which a hundred viral entities can be bound. It can then be used to trap viruses from different media such as water, food liquids, various types of surfaces, air, etc. This new product will respond to the problem of the occurrence of viruses in everyday life, which is of major interest to society. It was widely seen last year with the H1N1 Influenza virus on a global scale However it is also a daily concern for individuals with the herpes virus for example, or for the water industry, which is on high alert for public health issues and bioterror-ism, or again for the food industry. This clean-tech process, which INOFEA will commercialize, is simple, fast, environmentally friendly and highly cost-effici-ent. It enables the production of kilograms of this virus bin-ding nanomaterial in a few hours from simple, easily available and non-expensive raw material.

ResultsThe INOFEA project has advanced from the stage of a simple idea to a firmly established spin-off project in 2010. The status of INOFEA is described in terms of technology, products and business developments:– The production process is functional. It is described in a

patent and a scientific publication is in preparation for sub-mission,

– The nanomaterial can be produced on a regular basis at the lab scale;

– Process optimisation is currently a major R&D activity; – Work is ongoing to improve specificity of the nanomaterial

for distinguishing between two similar viruses. – The research project led to the creation of the spin-off IN-

OFEA, which is currently hosted at the newly created Basel Incubator (http://basel-inkubator.ch/companies);

– The company name and logo are protected by the Swiss Fe-deral Institute of Intellectual Property (MAREG sca/602193); the domain names corresponding to INOFEA (www.inofea.ch and www.inofea.com) and corresponding email addres-ses are reserved;

– An exclusive license pre-agreement for environmental ap-plications has been reached with the School of Life Sciences FHNW regarding use of the patent.

A preliminary survey of the potential market has been perfor-med and a range of potential customers have been identified such as:– The Diagnostics industry, – The Membrane industry

These primary customers will embed the product in new diagnostic tools and materials for their own products made for the drinking-water industry, the food industry, bioana-lytical laboratories, etc. Finally, the chemical industry and chemical retailers could also be a potential market. In numerous studies, a fast growing market for these appli-cations is expected to reach nearly $1 trillion. In 2008, BCC Research in the United Kingdom published a study entitled “Nanotechnology: a Realistic Market Assessment” describing a global market for nanotechnology of $25 billion by 2011, 86% dominated by nanomaterials. This study suggests a wide market for nanomaterials of which only 0.1% would re-present $21.5 million.

The INOFEA business model is based on the B to B marketing of virus binding nanomaterial, which is easy, fast and cost-efficient to produce. Quality-guaranteed, fully characterised and functional virus binding nanomaterials, designed for a wide range of relevant viruses, will be sold. Income will also come from the co-development of nanomate-rial-embedded products together with our customers.


Furthermore new products based on the patent will be deve-loped either by INOFEA itself or with the financial help of the CTI and acacemic institutions, such as the School of Life Sci-ences FHNW.R&D with the School of Life Sciences FHNW will be carried out to develop new imprinted materials targetingemerging pollutants for example.

Financial support for INOFEA is sought first through the Ven-ture Kick program of CTI, designed to foster spin-off creation in Switzerland. The project has been pre-selected for the early phase of the 3-stage program.

Conclusion and OutlookWe have identified the critical milestones leading to the foun-dation of the INOFEA company and to the business launch. Protecting intellectual property rights with a patent was an important first step. Identifying key market players and quan-tifying the market potential are crucial next steps to be con-tinued.A detailled INOFEA business plan will be finalized in 2011. Meanwhile the scientific and technical development continue at the School of Life Sciences FHNW: a prototype product will be developed in order to support proper business initiation. INOFEA forsees starting business in the second half of 2011.

References:[1] Ge Y, Turner AP. Too large to fit? Recent developments in macromolecular

imprinting. Trends Biotechnol 2008;26 (4):218–24.

[2] Janiak DS, Kofinas P. Molecular imprinting of peptides and proteins in

aqueous media. Anal Bioanal Chem 2007;389 (2):399–404.


Project Team: Alessandro Cumbo, Philippe Corvini, Patrick Shahgaldian, Yves Dudal (Institute for Chemistry and Bionanalytics and Institute for Ecopreneurship, School of Life Sciences FHNW)

Partner: Venture Kick (CTI), Basel Inkubator

Funding: Venture Kick Stage I by Confederation’s Innovation Promotion Agency (CTI)

Economic efficiency and benefit to society: As a spin-off, INOFEA aims at the economic valorizing of high potential research results. Benefit to society is foreseen in terms of new and efficient available products to detect and remove viruses many different matrices, and in terms of job creation.

INOFEA, Innovative Nanomaterials for Environmental Applications, an Ecopreneurship Venture

INOFEA is a spin-off creation project based on research into environmental applications for new nanomaterials at the School of Life Sciences FHNW. Initial valorization focuses on a recently patented innovation for the specific binding of viruses: virus imprinted polymers (VIP). INOFEA has been hosted at the new Basel Incubator since the second quarter of 2010 and has been pre-selected for the early phase of the CTI-Venture Kick process.

Alessandro Cumbo, Philippe Corvini, Patrick Shahgaldian, Yves DudalSchool of Life Sciences FHNW

Keywords: Spin-off Company – nanomaterials – environmental applications – Basel Incubator

40 | 41

Figure 1: Inofea Team

= 1 1 1

P Pdbl,d

q(qz+ 1– zVf )

+

( )z Pm,L

z+ Pm,d Pm,L Vf

=q ( )Dw,d

Dw,L

²/3

Introduction Self-microemulsifying drug delivery systems, (SMEDDS), can be used for the per-oral delivery of poorly water-soluble drugs which show low intestinal absorption owing to their insolubility in intestinal fluids. A large proportion of newly discovered active pharmaceutical ingredients exhibit this deficit in their biopharmaceutical properties. SMEDDS work by administering the drug in a dissolved form and preventing its precipitation in vivo by solubilising it through inclusion in colloidal lipid particles. While SMEDDS have been shown to improve the bioavailability of some drugs, the mechanism of intestinal absorption from the colloids remains poorly un-derstood and the rate of success is still variable. In the pre-sent work, the absorption kinetics of model poorly water-so-luble drug danazol formulated as a lipid based SMEDDS was investigated in the Caco-2 cell culture model. This is an es-tablished in vitro model for intestinal absorption. To impro-ve the prediction provided by the model, biorelevant media simulating the contents of the intestine under fasted and fed conditions were used. The combination of the Caco-2 model with simulated intestinal fluids is novel and the attributes of the system are not well described. The use of SMEDDS in combination with simulated intestinal fluids, in particular in the Caco-2 cell culture has not been reported before. The goal of this work was to investigate cell permeation kinetics of danazol formulated as SMEDDS under improved biore - levant in vitro conditions in order to establish the active principle of the technology and at the same time introduce an in vitro testing model with superior predictive power.

MethodsDanazol was formulated as SMEDDS consisting of tri-, di- and monoglyceride, Cremophor EL as surfactant and etha-nol as cosolvent. This formulation spontaneously produces

a microemulsion upon mixture with aqueous media. Caco-2 cells were cultured on Transwell inserts according to stan-dard protocols. Drug transport across the cell monolayer was measured in both the apical-to-basal and the basal-to-apical directions and data were evaluated with a kinetic model that provided drug permeability coefficients of the apical and the basal plasma membrane and the partition coefficient of drug between the media and the cell compartment (Fig. 1).

Permeation was studied with the microemulsion in media consisting of cell-compatible fasted and fed state simulating intestinal fluids, FaSSIFCaco and FeSSIFCaco. Control experi-ments were performed with the unformulated drug using the biorelevant media as well as with the microemulsion and the unformulated drug in a purely aqueous transport medium, TM. Furthermore, permeation was studied using SMEDDS with a wide range of lipid concentrations in purely aqueous media.

ResultsPermeability coefficient values of the drug with the SMEDDS were smaller than those of the unformulated drug. This was true when biorelevant media as well as when the purely aqueous transport medium were used. FaSSIFCaco and FeSSIFCaco themselves reduced permeability coefficients compared to TM (Fig. 2). On the other hand, both the microe-mulsion and the biorelevant media increased drug solubility in water (not shown). The model-deduced media-to-cell par-tition coefficients varied in accordance with the solubility of drug in the media. Apical permeability coefficients were generally 1.5 to 4 times greater than basal values.

To evaluate the effect of colloidal lipid particles originating from SMEDDS on drug absorption, a biophysical model was developed that distinguished the contribution of different


transport pathways in the cell culture model. This model resulted in the following equation expressing the apparent (measured) permeability coefficient as a function of the con-tributing factors. (Eq. 1)

solubility in the medium. The propensity of the drug, howe-ver, to permeate the cell membrane was generally reduced. This effect of SMEDDS was evident in fasted and fed state bio relevant media and did not seem to be influenced on a qualitative basis by the lipid components of the media. Fed state simulated intestinal fluid reduced in turn the permea-bility coefficient of the drug for the cell monolayer compared to the fasted state fluid. The increased drug solubility eli-cited by SMEDDS may, however, compensate or even over-compensate for the effect of permeability reduction in terms of drug absorption (mass transfer) rate from drug saturated media. This is because the lipophilic drug appears to exhibit the capacity to be absorbed by the cell membrane directly from its solubilized state in the colloidal lipid particles.

References:[1] Kang BK, Lee JS, Chon SK, Jeong SY, Yuk SH, Khang G, Lee HB, Cho SH. Deve-

lopment of self-microemulsifying drug delivery systems (SMEDDS) for oral bioa-

vailability enhancement of simvastatin in beagle dogs. Int J Pharm. 2004;274(1–

2):65–73.

[2] Jantratid E, Dressman J. Biorelevant Dissolution Media Simulating the Proxi-

mal Human Gastrointestinal Tract: An Update. Dissolut Technol. 2009;16(3):21–5.


Project Team: Georgios Imanidis, Daniel Preisig, Constantinos Markopoulos, Fabienne Thoennen (Institute for Pharma Technology, School of Life Sciences FHNW)

Partner: Christos Reppas (Department of Pharmaceutical Technolo-gy, National and Kapodistrian University of Athens, Greece); Jennifer Dressman (Institute of Pharmaceutical Technology, Johann Wolfgang Goethe University, Frankfurt am Main, Germany)

Funding: F. Hoffmann-La Roche Ltd.

Economic efficiency and benefit to society:Establishing in vitro models with an improved predictive power accurately simulating the in vivo situation will expe-dite drug development. Understanding the mode of action of drug delivery systems under these conditions will allow the introduction of potentially life saving drugs.

Absorption Mechanisms of Poorly Water-Soluble Drugs from Self-Emulsifying Formulations in the Caco-2 Cell Model Using Biorelevant MediaDrug development encompasses the formulation and manufacture of a delivery system which provides adequate bio-availability. For practical and technical reasons, testing of a product under development should be carried out in vitro using appropriate models. The predictive value of these models for the in vivo situation is essential for an efficient development process. Furthermore, understanding of the mechanism of action of the systems under development is crucial for the success of the endeavor.

Daniel Preisig, Fabienne Thoenen, Constantinos Markopoulos, Georgios Imanidis School of Life Sciences FHNW

Keywords: Drug absorption, intestinal, formulation, Caco-2, biorelevant media, self-emulsifying drug delivery system

42 | 43

Figure 1: Typical concentration profiles of bidirectional transport experiment of danazol in Caco-2 cell culture with biorelevant media. Experimental data points and fitted model curves are shown.

Figure 2: Deduced permeability coefficient of danazol for different lipid concentrations of SMEDDS in aqueous transport medium and biorelevant media FaSSIFCaco and FeSSIFCaco.

Figure 3: Apparent permeability coefficient of danazol versus free fraction of drug-saturated SMEDDS in aqueous transport medium. Fitted curve according to model of Eq. 1.

where,P is the measured permeability coefficientz is the free fraction of drug in the colloidal dispersionPdbl,d is the permeability coefficient of the diffusion boundary layer (unstirred water layer) for the free drugPm,d is the permeability coefficient of the free drug for the cell membranePm,L is the permeability coefficient of lipid particle-associa-ted drug for the cell membrane (due to direct transfer upon impact)Vf is the volume fraction of water (=1-φ), φ being volume frac-tion of the lipid phaseq accounts for the difference in diffusion boundary permea-bility between the free drug and the colloidal particle-asso-ciated drug and is given by

with D being the diffusion coefficient of the drug molecule and of the colloidal particle in water, respectively. Vf is com-monly >0.99, (5 mg/ml lipid ==> φ = 0.005) therefore it can be approximated by 1.

By fitting Eq.1 to the data (Fig. 3), the following values of the relevant permeability coefficients were deduced.Pm,d = 2.5E-4 ± 5.3E-5 cm/sPm,L = 1.1E-3 ± 3.6E-4 cm/sPdbl,d = 1.05E-3 ± 2.4E-4 cm/s

Hence, permeation by direct drug transfer from the lipid par-ticle to the cell membrane must be taking place in parallel with permeation of free drug.

ConclusionsIncorporation of the lipophilic drug in the colloidal struc-tures of SMEDDS was found to increase its overall drug

Introduction New drugs often exhibit low aqueous solubility, which can result in biopharmaceutical issues of incomplete drug ab-sorption as well as of high intra- and intersubject variability. These biopharmaceutical issues are major concerns for the development of novel drug products. Pharmaceutical techno-logy can here provide an important contribution to drug pro-ducts by introducing adequate drug delivery systems. Thus, lipid-based formulations can, for example, be developed to cope with poorly soluble drugs [1]. Lipid-based systems are categorized in simple oils and different types of self-emul-sifying systems [2]. The complex mixtures are named accor-ding to their dilution performance as self-emulsifying or self-microemulsifying drug delivery systems (SEDDS or SMEDDS, respectively).

Fig. 1 shows the fate of a lipid-based formulation following oral administration [3]. The formulation first undergoes dis-persion in the stomach and subsequently it is transferred into the intestine. Further dispersion occurs in the initial part of the gut, where bile salts and phospholipids are secreted into the lumen and the formulation is digested by lipolytic enzymes. Low water soluble drugs can move from the for-mulation droplets to lipolytic degradation phases to end in swollen micelles. These colloidal structures finally bring the drug to the site of absorption. During this passage, a drug should not crush. The absorption process can only take place in a limited time window of the gastro-intestinal passage so that precipitated drug is often too slowly re-dessolving for absorption. Consequently, this part of the dose is lost for oral bioavailability.

A formulation should therefore keep the drug in a solubi-lized form throughout the intestinal transit. This solubili-zation ability can be tested in vitro by using sophisticated dispersion/precipitation tests. It is further possible to ana-lyze the formulation digestion [4, 5]. These tests are current-ly not standardized and many experimental factors are still unknown. Moreover there is a need to better characterize the processes in vitro as well as to study the relevance for the in vivo situation. Thus, applied research is needed to transfer the research ideas to practical testing tools that can be used by the pharmaceutical industry.

ResultsA research consortium was formed and a website set up (www.lfcsconsortium.org) for internal communication of objectives and results. The consortium includes major pharmaceutical companies such as Sanofi Aventis, Merck, Capsugel and Gat-tefossé as main sponsors. Further partners come from acade-mia including eight universities. As one of the academic part-ners, the Institute of Pharma Technology (FHNW) is a member of the “Dispersion Working Group” within this consortium. The “Dispersion Group” is focusing on different in vitro tests that consider the dispersion/precipitation behavior of for-mulations. Some tests are targeted for an early development phase so that the experimental set-up as well as the analyti-cal technologies should bear the potential of high-throughput testing. Compendial dissolution vessels have to be studied as well, in order to asses their suitability for testing lipid formu-lation dispersion at a later development stage.

The main objective of the Institute of Pharma Technology (School of Life Sciences FHNW) is to evaluate new technolo-gies for in vitro dispersion testing of lipid-based formulati-ons. Simple dilution tests were studied to learn about experi-mental factors such as the choice of dilution medium or the optimal dilution level. An optical sensor system was used to detect drug precipitation upon dilution. Initial results were obtained with a formulation that was known to exhibit drug precipitation upon dilution. Using the optical sensor system, we observed that even at a rather high dilution of 1:100 or 1:200 (w/w) it was possible to detect drug particles.

In addition to dilution tests in an early development pha-se, we studied more sophisticated biopharmaceutical pre-cipitation tests. Fig. 2 illustrates such an assay including simulated gastric as well as simulated intestinal fluid. This biopharmaceutical method has the advantage that the tran-sit from the stomach to the intestinal environment can be simulated and it is therefore called a transfer test. For initial


experimental evaluation, the model drug dipyridamole was solubilized in simulated gastric medium and pumped at set rates into the simulated intestinal medium. The subsequent drug precipitation was monitored using different technolo-gies. In-line Raman spectroscopy enabled the quantification of drug crystal formation. The amount of precipitated drug was closely predicted by a multivariate model when it was compared to filtrated samples that were subsequently ana-lyzed by HPLC. The Raman spectroscopy enabled monitoring of the amount as well as the kind of crystalline particles. A further characterization was obtained by employing an on-line image analysis. It was possible to analyze the number of particles as well as their size over the entire experimental time span. We found that the kinetics of nuclei formation and growth depended on the transfer rate. The induction times of precipitation corresponded well between the spectroscopic and image analysis method. Further experiments are plan-ned to assess the suitability of the new in vitro test for a series of lipid-based drug formulations.

Conclusion and OutlookThe research consortium started in 2010, a website has been set up and some initial results have been obtained. Based on the studies of experimental factors, it is now planned to pro-vide protocols to the industrial partners. Test results will be compared by different laboratories to ensure the validity of the experimental data. However, the applied research is not only limited to in vitro test validation, but insights are also expected into the dispersion behavior of lipid-based formu-lations. Based on this characteristic behavior the existing categorization of lipid-based formulations will be reconsi-dered. A rational classification can be helpful in anticipating the formulation performance in vivo and such correlation of in vitro and in vivo results is currently foreseen in the third year of the research program. This will help to achieve the ultimate goal of the consortium: to provide in vitro tests for lipid-based formulations that are sensitive, reproducible and that can anticipate dosage form performance in humans.

References[1] Jannin V, Musakhanian J, Marchaud D. Approaches for the development

of solid and semi-solid lipid-based formulations. Adv Drug Deliv Rev 2008; 60:

734–46.

[2] Pouton CW. Formulations of poorly water-soluble drugs for oral admini-

stration: Physicochemical and physiological issues and the lipid classification

system. Eur J Pharm Sci 2006; 29: 278–87.

[3] Porter CHJ, Pouton CW, Cuine JF, Charman WN. Enhancing intestinal drug

solubilisation using lipid-based delivery systems. Adv Drug Deliv Rev 2008;

60: 673–691.

[4] Arnold Y, Bravo Gonzalez R, Versace H, Kuentz M. Comparison of different

in vitro tests to assess oral lipid-based formulations using a poorly soluble

acidic drug. J Drug Del Sci Tech 2010; 20(2): 143–148.

[5] Porter CHJ, Kaukonen AM, Taillardat-Bertschinger A, Boyd BJ, O’Connor

JM, Edwards GA. Use of in vitro lipid digestion data to explain the in vivo

performance of triglyceride-based oral lipid formulations of poorly water-

soluble drugs: Study with halofantrine. J Pharm Sci 2004; 93 (5): 1110–21.

Research Focus Area:Therapeutic Technologies (TT)

Project Team: Yvonne Arnold, Cordula Stillhart, Martin Kuentz (Institute for Pharma Technology, School of Life Sciences FHNW)

Partner: Goethe Universität (Frankfurt/Main, Germany), National and Kapodistrian University of Athen (Greece), University College of Cork (Ireland), School of Life Sciences FHNW and Industrial Partners (Capsugel, Merck Sereno GmbH, Sanofi Aventis GmbH, Gattefossé).

Funding: The Lipid Formulation Classification System Consortium (LFCS)

Economic efficiency and benefit to society: Industrial protocols will be provided for in vitro tests of lipid-based drug formulations. These drug delivery systems are pivotal for challenging drugs so that development can be performed in a more efficient and cost-effective manner.

New Technologies for In Vitro Testing of Lipid-Based Drug Formulations

Lipid-based formulations are becoming increasingly important and there is a need for standardized in vitro tests for this type of system. The current consortium project aims to provide testing protocols to industry. Such tests should ultimately help to anticipate how lipid-based formulations perform in humans.

Yvonne Arnold, Cordula Stillhart, Martin Kuentz School of Life Sciences FHNW

Keywords: Pharmaceutical technology, lipid-based formulation, in vitro test, dispersion, drug precipitation

44 | 45

Figure 1: Fate of a lipid-based drug formulation in the gastro-intestinal tract.

Figure 2: Scheme of a biopharmaceutical transfer test and installed monitoring tools. Initial results are shown using dipyridamole as model drug.

IntroductionThe number of medicinal implants introduced into patients worldwide is increasing year by year. Although the failure rate of implants has decreased significantly in recent years, too many revisions are still necessary. The main reason for failure is implant loosening under mechanical stress due to insufficient implant-bone integration and/or due to develop-ment of infections around the implant. Generating roughness and porosity on the implant surface at the nano- and mi-crometer scale is the method of choice to initiate good bone ingrowth [1]. However, rough and porous surfaces also foster adhesion of bacteria leading to increased risk of infection. Bacterial infections and the building of a biofilm after imp-lant placement have been observed to be a serious problem in implantology in general. In particular in knee, hip and oral environments, infections have serious consequences for the patients [2]. Therefore it is a challenge to develop implant surface treatments which produce surface properties for good osseointegration and which possess properties for re-ducing infection risk as well [3]. The goal of the project NAP-TIS is to develop surfaces of titanium implants which fulfill both requirements focussing on the development of new po-rous surfaces of titanium implants by spark assisted ano-dizing. Optimizing the electrolytes used, anodization offers the unique ability to micro- and nanostructure the titanium implant surface allowing better osseointegration and at the same time to incorporate active antimicrobial metal ions.

ResultsThe surface treatments applied in the course of this project include precleaning, spark-assisted anodizing in proprieta-ry electrolytes* and with proprietary anodizing parameters* and final cleaning. The spark-assisted anodizing process [4] produces different layers (Fig. 1), which can be affected by

different post-anodization treatments. Experiments with specifically designed “test-samples” will clarify whether the different mechanical pretreatments of the samples for de-

burring may significantly change the surface, which could affect and change the anodizing process. It has been discove-red that the surface roughness of the anodized layers is very similar for the different mechanical pretreatments, as well as the elemental composition of the anodized layers. This de-monstrated that the mechanical pretreatment for deburring has no significant effect on the overall anodization process. Although similar in chemical composition and morpholo-gical appearance, the thickness of the porous layer can be tuned, by modifications of the sample preparation protocol under the conditions tested between 1.5 and 3.5 µm (Fig. 2).

Samples treated according to the protocol developed* have been subjected to test methods for the further characteriza-tion of the modified titanium surfaces e.g. confocal microsco-py to determine the surface roughness (SRa), biocompatibi-lity and stem cell differentiation assays and assays for the determination of the antimicrobial activity of the samples. In the same context the first samples with antimicrobial ac-tive copper deposits were prepared and tested. The samples containing copper, which were introduced during the elec-trochemical process in the presence of the proprietary elec-trolyte* were analyzed by SEM and EDX to reveal the copper distribution. Clusters and submicron sized copper particles were found in the treated samples.

In order to determine the antimicrobial activity of the samples generated during the project, an appropriate assay was developed. In the literature concerning the antimicrobial activity of copper contradicting results can be found: in cer-tain cases bacterial growth is inhibited by extracts from me-tallic copper surfaces whereas in other cases direct contact of the microorganism with the copper surface seems to be re-quired. Using copper discs we found that E.coli bacteria die


both when being cultured in simulated body fluid (SBF) in the presence of metallic copper and in the extract after removal of the copper disc. According to these results, two different types of assays were performed: (i) anodized test samples were extracted for 16hrs with SBF and the extract incubated with 104 E. coli (liquid assay) or (ii) the anodized test samples were extracted for extended time periods (32 days) and 105 E.coli were incubated with the extracted titanium disc to de-monstrate the long-term efficacy of the antimicrobial active sample (solid assay). First results of the antimicrobial acti-vity showed very promising and encouraging effects. These will be further followed and extended in the second year of the project with regard to copper and other incorporated ele-ments in the course of the anodizing process, as well as with regard to other microorganisms.

To determine the biocompatibility and incorporation po-tential of the titanium implant surfaces developed, several bioassays were performed. Bone marrow derived mesenchy-mal stem cells were plated on titanium sample discs and cultured for 20 days under conditions either favouring (OM) or preventing (CM) differentiation towards osteoblastic li-neage differentiation. Subsequently they were analyzed for cell proliferation and the presence of differentiation markers (alkaline phosphatase activity, aPa). While the proliferation of cells (Fig. 3a) shows the cytocompatibility of the substrate, the expression of differentiation markers indicates the suit-ability of the material to serve as bone generation sites. Cell growth was similar on both the non-anodized reference and the treated sample surfaces, however cell differentiation was only observed in the anodized samples upon culture in diffe-rentiation inducing medium (Fig. 3b).

Conclusion and OutlookIn the course of the Nano Argovia funded project NAPTIS, samples of titanium grade 4 used in the implant industry were fully characterized by means of microscopic, structural and chemical surface analysis technologies. The samples were subjected to the electrochemical spark-assisted anodizing process and the layers formed were also analyzed with regard to their structural and chemical composition. In initial expe-riments antimicrobial activity could be observed in samples containing copper. Differentiation of stem cells could be only observed in case of the electrochemically treated samples. Based on these very promising results other electrochemi-cal treatment processes utilizing electrolytes with different

elemental conditions will be studied for their antimicrobial activity and effects on stem cell differentiation. The most pro-mising candidates will be followed up in collaboration with the industrial partners in in vitro and in vivo models.

References:[1] Brunette, DM, Tengvall, P, Textor, M, Thomsen, P. Titanium in Medicine: ma-

terial science, surface science, engineering, biological responses and medical

applications. Berlin: Springer-Verlag 2001.

[2] Fitzgerald R, Jr. Infected Total Hip Arthroplasty: Diagnosis and Treatment.

J Am Acad Orthop Surg 1995;3 (5):249–62.

[3] Ryu HS, Hong S-H. Anti-bacterial and corrosion properties of Ag-containing

oxide coating on AZ31 magnesium alloy formed by micro-arc oxidation. 216th

ECS Meeting October 4–9, 2009, Vienna, Austria.

[4] Jung, C. Surface properties of titanium implants treated by spark-assisted

anodizing; European Cells and Materials, 2010;19 (Suppl 2):4.

* For confidentially reasons no details can be given here


Project Team: Uwe Pieles, Joachim Köser, Michael de Wild, Waldemar Hoff-mann (Institute of Chemistry and Bioanalytics, Institute of Medicinal and Analytical Technology, School for Life Sciences FHNW) Ivan Martin (Department of Biomedicine, University of Basel)

Partner:Falko Schlottig (Thommen Medical AG) Christiane Jung (KKS Ultraschall AG)

Funding:Swiss Nanoscience Institute (SNI)

Economic efficiency and benefit to societyMedical implants with better surface properties, causing fewer problems with rejection or inflammation, will finally save costs for the public health system and will be beneficial for the health of the patients.

NAPTIS: Nano- and Micro-Scaled Porous Surfaces of Titanium Implants Produced by Spark-Assisted Anodizing

Utilizing electrochemical spark-assisted anodizing processes in combination with a variety of different electrolytes, porous layers can be produced on titanium implant surfaces. They exhibit structural and antimicrobial properties which allow proper cell adhesion, cell differentiation and significantly reduce the risk of infections.

Uwe Pielesa, Michael de Wilda, Joachim Kösera, Ivan Martinb aSchool of Life Sciences FHNW, bUniversity of Basel

Keywords: Implants, dental, nanoporous surface, spark assisted anodization, titanium

46 | 47

Figure 1: Layers produced by the spark-assisted anodizing process

Figure 2: Scanning electron micrographs of different surface modified samples (sample 13A, 13B, 13C)* (buttom: results of the Calo- test)

Figure 3a: Stem cell proliferation on reference samples (Ti ref) and samples C' (Ti 1) and D' (Ti 2) in normal (CM) or differentiation inducing (OM) cell culture medium.

Figure 3b: Expression of the differentiation marker alkaline phosphatase in stem cell cultures growing on untreated Ti reference discs (Ti ref) and samples C' (Ti 1) and D' (Ti 2) in normal (CM) or differentiation inducing (OM) cell culture medium.

IntroductionIn the industrial countries one million patients suffering from severe heart disease with life expectancy of less than 12 months enter end-stage heart failure each year. Heart trans-plantation (HTX) remains the only therapeutic option for these patients. However, HTX is limited by the availability of donor hearts for the right patient at the right time. An alternative for patients with no other perspectives of survival are ventricular assist devices (VAD). Current axial-flow blood pumps contain a conveying spiral with central rotating body. The high speed impact of blood on the central body generates adverse effects such as friction and shear stress. Hemolysis and thrombus formation are inevitable results, a source of major clinical complication. Hence the majority of R&D effort in this field is focused on significant reduction of blood damage and down-sizing of the devices for convenient implantation.

MethodSince there is no existing model for a hollow-rotor axial pump, only basic principles from the theory of traditional turbo pumps can be transferred. Based on this theory, basic design guidelines such as in- and outlet blade angles can be used to create an initial rotor design. Further design parameters of the hollow rotor to be considered for first experiments are rotor length, diameter of outer and inner holes, blade outlet angle, blade inlet angle, blade unrolling angle and the ratio between the blade unrolling angle and rotor length. These points arise as a result of the medical specifications, literature research and empirical findings during the experiments. The rotors were created with a parameterized model so that the above-mentioned points are fully considered and constrained. The CAD-model allows description of all the parameters as well as the non-linear slope of the helix. Considering that the role of a diffuser is important for overall performance, this project focused on the hydraulic characteristics of the rotor-principle. The prototypes were manufactured with a rapid prototyping printer (Objet Geometries Ltd.). The material used was Vero-Blue™/ Fullcure™ 840 (acryl based photopolymere) and the models were manufactured with a precision of 0.05mm.

Hydraulic performance was tested experimentally using a pump test bench as shown in fig.1. The fluid used was dema-terialized and degassed water and a bypass- pump was in-stalled to test the gauges and to remove air from the piping. The laboratory prototypes were mounted with the rotor socket and drag was simulated with a restriction valve. Several pa-rameters such as pressure output, mass flow and rotational speed could be recorded digitally for immediate evaluation using measuring instruments from Endress+Hauser Flowtec.

A Promag 55S flow meter detected volume flow rate using magnetic induction while the differential pressure gauge was a Deltabar S PMD 70. The output signal was in both cases a standard current signal from 4 to 20mA. A National Instru-ments analogue to digital signal converter was used; measure-ment data were detected in real time and processed with a measuring program based on LabView®.

ResultsAn axial-flow pump with circumferentially positioned rotor blades was designed. This unique “hollow-rotor” design elimi-nates the central body without reducing pumping perfor m-ance [fig. 2]. By diminishing blood friction surfaces, a re duc-tion of bleeding complications and thromboembolic events is expected. As per the main design parameters, derived from the theory of traditional turbo pumps [1,2], a range of rotor geo-metries were designed and manufactured using an additive manufacturing method. For hydraulic evaluation, a complex pump test stand allowing speeds up to 15000 min-1 was deve-loped [fig. 1]. The functionality and hydraulic performance of the different designs were tested and analysed. The most ap-propriate hydraulic performance [3] in relation to geometrical and surgical conditions is achieved with a speed of between 11000 and 12000 min-1. Head-capacity curves [fig.3] indicate good pumping characteristics of between 5–6 l/min with a pressure output of 100–150 mmHg.

Conclusion and OutlookThe experiments showed that even in this early phase of deve-lopment, this novel “hollow-rotor” principle would fulfill the hydraulic specifications required for implantable ventricular assist devices. It demonstrated that the hollow-rotor has a high potential for successful clinical use. Especially the effi-ciency factor in comparison to volume is promising for intra-corporal blood conveyance.

Based on the verified functionality of the hollow-rotor concept, an extracorporeal operating VAD for in vitro tests will be de-veloped. The next steps comprise active/passive magnetic levi-tation, electrical drive, hydrodynamic design and haemocom-patibility. For an accurate prediction of flow field and shear stress Computational Fluid Dynamics (CFD) will be used.

References:[1] Marseille O. Entwicklungs- und Bewertungsverfahren für Rotationsblut-

pumpen. Dissertation. RWTH Aachen, 2001.

[2] Stepanoff AJ. Centrifugal and Axial Flow Pumps: Theory, Designs and

Application. 2nd ed., Florida: Krieger Publishing Company, 1992.

[3] Behbahani M, Behr M, Hormes M, Steinseifer U, Arora D, Coronado O, Pas-

quali M. A Review of Computational Fluid Dynamics. Euro J Applied Mathe-

matics 2009;20 363–397

Research Focus Area:Therapeutic Technologies (TT)

Project Team:Erik Schkommodau, Sandro Fabbri, Mathias Jeker (Institute for Medical and Analytical Technologies, School of Life Sciences FHNW)

Partner:Doan Baykut, Rudi Pfluger, Thomas Ruppli (Coras Medical AG)

Funding:Confederation’s Innovation Promotion Agency (CTI)

Economic efficiency and society benefit:In the industrial countries cardiovascular diseases are the most common causes of death. If the technology of LVADs is successful in terms of long time heart support the trans-plantation problems including their waiting procedures would become less severe.

Development of a Functional Model of a Hollow Rotor Axial Flow Pump for Cardiac Assistance

Left Ventricular Heart Assist Devices (LVAD) are becoming more and more important as a therapy option for severe heart desease. A novel pumping concept was developed and its operational effectiveness proved experimentally. Furthermore the hydraulic performance of this particular principle was compared quantitatively with a conventional axial blood pump.

Erik Schkommodaua, Sandro Fabbria, Matthias Jekera, Doan Baykutb

aInstitute for Medical and Analytical Technologies, School of Life Sciences FHNW, bCorasMedical AG

Keywords: Silica core-shell nanoparticles, 14C-labelling, fluorescent, polyethylene glycol

48 | 49School of Life Sciences

Figure 1: Electromechanical pump test stand

Figure 2a: Drive unit

Figure 2b: Hollow-rotor

Figure 3: Head-capacity curve of rotor #26

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Environmental Management (EM)

Holistic environmental management for the sustainable use of resources and the preservation of an environment worth living in.

IntroductionUntil now, handling human waste in all developed countries was based on flush toilets, using water as transporting, di-luting and flushing medium and at the same time reducing the nasty odors emanating especially from feces. The effort needed to clean waste water is enormous and increasing be-cause of more and more environmental constraints required by authorities. At the same time ‚new’ substances are being used which are not decomposed by the sewage station/plant, causing an increasing contamination of superficial as well as groundwater with antibiotics, medicines, hormones, etc. causing ecological problems. Until now there were no alter-natives to the flush toilet, besides the simple latrine, the chem ical toilet used on boats/yachts or the compost toilets in remote housing, which are all not fit for modern societies’ demand. Research has been done especially by Eawag, the Swiss Federal Institute of Aquatic Science and Technology, in the field of urine separating technologies [1]; the practical testing of the toilets had been undertaken mainly by the for-mer High School of Applied Sciences FHBB [2].

In the area of waste management, preventive resource pro-tection is of foremost importance. In the Research Focus Area of Environmental Management our School of Life Sciences FHNW is also concerned with innovative concepts in the fields of technology and strategy development of water and waste management, not only in industrialized countries, but

also in emerging and developing countries. Increasing water pollution caused by micropollutants, a greater organic-toxic burdening of ground and surface water and an aggravation of the water crisis in arid and semi-arid countries make sustain-able use of water essential. The development of a waterless toilet fits perfectly in that scheme. It is therefore a must, if we want to save water.

ResultsThe only solution to this challenge is a device where waste is automatically packed in plastic bags to avoid contact of humans with their own waste. There are no alternatives to bags: neither latrines nor compost toilets can be considered as modern means for a basic human need. Deep freezing or vacuuming all excrements is technically possible but extreme-ly expensive and energy consuming. So we had to solve the problem by using bags made of plastic or paper: the challenge was to put the bag in place, without using the hands. At the same time the bag has to be held in place, without being bound with pinching devices that could come into contact with urine or feces. The defecation act has to be as clean as possible. As one solution the bag could be prefabricated and then put in place for each use; technically very challenging. The bet-ter solution was to be formed on the spot, starting with an endless tube being stored under, but wrapped over the seat of the bowl and then stretched and pulled into and through

the bowl. After every use the portion of tube has to be sealed by heat, forming tight bags containing the waste. One of the main difficulties, beside the technical problems, was to find a plastic material which kept nasty odours from seeping or evaporating through the film. With the aid of renowned in-dustrial partners, and having tested many types of plastics, finally a nearly tight material was developed. It keeps back odours no longer than 8 to 10 days.

Conclusion and OutlookThe new toilet has been developed for use in hospitals, being mounted on wheels that enable the device to be moved from room to room, or more precisely from bed to bed. In this way patients can easily urinate or defecate without needing to be moved or helped to the ordinary washroom by personnel, thus avoiding embarrassing situations. The seat height can be adjusted individually, so every person can accommodate themselves. Between the hidden location of the seat at the beginning and the main seat position, an intermediate po-sition is intended where the seat is mounted in a slightly inclined position so that the toilet can be used as a urinal for men. The toilet is independent of electrical supply, two accumulators covering the supply needed especially for the sealing of the bags.

First hospital tests are being done during winter 2010/11. The results will show whether people are willing to use such a new toilet and whether they appreciate sitting on a plastic film covering the seat ring. Defecating is a very intimate hu-man necessity and it is not yet clear to what extent one can interfere in those habits.

There are many other possible applications where a water -less toilet could replace the ordinary flush toilet: e.g. in remote housing, buses, trains, yachts/boats etc. Early tests were undertaken e.g. in airplanes, showing the practical techno-logical difficulties such a new device has to cope with [3]. Contacts with entrepreneurs in the above mentioned fields show that there is reason to be optimistic concerning a real but slow change from using water toilets and moving to wa-terless techniques. It is clear that we will succeed only in the very long term and financial support for further testing will be needed.

References:[1] Larsen TA, Lienert J. Novaquatis final report: NoMix – A new approach to

urban water management. Eawag, Dübendorf, 2007

[2] Mayer M, Lüscher C. Urinseparation – Praktische sanitärtechnische Erfah-

rungen aus 2-jährigem Betrieb einer Anlage zur separaten Gewinnung von

Urin; Amt für industrielle Betriebe AIB, Liestal, 2004

[3] Wilfert SA. Konzeption einer wasserlosen Toiletteneinrichtung für den

Luftverkehr. Dissertation. RWTH Aachen, Fakultät für Maschinenwesen,

Aachen, Germany, 2005

Waterless Mobile Toilet for Hospital Use

Within this research project an entirely new toilet concept has been designed and developed. It works without water as flushing and transporting agent but uses a plastic sheet forming an endless tube, tightly rolled under the seat and being sealed after every use creating a sort of plastic bag. The new toilet saves water, and reduces or prevents the discharge of micropollutants in the environment.

Claude Lüscher, Julien FurstosSchool of Life Sciences FHNW

Keywords: Waterless toilet, water saving, micro-pollutant free sewage water


Research Focus Area: Environmental Management (EM)

Project Team:Claude Lüscher, Julien Furstos (Institute for Ecopreneurship, School of Life Sciences FHNW) and Henri Zinsli, München-stein

Partner: FOLAG AG Sempach; Zim-Clip GmbH Neuchâtel; espro AG Stallikon

Funding: Federal Office for the Environment (FOEN), Förderfonds Aar-gau, canton Aargau and Amt für Umwelt, canton Solothurn

Economic efficiency and benefit to society:Reduced water consumption and discharge of sewage water, thus reduced costs of water sanitation. Cleaner toilet, thus better hygiene and less danger of infection and poor hygiene.

Figure 1: Waterless toilet ready for move (left); ready for use (right)

IntroductionPhosphorus in the form of phosphates plays a crucial role as a building block of genes and in cell metabolism. For plants, phosphorus – after nitrogen – is the most important nutrient. Soil which is poor in phosphates limits plant growth, which leads to smaller crops. In order to sustain high harvests, the phosphates consumed by the crops must be continuously re-plenished. Traditionally the phosphates are brought back to the fields in the form of human and animal excrement as well as with compost. In industrialised agriculture, farmers also supply phosphates in the form of synthetic fertilizers such as triplesuperphosphate.

Fertilizer production consumes 90% of the annual raw phos-phate production today. Morocco, including Western Sahara, has large deposits and delivers almost half the fertilizer on the export market. In 2008 demand was higher than supp-ly and the world market price of phosphate rock surged by 700%. Phosphorus demand is increasing by some 2% per year (International Fertilizer Industry Association) due to rising world population, meat consumption and biofuel consump-tion. The phosphate ore that can be economically extracted will last another 50 years if the trend continues (approximated with data from U.S. Geological Survey [1]). Other deposits are available but mining will be more difficult and more costly. These other deposits also contain higher concentrations of heavy metals – in particular cadmium and uranium [2].

Sewage sludge is in general an important source of phospho-rus in European countries and its importance is rising as the number of households connected to wastewater treatment plants (WWTP) rises and the number of plants with elimina-tion of phosphorus increases. The most recent statistics indi-cate that sludge is produced in Europe containing 10 million tonnes of dry matter [3]. For cost reasons the main solution (40%) is to use the sludge directly on fields.

Several European countries, Switzerland among others, are developing processes to recover phosphorus from the waste water system, either from the sludge itself or from the ashes of the sludge. The Canton of Zürich wants to recover phos-phorus from sewage sludge while using the latter's energy content. We were asked to evaluate industrially implemented processes for recovery and energy use and thus to provide in-formation necessary for implementing a new sewage sludge treatment in 2015.

Results15 processes ready for full-scale operation were identified from the many options available [4]. 11 concerned phosphorus recovery and 4 the use of the energy in the sludge (mineraliza-tion). The process chains for recovery from a certain material (water, sludge or the residue after mineralization) are similar.After a preliminary evaluation the most advantageous pro-cesses of every type were chosen: for recovery from water,

sludge or the residue after mineralization. Process chains were formed reaching from the waste water treatment plant to landfill (Fig. 1). The process chains produce water which fulfils discharge criteria, phosphorus products suitable for agricul-ture or further processing and a solid suitable for landfill.

Process chain 0 is the reference chain without phosphorus re-covery. In process chain 1 the phosphorus is precipitated from the liquor from the sludge dewatering; the sludge is incinera-ted in a Municipal Solid Waste Incineration Plant (MSWI). In process chain 2 the dewatered sludge is dissolved in acid, the residue separated and phosphate precipitated from the liquid phase; the residue is incinerated in an MSWI.

Only some types of mineralization allow recovery of phospho-rus from the residue. Two unsuitable types are incineration in a cement plant, since the phosphorus is incorporated in the cement, and co-incineration in an MSWI, since the resulting ash has a low phosphorus content making recovery expensive. Suitable processes are either separate incineration of sludge (monoincineration) or transformation of the sludge energy to heat directly in the liquid phase (wet oxidation), both of which yield a fairly concentrated mineral residue. In process chains 3 to 6 monoincineration and wet oxidation are combined with two different recovery processes: the extraction of phosphorus from the mineral residue and the production of a phosphoric fertilizer raw material through removal of heavy metals.

The recovery potential, specific cost and specific energy de-mand of the process chains was evaluated (Picture 2):– The cost and energy demand of the recovery from process

liquid is lower than the reference chain since the advantage of co-incineration outweighs the rather small demands of this type of recovery. However, only a quarter of the availa-ble phosphorus can be recovered.

– Recovery from sludge or extraction from the mineral resi-due (process chains 2, 4 and 6) yields three quarters of the available phosphorus. However, those processes are expen-sive because of the high chemicals demand.

– The production of a phosphoric fertilizer raw material through removal of heavy metals from the residue (process chains 3 and 5) have a yield of almost 90% and the cost and energy demand is moderate, in the same range as the mining and production cost for the common fertiliser triplesuper-phosphate. The combination with monoincineration has higher cost, but is a more well-known technology with lower energy demand than wet oxidation. The product of the pro-cess is not sufficiently soluble for plants; further develop-ment is needed.

Conclusion and OutlookSwitzerland imports 16'000 t of phosphorus every year, main-ly in the form of fertilizers and fodder [5]. This is about 15% of the total agricultural demand for phosphorus. The surplus from imports ends up in three places: in cement, as MSWI-ash on a landfill or in the soil of the farmland as unused fertilizer. Switzerland could use the limited phosphorus resources in a more sustainable way. A reuse of phosphorus in the residues of monoincineration or wet oxidation would be one measure. Another would be reduced fertilizer consumption. In the Can-ton of Zürich a monoincineration plant is planned. The Federal Office for the Environment is revising waste disposal regulati-ons and is considering obligatory recovery of the phosphorus in wastewater.

References:[1] U.S. Geological Survey, Mineral Commodity Summaries, 2010. Available un-

der: http://minerals.usgs.gov/minerals/pubs/commodity/phosphate_rock/mcs-

2010-phosp.pdf

[2] Gilbert, N. Envrionment: The disappearing nutrient. Nature 2009; 461 (7265):

716–8

[3] RPA, Milieu, WRc. Environmental, economic and social impacts of the use of

sewage sludge on land. Consultation Report on Options and Impacts. Report for

the European Commission. 2008

[4] Hermann, L. Rückgewinnung von Phosphor aus der Abwasserreinigung.

Eine Bestandsaufnahme. Umwelt-Wissen Nr. 0929. Bern:Bundesamt für Um-

welt, 2009

[5] Binder CR, de Baan L, Wittmer D. Phosphorflüsse in der Schweiz. Stand,

Risiken und Handlungsoptionen. Umwelt-Wissen Nr. 0928. Bern:Bundesamt für

Umwelt, 2009

Research Focus Area:Environmental Management (EM)

Project Team:Anders Nättorp, Claude Lüscher, Thomas Wintgens (Institute for Ecopreneurship, School of Life Sciences FHNW)

Partner:Fredy Dinkel, Carbotech Ltd, Basel

Funding:Amt für Abfall, Wasser, Energie und Luft (AWEL)

Economic efficiency and benefit to society:The project makes an economic and ecological comparison of different processes which provides institutions and enter-prises with a good basis for sustainable decisions. The study shows that phosphorus recovery is, or will soon become, eco-nomically and ecologically advantageous.

Recovering Phosphorus and Closing the Nutrient Cycle

Today, the finite phosphorus deposits are used for production of industrial fertilizer for agricultural yields. In this project, processes for recovery of the phosphorus in wastewater, the most important as yet untapped phosphorus source, were evaluated from an economic and ecological point of view. The results contribute to the planning and implementation of sustainable disposal of sewage sludge in the Canton of Zürich

Anders Nättorp, Claude LüscherSchool of Life Sciences FHNW

Keywords: Phosphorus, Sustainable, Resource Management, Recovery, Incineration, Technology


WWTP

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Figure 1: The evaluated process chains: mineralization processes in orange and processes for phosphorus recovery in white. Figure 2: Evaluation of the process chains.

BackgroundSustainable and integrated management of water resources is one of the biggest global challenges. Clean freshwater is a limited and in many regions, scarce resource and rapid global changes such as population growth, economic development, migration and urbanization are placing new strains on wa-ter resources and on the infrastructure that supplies drin-king water to citizens, businesses, industry and institutions [1]. Ensuring a safe, sufficient and affordable water supply is therefore becoming an ever more demanding issue for politi-cians, researchers and water professionals.An aggravating factor, especially in developing and transi-tion countries, is the huge amount of water being lost through leaks in water distribution networks, called physical or ‘real’ water losses, and the volumes of water which are distribut-ed without being invoiced, called ‘apparent’ water losses. The sum of real and apparent water losses and unbilled authori-sed consumption constitute the so called Non-Revenue Water (NRW) of a water distribution network (see Fig. 1). In 2006, the World Bank estimated that in developing countries, non-revenue water accounts on average for 40–50% of the water produced.

Despite progress achieved in the past, water utilities are often still not aware of innovative management methods for water supply networks. They focus rather on the supply-side by de-veloping new resources, and give only second priority to the efficient distribution of water to the users. Demand-oriented water distribution requires good management of water supp-ly networks, including, amongst other aspects, the creation of network registers, hydraulic modelling, zoning of the network and the development of water demand scenarios. Based on this information, the technologies and design of the networks can be adapted to the needs with the selection of appropriate technologies for network adaptation and improvement. While efficient and effective solutions enabling water loss reduc-

tions are available, there is an obvious need for awareness raising, knowledge transfer, financing solutions, reference materials and capacity building.

Project ApproachFrom an integrated water resource management view, there is an obvious need to reduce water losses. In order to tackle this challenge, a Development Partnership with internatio-nal partners has been created. Within this project, FHNW is responsible for project management and coordination, orga-nisation of stakeholder dialogues, the project homepage and parts of the developed technical manual. The main objective of the development partnership is the red-uction of water losses through improved management of exi-sting water supply networks at different levels of intervention:

Development of guidelines for water loss reduction – a focus on pressure managementThese guidelines are meant to provide various stakeholders with relevant information on PM in order to understand the different types, reasons for and impacts of water losses, to develop a strategy for water loss reduction in water supply systems and to understand prerequisites, methods and in-struments for water loss reduction. This ambitious task is based on intensive dialogues, literature research, field expe-rience and working sessions. The guidelines are composed of three documents: a summary brochure for decision-makers, a technical manual for planning and design as well as comple-mentary working materials.

Capacity development through dialogues, training and on-the-job instructions To allow the implementation of the guidelines, target group oriented workshops and training sessions are necessary. Based on an assessment of needs for three target groups (1. policy and management; 2. planning and design; 3. imple-mentation, operation and maintenance), tailor-made training plans and materials have been developed for the design, in-stallation and management of pressure management areas including valve design, maintenance and repair. The content of the guidelines provides the framework for a comprehen-sive training programme for technical directors and techni-cians of water utilities as well as potential trainers for local training institutions. Capacity building plays a key role within the project, as it can ensure education of staff and sustain the technology implementation and operation. It also raises the quality of the system and the understanding and commitment of the staff.

Furthermore, new pipe breaks have been reduced by approxi-mately 50% with a project payback time of 4–5 months.

Conclusion and OutlookKnowledge is an important precondition for managing water losses effectively and efficiently. The guidelines developed for water loss reduction should help to transfer knowledge about recent developments for efficient water loss management. Of-fering the guidelines with working materials in combination with training measures and pilot projects for pressure man-

agement implementation is seen as an effective approach for a long-term reduction of leakage from water distribution net-works and more sustainable water resources management. References:[1] World Water Council, Istanbul Water Consensus for Local and Regional

Authorities. Proceedings of the 5th World Water Forum, Istanbul, Turkey, 2009.

[2] Lambert AO, Hirner W. Losses from Water Supply Systems: Standard Ter-

minology and Recommended Performance Measures. International Water As-

sociation (IWA), 2000.

[3] Knobloch A, Klingel P, Oertlé E, Mutz D, Fallis P, Sorg F, Ziegler D. Guide-

lines for Water Loss Reduction – A Focus on Intelligent Pressure Management.

Proceedings of the IWA International Specialised Conference ‘Water Loss

2010‘, São Paulo, Brazil 2010.

[4] Thornton J, Lambert AO. Progress in practical prediction of pressure: leak-

age, pressure: burst frequency and pressure: consumption relationships.

Proceedings of the IWA Specialised Conference 'Leakage 2005', Halifax, Nova

Scotia, Canada, 2005.

[5] Oppinger P. Reducing Water Losses by Intelligent Pressure Management,

Water & Wastewater International, 2009.


Project Team: Emmanuel Oertlé, Dieter Mutz (Institute for Ecopreneurship IEC, School of Life Sciences FHNW)

Partner: Deutsche Gesellschaft für Internationale Zusammenarbeit (GIZ) GmbH, VAG-Armaturen GmbH (VAG), Karlsruhe Insti-tute of Technology (KIT).

Funding: German Ministry for Economic Cooperation (BMZ) and VAG-Armaturen GmbH (VAG).

Economic efficiency and benefit to society:Pressure management can be an immediate and cost effective solution: Savings can range from 0.6 up to 3.4 million USD/ year/network, and payback times are normally a few months. Furthermore, “Reducing the amount of water losses by half would generate enough water to supply an additional 90 mil-lion people in the developing countries.” (World Bank, 2006)

Water Loss Reduction – A Focus on Pressure Management

Customers worldwide expect to be supplied with affordable drinking water in sufficient quantity, quality and con-tinuity, but water losses often represent a major challenge to achieving these aims. Thus, we are involved in a part-nership to introduce, promote and support Pressure Management (PM) as a best practice instrument for water loss reduction, especially for developing countries and emerging economies.

Emmanuel OertléSchool of Life Sciences FHNW

Keywords: Water Loss Reduction, Pressure Management, Guidelines, Technical Cooperation.


Demonstration of improved management in pilot areas The technical implementation of the guidelines will be exa-mined together with the implementation of the required tech-nical installations for pressure management in selected pilot areas (Fig. 2). The pilot projects will serve as practical exam-ples for the discussion on best practices regarding design and management of water supply networks.

The Pressure Management Homepage – www.waterloss-reduction.comIn order to exchange information, facilitate contacts and al-low downloading the guidelines free of charge, the project homepage is available at the following URL: www.waterloss-reduction.com.

Pressure Management (PM)The guidelines developed highlight pressure management as an efficient and effective measure to reduce real water losses. Pressure management can be defined as the ‘practice of man-aging distribution network pressures to the optimum levels of service while ensuring sufficient and efficient supply to legitimate uses’ [4]. The goal is to reduce unnecessary pressure and to even out pressure variations in order to reduce leak-age flow rates and to prevent pipes from bursting. Pressure management uses advanced valve technologies that require advanced design, installation and management skills. The Example of Santo Amaro, São Paulo, BrazilIn the metropolitan area of São Paulo, with continuously in-creasing water consumption, it has become a challenge for the local water utility (namely the state-owned Companhia de Saneamento Básico do Estado de São Paulo, SABESP) to provide a constant supply of water to more than 17 million inhabitants. One reason for the difficulty of continuous water supply is the high volume of water losses that occur during operation due to leakage. If the pressure can be managed ef-fectively, then water loss will be reduced [5].In the District of Santo Amaro, VAG and SABESP has installed a pilot site consisting of a sophisticated water loss reduc-tion system that includes pressure regulating valves, control panels, telemetry installations and the supporting software [5]. Two technologies were implemented: in a first stage, time-based modulation and in a second stage, remote node based modulation, which resulted in the benefits shown in Fig. 3.

Figure 2: Implementation of pressure management component in Ain Al Basha, Jordan, where water losses were reduced by up to 40%

Figure 1: Standard terminology for the water balance according to IWA [2]

Figure 3: Results obtained in the district of Santo Amaro in Brazil between January 2005 and June 2006

Technology Period Water losses Water savings (m3/month) (%)

WithoutPM Jan.2005–Sep.2005 301.7 –

Timebasedmodulation Oct.2005–Jan.2006 203.9 -33%

Remotenodebasedmodulation Feb.2006–Jun.2006 178.0 -41%

IntroductionNitrification (biological oxidation of ammonia nitrogen to ni-trate) is carried out by autotrophic bacteria which are more sensitive to different inhibitory effects compared to hetero-trophic bacteria. Inhibitory effects of various compounds on nitrification can occur with some industrial wastewaters, es-pecially those from the pharmaceutical or chemical industry. To prevent such problems, industrial wastewaters are often treated together with municipal wastewaters which leads to dilution of the active substance of industrial waters. In many cases the desired treatment efficiency is only reached after sufficiently long adaptation of activated sludge. Industrial or pharmaceutical wastewater can contain high amounts of organic matter and salts, color, poorly biodegra-dable organic compounds or substances which inhibit the activity of nitrifiying microorganisms [1]. The application of MBR technology can be a promising option for the treatment of industrial wastewaters, particularly if nitrification is re-quired [2,3]. Legislation both in Switzerland and the EU requires nitrogen removal from municipal wastewater before its discharge into water bodies. For example, the Swiss water protection ordi-nance (Gewässerschutzverordnung) requires a nitrification efficiency of up to 90% depending on local conditions and an effluent concentration of ammonia nitrogen below 2 mg/L [4]. In this research project a membrane bioreactor (120 L, hollow fibres; Fig. 1) was continuously fed by a mixture of munici-pal and industrial wastewater and served as an adaptation reactor. The loading of the MBR with industrial wastewater was increased in steps, so that its proportion of the mixed wastewater was 0, 5, 10, 20 and 30% respectively. The length of each step was 3–4 weeks. Adaptation of activated sludge to industrial wastewater was evaluated based on the chemi-cal analysis of influent and effluent and measurement of the respiration activity of activated sludge. At regular intervals the influence of the industrial wastewater was compared to a non-adapted population from another municipal nitrifying wastewater treatment plant (reference activated sludge).

Results of respirometric testsThe respirometric tests served for the evaluation of nitrifica-tion activity. At weekly intervals a sample of activated sludge was withdrawn from the MBR and subjected to a series of respirometric tests with different ratios of municipal to in-dustrial wastewater ranging from 0 to 100%. Results of respi-rometric tests relating to activated sludge from the MBR and referential sludge are shown in figures 2 and 3 respectively. Samples of reference activated sludge were taken from ano-ther wastewater treatment plant and were not exposed to the

influence of industrial wastewater before the test. The maximum inhibition effect of the industrial wastewater on each sample was evaluated by the decrease in the nitrifica-tion respiration rate in the test with 100% industrial wastewa-ter compared to the test with 0% industrial wastewater. Before the start-up of the adaptation reactor the inhibition rate was

about 95%; after 164 days of adaptation and stepped increase of industrial wastewater in the influent into the MBR, the in-hibition was only 61%. In contrast, the inhibition effect on the samples of reference activated sludge did not change substan-tially during the whole experimental period and remained in the range of 83–92% (Fig. 3).Noticeable peaks in the Ammonia Utilization Rate (AUR) were observed during most of the tests for tested ratios of 15%. This

fact can be explained by the possible presence of microorga-nisms which have similar metabolism compared to nitrifying bacteria. It means that their activity could be inhibited by al-lylthiourea although they were able to oxidize some organic compounds of industrial wastewater. Thus their respiration

activity will be increased after the addition of a small amount of industrial wastewater; due to the respiration test methodo-logy, their activity will be measured together with the nitri-fiers. With higher proportions of industrial wastewater (more than 30%), the inhibitory effect of industrial wastewater was more pronounced and thus a decrease in AURs by all kind of microorganisms was observed.

Results of chemical analysesThe results of regular chemical analyses showed that the in-crease in the content of industrial wastewater in the MBR influent had almost no influence on the concentration of am-monia nitrogen in the effluent from the MBR. The average con-centration of ammonia nitrogen in the effluent was 0.38 mg/L . In addition, the concentration of ammonia nitrogen was very stable during the whole adaptation period, which also contri-buted to the overall high efficiency of ammonia nitrogen remo-val – on average 98%. In terms of nitrite nitrogen concentration, its very high con-centration (up to 10 mg/L nitrite nitrogen) was detected in the initial period of MBR operation, probably as a consequence of transition conditions after the transfer of the seed into the new conditions in the pilot plant. However after the stabili-zation of MBR operation, the nitrite nitrogen concentration decreased to 0.6 mg/L and remained stable. The Chemical Oxygen Demand (COD) concentration in the effluent was influenced by the organic load in the influent wastewater. Its slight long-term increase was observed during the whole MBR operation. A sharp increase in COD concentra-tion in the effluent took place after each proportional increase in industrial wastewater in the influent. However, during the individual adaptation intervals (constant share of industrial wastewater in the MBR influent) a slow decline of the COD concentration was observed. On average the COD removal ef-ficiency was above 88%. From the results mentioned above it is obvious that the ap-plication of membrane technology is promising for industri-al wastewater treatment, particularly if good effluent quality and high efficiency of nitrification should be achieved.

Conclusion and OutlookFrom the study it can be concluded that a long-term (164 day) and stepped adaptation of activated sludge resulted in a 34% decline in the inhibitory effect of industrial wastewater on nitrifying bacteria. A steady increase in AUR during the ni-trification tests conducted under the addition of industrial wastewater only (100% share of industrial wastewater) was observed during the whole MBR operation period. The increase in the industrial wastewater proportion in the MBR influent had no significant influence on the concentra-tion of ammonia nitrogen in the effluent with an average of 0.38 mg/L , corresponding to an approximate removal rate of 98%. A slow increase in the COD concentration in the effluent was observed during the whole MBR operation with increa-sing industrial wastewater loading; the average COD removal efficiency was 88%. The project will continue with a further increase in industrial wastewater load to 50, 75 and 100%. Because of the higher effluent COD concentration resulting from a higher propor-tion of industrial wastewater in the influent, post-treatment processes, such as sorption on activated carbon or ozonation will be tested.

References:[1] Wei X, Wang Z, Fan F, Wang J, Wang S. Advanced treatment of a complex

pharmaceutical wastewater by nanofiltration: Membrane foulant identifica-

tion and cleaning, Desalination 2010; 251 (1-3): 167–175

[2] Li H, Yang M, Zhang Y, Yu T, Kamagata Y. Nitrification performance and

microbial community dynamics in a submerged membrane bioreactor with

complete sludge retention. J. Biotechnol. 2006; 123 (1): 60–70

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[4] Swiss Federal Council. Gewässerschutzverordnung des Schweizerischen

Bundesrates vom 28. Oktober 1998.


Project Team: Lukas Dvorak (Institute for Ecopreneurship, School of Life Sciences FHNW and Department of Water Technology and Environmental Engineering, ICT Prague)Jan Svojitka, Thomas Wintgens (Institute for Ecopreneurship, School of Life Sciences FHNW)

Partner: ProRheno AG, Basel, SwitzerlandInstitute of Chemical Technology Prague, Czech Republic

Funding: Sciex-NMSch (Scientific Exchange Programme between Swit-zerland and the New Member States of the EU),

Economic efficiency and benefit to society: Nitrifying bacteria adapted to the treatment of industrial wastewater represent a cheaper option for the removal of nitrogen from water compared to physical or chemical meth-ods. The application of a membrane bioreactor contributes to overall higher quality of effluent as well. The good qual-ity of effluent means reduction in fees for discharging of wastewater into water bodies and has undisputed ecological benefits.

Adapting a Membrane Bioreactor (MBR) to Industrial Wastewater Treatment

The influence of industrial (pharmaceutical and chemical) wastewater on MBR performance was investigated. A key parameter was the process of nitrification, which was evaluated based on influent and effluent parameters and kinetic testing of nitrification. The loading of the MBR with industrial wastewater was gradually increased to study the adaptation effect of nitrifying bacteria.

Lukas Dvorak, Jan Svojitka, Thomas Wintgens School of Life Sciences FHNW

Keywords: membrane bioreactor, industrial wastewater, activated sludge, nitrification, adaptation


Figure 1: Photo of adaptation membrane bioreactor

Figure 2: AUR for tested shares of industrial wastewater (IWW) under diffe-rent IWW concentrations in the MBR influent

Figure 3: AUR for tested shares of IWW - reference activated sludge

Appendix

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Institute for Chemistry and Bioanalytics (ICB)

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Beck K, Vannini A, Cramer P, Lipps G. The archaeo-eukaryotic primase of plasmid pRN1 requires a helix bundle domain for faithful primer synthesis. Nucleic Acids Res 2010;38 (19):6707–18.

Berkner S, Lipps G. Genetic tools for Sulfolobus spp.: vectors and first applica-tions. Arch. Microbiol. 2008;190 (3):217–30.

Berkner S, Lipps G. Mutation and reversion frequencies of different Sulfolobus species and strains. Extremophiles 2008;12 (2):263–70.

Berkner S, Wlodkowski A, Albers SV, Lipps G. Inducible and constitutive promoters for genetic systems in Sulfolobus acidocaldarius. Extremophiles 2010;14 (3):249–59.

Coleman AW, Jebors S, Shahgaldian P, Ananchenko GS, Ripmeester JA. Para-Acylcalix[n]arenes from molecular to macrosco-pic assemblies. Chem. Commun. (Cambridge, U. K.) 2008 (20):2291–303.

Detsch R, Dieser I, Deisinger U, Uhl F, Hamisch S, Ziegler G, Lipps G. Biofunctionalization of dispense-plotted hydroxyapatite scaffolds with peptides: Quantification and cellular response. Journal of Biomedical Materials Research Part A 2010;92A (2):493–503.

Ehrler S, Pieles U, Wirth-Heller A, Shahgaldian P. Surface modification of resorcinarene based self-assembled solid lipid nanoparticles for drug targeting. Chem. Commun. (Cambridge, U. K.) 2007 (25):2605–7.

Elend D, Pieles U, Shahgaldian P. Para-Carboxy Modified Amphiphilic Calixarene, Self-As-sembly and Interactions with Pharmaceutically-Relevant Molecules. CHIMIA International Journal for Chemistry 2010;64:45–8.

Escher C, Lochmüller H, Fischer D, Frank S, Reimann J, Walter MC, Ehrat M, Ruegg MA, Gygax D. Reverse protein arrays as novel approach for protein quanti-fication in muscular dystrophies. Neuromuscular Disorders 2010;20 (5):302–9.

Escher CA, Lochmüller H, Walter MC, Ehrat M, Reimann J, Ruegg MA, Gygax D. M.P.4.07 Reverse protein arrays for efficient protein diagno-sis of muscular dystrophies in less than 10 mg muscle tissue. Neuromuscular Disorders 2009;19 (8–9):606.

Fasler-Kan E, Barteneva n, Ketterer S, Wunderlich K, Huwyler J, Gygax D, Flammer J, Meyer P. Activation of the JAK–STAT intracellular pathway in human retinal pigment epithelial cell line ARPE-19. International Journal of Interferon, Cytokine and Mediator Research 2010;2:127–36.

Fasler-Kan E, Suenderhauf C, Barteneva N, Poller B, Gygax D, Huwyler J. Cytokine signaling in the human brain capillary endothelial cell line hCMEC/D3. Brain Research 2010;1354:15–22.

Geranio L, Hommes G, Shahgaldian P, Wirth-Heller A, Pieles U, Corvini P. Radio (14C)- and fluorescent-doubly labeled silica nanopar-ticles for biological and environmental toxicity assessment. Environmental Chemistry Letters 2010 (8):247–51.

Gerspacher C, Scheuber U, Schiera G, Proia P, Gygax D, Di Liegro I. The effect of cadmium on brain cells in culture. Int J Mol Med 2009;24 (3):311–8.

Helttunen K, Nauha E, Kurronen A, Shahgaldian P, Nissinen M. Conformational polymorphism and amphiphilic properties of resorcinarene octapodands. Organic & Biomolecular Chem-istry 2011;9 (3):906–14.

Helttunen K, Shahgaldian P. Self-assembly of amphiphilic calixarenes and resorcinare-nes in water. New J. Chem. 2010;34 (12):2704–14.


Joesch C, Guevarra E, Parel SP, Bergner A, Zbinden P, Konrad D, Albrecht H. Use of FLIPR membrane potential dyes for validation of high-throughput screening with the FLIPR and micro ARCS technologies: identification of ion channel modulators ac-ting on the GABAA receptor. J. Biomol. Screening 2008;13 (3):218–28.

Köser J, Shahgaldian P, Bammerlin M, Battiston FM, Pieles U. Time resolved analysis of molecular interactions using nano mechanical cantilever sensors. J. Phys.: Conf. Ser. 2007;61:612–7.

Kruse A, Pieles U, Riener MO, Zunker C, Bredell MG, Gratz KW.Craniomaxillofacial fibrous dysplasia: a 10-year database 1996–2006. Br J Oral Maxillofac Surg 2009;47 (4):302–5.

Leder L, Stark W, Freuler F, Marsh M, Meyerhofer M, Stettler T, Mayr LM, Britanova OV, Strukova LA, Chudakov DM, Souslova EA. The Structure of Ca2+ Sensor Case16 Reveals the Mechanism of Reaction to Low Ca2+ Concentrations. Sensors 2010;10 (9):8143–60.

Legrand S, Catheline A, Kind L, Constable EC, House-croft CE, Landmann L, Banse P, Pieles U, Wirth-Heller A. Controlling silica nanoparticle properties for biomedical applications through surface modification. New J. Chem. 2008;32 (4):588–93.

Leparoux M, Leconte Y, Wirth A, Buehler T.In Situ Treatment of Thermal RF Plasma Processed Nano-powders to Control their Agglomeration and Dispersability. Plasma Chemistry and Plasma Processing 2010;30 (6):779–93.

Lipps G. Molecular biology of the pRN1 plasmid from Sulfolobus is-landicus. Biochem. Soc. Trans. 2009;37 (1):42–5.

Meili-Butz S, Niermann T, Fasler-Kan E, Barbosa V, Butz N, John D, Brink M, Buser PT, Zaugg CE. Dimethyl fumarate, a small molecule drug for psoriasis, inhi-bits Nuclear Factor-kB and reduces myocardial infarct size in rats. Eur. J. Pharmacol. 2008;586 (1–3):251–8.

Nault L, Cumbo A, Pretot RF, Sciotti MA, Shahgaldian P. Cell transfection using layer-by-layer (LbL) coated calixarene- based solid lipid nanoparticles (SLNs). Chemical Communi-cations 2010;46 (30):5581–3.

Noser J, Schmutz H-R, Schmid S, Schneider P. Bestimmung von Enniatinen in Getreideprodukten aus dem Schweizer Markt. Lebensmittelchemie 2007;61 (3):66–7.

Prato S, Vitale RM, Contursi P, Lipps G, Saviano M, Rossi M, Bartolucci S. Molecular modeling and functional characterization of the monomeric primase-polymerase domain from the Sulfolobus solfataricus plasmid pIT3. Febs J. 2008;275 (17):4389–402.

Saxer S, Pieles U, Elsener M, Horisberger M, Tosatti S, Textor M, Gademann K, Zürcher S. Design of a high-throughput device for screening surface mo di fication protocols. Progress in Organic Coatings 2009;67 (1):20–7.

Shahgaldian P. Macrocyclic Molecules for the Design of Self-Assembling Amphiphiles. CHIMIA International Journal for Chemistry 2010;64:427.

Shahgaldian P, Sciotti MA, Pieles U. Amino-Substituted Amphiphilic Calixarenes: Self-Assembly and Interactions with DNA. Langmuir 2008;24 (16):8522–6.

Shahgaldian P, Wirth A, Pieles U. Nanoparticulate systems: a new competence platform at the University of Applied Sciences Northwestern Switzerland (FHNW). Chimia 2008;62 (5):441–3.

Spies P, Chen GJ, Gygax D. Establishment of a miniaturized enzyme-linked immuno-sorbent assay for human transferrin quantification using an intelligent multifunctional analytical plate. Anal. Biochem. 2008;382 (1):35–9.

Spies P, Mueller R, Chen GJ, Gygax D. A simple approach to improve the sensitivity of enzyme-linked immunosorbent assay using the IMAPlate 5RC96 for result readout. Anal. Biochem. 2010;397 (1):48–52.

Toan V, Thao V, Walder J, Schmutz H-R, Ha C. Contamination by Selected Organochlorine Pesticides (OCPs) in Surface Soils in Hanoi, Vietnam. Bulletin of Environmental Contamination and Toxicology 2007;78 (3):195–200.

Toan V, Thao V, Walder J, Schmutz H-R, Ha C. Level and Distribution of Polychlorinated Biphenyls (PCBs) in Surface Soils from Hanoi, Vietnam. Bulletin of Environ-mental Contamination and Toxicology 2007;78 (3):211–6.

Vonwil D, Wendt D, Stroebel S, Wallny HJ, Gygax D, Heberer M, Martin I. Assessment of the stability of TGFbeta 3 bioactivity for poten-tial bioreactor applications. Biochem. Eng. J. 2008;39 (3):586–9.

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Waser M, Siebenhaar C, Zampese J, Grundler G, Constable E, Height M, Pieles U. Novel Grafting Procedure of Ruthenium 2,2':6',2-Terpyridine Complexes with Phosphonate Ligands to Titania for Water Splitting Applications. CHIMIA International Journal for Chemistry 2010;64 (5):328–9.

Zenker A, Schmutz H, Fent K. Simultaneous trace determination of nine organic UV-ab-sorbing compounds (UV filters) in environmental samples. J. Chromatogr., A 2008;1202 (1):64–74.

Publications

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Institute for Ecopreneurship (IEC)

Bayer C, Follmann F, Melin T, Wintgens T, Larsson K, Almemark M. The ecological impact of membrane-based extraction of phe-nolic compounds – a life cycle assessment study. Water Sci-ence & Technology–WST 2010;62 (4):915–9.

Boeni H, Mutz D. Cleaner Production und Transfer von umweltfreundlichen Technologien. Die Volkswirtschaft 2008;4:51–4.

Bütehorn S, Carstensen F, Wintgens T, Melin T, Volmering D, Vossenkaul K. Permeate flux decline in cross-flow microfiltration at constant pressure. Desalination 2010;250 (3):985–90.

Bütehorn S, Brannock M, Pierre-Le-Clech, Leslie G, Volmering D, Vossenkaul K, Wintgens T, Melin T. Observation of cake layer formation and removal on micropo-rous hollow-fibre membranes. Desalination and Water Treat-ment 2009 (9):82–5.

Caminada D, Zaja R, Smital T, Fent K. Human pharmaceuticals modulate P-gp1 (ABCB1) transport activity in the fish cell line PLHC-1. Aquat. Toxicol. 2008;90 (3):214–22.

Christen V, Caminada D, Arand M, Fent K. Identification of a CYP3A form (CYP3A126) in fathead minnow (Pimephales promelas) and characterisation of putative CY-P3A enzyme activity. Analytical and Bioanalytical Chemistry 2010;396 (2):585–95.

Christen V, Crettaz P, Oberli-Schrämmli A, Fent K. Some flame retardants and the antimicrobials triclosan and triclocarban enhance the androgenic activity in vitro. Che-mosphere 2010;81 (10):1245–52.

Christen V, Hickmann S, Rechenberg B, Fent K. Highly active human pharmaceuticals in aquatic systems: A concept for their identification based on their mode of action. Aquatic Toxicology 2010;96 (3):167–81.

Christen V, Oggier DM, Fent K. A Microtiter-Plate Based Cytochrome P4503A Activity Assay in Fish Cell Lines. Environmental toxicology and chemistry/SETAC 2009.

Cirja M, Hommes G, Ivashechkin P, Prell J, Schäffer A, Corvini P, Lenz M. Impact of bio-augmentation with Sphingomonas sp. strain TTNP3 in membrane bioreactors degrading nonylphenol. Ap-plied Microbiology and Biotechnology 2009;84 (1):183–9.

Cirja M, Ivashechkin P, Schaeffer A, Corvini PFX. Factors affecting the removal of organic micropollutants from wastewater in conventional treatment plants (CTP) and membrane bioreactors (MBR). Rev. Environ. Sci. Bio/Technol. 2008;7 (1):61–78.

Cirja M, Zuehlke S, Ivashechkin P, Hollender J, Schaeffer A, Corvini PFX. Behavior of two differently radiolabeled 17alpha -ethinyles-tradiols continuously applied to a laboratory-scale membrane bioreactor with adapted industrial activated sludge. Water Res. 2007;41 (19):4403–12.

Corvini P, Shahgaldian P. LANCE: Laccase-nanoparticle conjugates for the elimination of micropollutants (endocrine disrupting chemicals) from wastewater in bioreactors. Reviews in Environmental Science and Biotechnology 2010;9 (1):23–7.

Corvini PFX, Meesters R, Mundt M, Schaeffer A, Schmidt B, Schroeder HF, Verstraete W, Vinken R, Hollender J. Contribution to the detection and identification of oxidation metabolites of nonylphenol in Sphingomonas sp. strain TTNP3. Biodegradation 2007;18 (2):233–45.

Evangelou M, Ebel M, Hommes G, Schaeffer A. Biodegradation: The Reason for the Inefficiency of Small Or-ganic Acids in Chelant-Assisted Phytoextraction. Water, Air, & Soil Pollution 2008.

Fent K. Ecotoxicology of engineered nanoparticles. In: Frimmel FH, Niessner R, editors. Nanoparticles in the Water Cycle: Sprin-ger, Berlin Heidelberg, 2010.

Fent K. Effects of Pharmaceuticals on Aquatic Organisms. In: Küm-merer K, editor. Pharmaceuticals in the Environment: Sprin-ger, Berlin Heidelberg, 2008. pp. 175–203.

Fent K. Ökotoxikologie: Georg Thieme, Stuttgart, 2007.

Fent K. Permanent Fisch Cell Cultures as Important Tools in Ecotoxi-cology. ALTEX 2007;24 (Special Issue):26–8.

Fent K, Kunz PY, Gomez E. UV Filters in the Aquatic Environment Induce Hormonal Ef-fects and Affect Fertility and Reproduction in Fish. CHIMIA International Journal for Chemistry 2008;62 (5):368–75.

Fent K, Kunz PY, Zenker A, Rapp M. A tentative environmental risk assessment of the UV-filters 3-(4-methylbenzylidene-camphor), 2-ethyl-hexyl-4-trimetho-xycinnamate, benzophenone-3, benzophenone-4 and 3-benzyli-dene camphor. Mar. Environ. Res. 2010;69 (Supplement 1):S4–S6.

Fent K, Kunz PY, Zenker A, Rapp M. A tentative environmental risk assessment of the UV-filters 3-(4-methylbenzylidene-camphor), 2-ethyl-hexyl-4-trimetho-xycinnamate, benzophenone-3, benzophenone-4 and 3-benzy-lidene camphor. Mar Environ Res 2009;10:10.

Fent K, Weisbrod CJ, Wirth-Heller A, Pieles U. Assessment of uptake and toxicity of fluorescent silica nano-particles in zebrafish (Danio rerio) early life stages. Aquatic Toxicology 2010;100 (2):218–28.

Fent K, Zenker A, Rapp M. Widespread occurrence of estrogenic UV-filters in aquatic ecosystems in Switzerland. Environmental Pollution 2010;158 (5):1817–24.

Geranio L, Hommes G, Shahgaldian P, Wirth-Heller A, Pieles U, Corvini P. Radio (14C)- and fluorescent-doubly labeled silica nanopar-ticles for biological and environmental toxicity assessment. Environmental Chemistry Letters 2010 (8):247–51.

Goksoeyr A, Tollefsen KE, Grung M, Loeken K, Lie E, Zenker A, Fent K, Schlabach M, Huber S. Balsa Raft Crossing the Pacific Finds Low Contaminant Le-vels. Environ. Sci. Technol. 2009;43 (13):4783–90.

Heim T, Hengevoss D. Mit Cleaner Production zu mehr Ressourceneffizienz. io new management 2010 (1004).

Hengevoss D. Umweltverträgliche Abfallentsorgung. Umwelt Perspektiven 2007 (1).

Hengevoss D. Umweltverträgliche Entsorgung von Abfällen. Blickwechsel – das Magazin des Lichtensteinischen Entwicklungsdienstes (LED) 2007 (2):13–4.

Hochstrat R, Wintgens T, Kazner C, Jeffrey P, Jefferson B, Melin T. Managed aquifer recharge with reclaimed water: approaches to a European guidance framework. Water Science & Techno-logy-WST 2010;62 (6):1265–73.

Hochstrat R, Wintgens T, Kazner C, Melin T, Gebel J. Options for water scarcity and drought management – the role of desalination. Desalination and Water Treatment 2010;18:96–102.

Hullebusch EDv, Rossano S, Farges F, Lenz M, Labanowski J, Lagarde P, Flank AM, Lens PNL. Sulfur K-edge XANES spectroscopy as a tool for understan-ding sulfur chemical state in anaerobic granular sludge. J. Phys.: Conf. Ser. 2009;190:012184.

Kazner C, Meier J, Wintgens T, Melin T. Capillary nanofiltration coupled with powdered activated carbon adsorption for high quality water reuse Water Science and Technology 2009;60 (1):251–9.

Kochan J, Wintgens T, Hochstrat R, Melin T. Impact of wetting agents on the filtration performance of polymeric ultrafiltration membranes. Desalination 2009;241 (1–3):34–42.

Kochan J, Wintgens T, Melin T, Wong J. Characterization and filtration performance of coating-mo-dified polymeric membranes used in membrane bioreactors. Chemical Papers 2009;63 (2):152–7

Kochan J, Wintgens T, Wong JE, Melin T. Polyelectrolyte-modified polyethersulfone ultrafiltration membranes for wastewater treatment applications. Desali-nation and Water Treatment 2009 (9):175–80.

Kochan J, Wintgens T, Wong JE, Melin T. Properties of polyethersulfone ultrafiltration membranes mo-dified by polyelectrolytes. Desalination 2010;250 (3):1008–10.

Kolvenbach B, Schlaich N, Raoui Z, Prell J, Zuehlke S, Schaeffer A, Guengerich FP, Corvini PFX. Degradation pathway of bisphenol A: does ipso substituti-on apply to phenols containing a quaternary alpha -carbon structure in the para position? Appl. Environ. Microbiol. 2007;73 (15):4776–84.

Kouloumbos VN, Schaeffer A, Corvini PFX. Impact of sewage sludge conditioning and dewatering on the fate of nonylphenol in sludge-amended soils. Water Res. 2008;42 (14):3941–51.

Kouloumbos VN, Schaeffer A, Corvini PFX. The role of sludge conditioning and dewatering in the fate of nonylphenol in sludge-amended soils. Water Sci. Technol. 2008;57 (3):329–35.

Kunz PY, Fent K. Estrogenic activity of ternary UV filter mixtures in fish (Pi-mephales promelas) – An analysis with nonlinear isobolo-grams. Toxicology and Applied Pharmacology 2009;234 (1):77–88.

Lenz M, Aelst ACv, Smit M, Corvini PFX, Lens PNL. Biological Production of Selenium Nanoparticles from Waste Waters. Advanced Materials Research 2009;71–73:721–4.

Lenz M, Enright A, O’Flaherty V, van Aelst A, Lens P. Bioaugmentation of UASB reactors with immobilized Sulfuro-spirillum barnesii for simultaneous selenate and nitrate remo-val. Applied Microbiology and Biotechnology 2009;83 (2):377–88.

Lenz M, Hullebusch EDv, Farges F, Corvini PFX, Lens PNL. Biological Selenium Remediation – A Simple Process Compli-cated. 19th Annual V.M. Goldschmidt Conference. Davos: Geo-chimica et Cosmochimica Acta, 2009. pp. A743.

Lenz M, Lens PNL. The essential toxin: the changing perception of selenium in environmental sciences. The Science of the total environment 2009;407 (12):3620–33.

Lenz M, van Hullebusch ED, Farges F, Nikitenko S, Borca CN, Grolimund D, Lens PNL.Selenium Speciation Assessed by X-Ray Absorption Spectro-scopy of Sequentially Extracted Anaerobic Biofilms. Environ. Sci. Technol. 2008;42 (20):7587–93.


Lenz M, van Hullebusch ED, Farges Fo, Nikitenko S, Corvini PFX, Lens PNL. Combined Speciation Analysis by X-ray Absorption Near-Edge Structure Spectroscopy, Ion Chromatography, and So-lid-Phase Microextraction Gas Chromatography−Mass Spec-trometry To Evaluate Biotreatment of Concentrated Selenium Wastewaters. Environ. Sci. Technol. 2011;45 (3):1067–73.

Lenz M, Hullebusch EDV, Hommes G, Corvini PFX, Lens PNL.Selenate removal in methanogenic and sulfate-reducing up-flow anaerobic sludge bed reactors. Water Research 2008;42 (8–9):2184–94.

Lenz M, Janzen N, Lens PNL. Selenium oxyanion inhibition of hydrogenotrophic and ace-toclastic methanogenesis. Chemosphere 2008;73 (3):383–8.

Lenz M, Smit M, Binder P, van Aelst AC, Lens PNL. Biological Alkylation and Colloid Formation of Selenium in Methanogenic UASB Reactors. J Environ Qual 2008;37 (5):1691–700.

Levantesi C, La Mantia R, Masciopinto C, Böckelmann U, Ayuso-Gabella MN, Salgot M, Tandoi V, Van Houtte E, Wintgens T, Grohmann E. Quantification of pathogenic microorganisms and microbial indicators in three wastewater reclamation and managed aquifer recharge facilities in Europe. Sci. Total Environ. 2010;408 (21):4923–30.

Li C, Ji R, Vinken R, Hommes G, Bertmer M, Schäffer A, Corvini PFX. Role of dissolved humic acids in the biodegradation of a sin-gle isomer of nonylphenol by Sphingomonas sp. Chemosphere 2007;68 (11):2172–80.

Liu Q, Ji R, Hommes G, Schäffer A, Corvini PFX. Fate of a branched nonylphenol isomer in submerged pad-dy soils amended with nitrate. Water Research 2008;42 (19):4802–8.

Martin C, Corvini PFX, Vinken R, Junghanns C, Krauss G, Schlosser D. Quantification of the influence of extracellular laccase and intracellular reactions on the isomer-specific biotransforma-tion of the xenoestrogen technical nonylphenol by the aquatic hyphomycete Clavariopsis aquatica. Appl Environ Microbiol 2009;75 (13):4398–409.

Mesitschek D, Fent K. Chromexposition und Galvanikbetriebe. Galvanotechnik 2010 (5):1167–80.

Mutz D. Mitverwertung von Abfällen in der Zementproduktion – Er-fahrungen aus Entwicklungsländern Co-processing waste material in cement production – Experience from developing countries. ZKG International 2007;60 (1):68–78.

Mutz D, Hengevoss D, Dubach B, Degré J, Wehenpohl G, Schimpf W, Rubertus E. The GTZ-Holcim strategic alliance on co-processing waste material in cement production – A success story we can build on. 2009;http://www.coprocem.com.

Nättorp A. Vorteile bei Verwendung von Wachsbitumen im Strassenbau. Strasse und Verkehr 2009;95:23–6.

Niewersch C, Meier K, Wintgens T, Melin T. Selectivity of polyamide nanofiltration membranes for ca-tions and phosphoric acid. Desalination 2010;250 (3):1021–4.

Nowak KM, Kouloumbos VN, Schaeffer A, Corvini PFX. Effect of sludge treatment on the bioaccumulation of no-nylphenol in grass grown on sludge-amended soil. Environ. Chem. Lett. 2008;6 (1):53–8.

Oertlé E, Mutz D, Knobloch A, Klingel P, Fallis P, Sorg F, Ziegler D. Guidelines for Water Loss Reduction – A Focus on Intelligent Pressure Management. IWA International Specialised Confe-rence "Water Loss 2010". Sao Paulo, 2010.

Oggier DM, Weisbrod CJ, Stoller AM, Zenker AK, Fent K. Effects of diazepam on gene expression and link to physiolo-gical effects in different life stages in zebrafish Danio rerio. Environ Sci Technol 2010;44 (19):7685–91.

Page D, Dillon P, Toze S, Bixio D, Genthe B, Jiménez Cisneros BE, Wintgens T. Valuing the subsurface pathogen treatment barrier in water recycling via aquifers for drinking supplies. Water Research 2010;44 (6):1841–52.

Riefer P, Klausmeyer T, Schwarzbauer J, Schäffer A, Schmidt B, Corvini P. Rapid incorporation and short-term distribution of a nonyl-phenol isomer and the herbicide MCPA in soil-derived organo-clay complexes. Environmental Chemistry Letters 2010:1–5.

Sandoval Soto L, Jutz M, Langbein A, Wintgens T. Gras effizient trocknen. Swiss engineering STZ 2009 (6):30–1.

Schmidt B, Ebert J, Lamshoeft M, Thiede B, Schumacher-Buffel R, Ji R, Corvini PFX, Schaeffer A. Fate in soil of 14C-sulfadiazine residues contained in the ma-nure of young pigs treated with a veterinary antibiotic. J. En-viron. Sci. Health, Part B 2008;43 (1):8–20.

Schlumpf M, Kypke K, Vokt CC, Birchler M, Durrer S, Faass O, Ehnes C, Fuetsch M, Gaille C, Henseler M, Hofkamp L, Maerkel K, Reolon S, Zenker A, Timms B, Tresguerres JAF, Lichtensteiger W. Endocrine active UV filters: developmental toxicity and expo-sure through breast milk. Chimia 2008;62 (5):345–51.

Seibt U, Kesselmeier J, Sandoval-Soto L, Kuhn U, Berry JA. A kinetic analysis of leaf uptake of COS and its relation to transpiration, photosynthesis and carbon isotope fractiona-tion. Biogeosciences Discuss. 2009;6 (5):9279–300.


Seibt U, Kesselmeier J, Sandoval-Soto L, Kuhn U, Berry JA. A kinetic analysis of leaf uptake of COS and its relation to transpiration, photosynthesis and carbon isotope fractiona-tion. Biogeosciences 2010;7 (1):333–41.

Svoijtka J, Wintgens T, Melin T. Treatment of landfill leachate in a bench scale MBR.Desalina-tion and Water Treatment 2009 (9):136–41.

Toan V, Thao V, Walder J, Schmutz H-R, Ha C. Contamination by Selected Organochlorine Pesticides (OCPs) in Surface Soils in Hanoi, Vietnam. Bulletin of Environmental Contamination and Toxicology 2007;78 (3):195–200.

Toan V, Thao V, Walder J, Schmutz H-R, Ha C. Level and Distribution of Polychlorinated Biphenyls (PCBs) in Surface Soils from Hanoi, Vietnam. Bulletin of Environmental Contamination and Toxicology 2007;78 (3):211–6.

Von Hobe M, Kuhn U, Van Diest H, Sandoval-Soto L, Kenntner T, Helleis F, Yonemura S, Andreae MO, Kesselmeier J. Automated in situ analysis of volatile sulfur gases using a Sulfur Gas Analyser (SUGAR) based on cryogenic trapping and gas-chromatographic separation. International Journal of Environmental Analytical Chemistry 2008;88 (5):303–15.

Weisbrod CJ, Kunz PY, Zenker AK, Fent K. Effects of the UV filter benzophenone-2 on reproduction in fish. Toxicol. Appl. Pharmacol. 2007;225 (3):255–66.

Weston A, Caminada D, Galicia HF, Fent K. Effects of lipid-lowering pharmaceuticals bezafibrate and clofibric Acid on lipid metabolism in fathead minnow (pime-phales promelas). Environ Toxicol Chem 2009;28 (12):2648–55.

Wintgens T, Kazner C, Melin T. RECLAIM WATER – manage aquifer recharge for safe indirect potable reuse. In: Hoven Tvd, Kazner C, editors. TECHNEAU: Safe Drinking Water from Source to Tap: IWA Publishing, London, 2009.

Wintgens T, Hochstrat R, Kazner C. Water recycling in Europe and the Mediterranean: framework conditions and technologies for future development. WORK-SHOP Membrane technologies for alternative water resources. Thessaloniki, 2009. pp. 7–10.

Yu L, Fink G, Wintgens T, Melin T, Ternes TA. Sorption behavior of potential organic wastewater indicators with soils. Water Research 2009;43 (4):951–60.

Zaja R, Caminada D, Loncar J, Fent K, Smital T. Development and characterization of P-glycoprotein 1 (Pgp1, ABCB1)-mediated doxorubicin-resistant PLHC-1 hepatoma fish cell line. Toxicology and Applied Pharmacology 2008;227 (2):207–18.

Zenker A, Schmutz H, Fent K. Simultaneous trace determination of nine organic UV-ab-sorbing compounds (UV filters) in environmental samples. J. Chromatogr., A 2008;1202 (1):64–74.

Institute for Medical and Analytical Technologies (IMA)

Belei P, Schkommodau E, Frenkel A, Mumme T, Radermacher K.Computer-assisted single- or double-cut oblique osteotomies for the correction of lower limb deformities. Proceedings of the Institution of Mechanical Engineers, Part H: Journal of Engi-neering in Medicine 2007;221 (7):787–800.

Belei P, Schkommodau E, Frenkel A, Mumme T, Radermacher K. Evaluation of an optimization module for single- and double-cut oblique osteotomy based correction of deformed long bone. 7th Annual meeting of the International Society for Computer Assisted Orthopaedic Surgery. Heidelberg: CAOS International, 2007. pp. 687–90.

Belei P, Skwara A, De La Fuente M, Schkommodau E, Fuchs S, Wirtz Dieter C, Kamper C, Radermacher K. Fluoroscopic navigation system for hip surface replacement. Comput Aided Surg 2007;12 (3):160–7.

Bormann T, Friess S, de Wild M, Schumacher R, Schulz G, Müller B. Determination of strain fields in porous shape memory alloys using micro-computed tomography. Proc. SPIE 2010;7804:78041M.

Bormann T, Schumacher R, Müller B, Mertmann M, Pieles U, de Wild M. Properties of NiTi-structures fabricated by selective laser melting Annual meeting of the Swiss Society for Biomaterials. Empa Dübendorf: European Cells and Materials, 2010. pp. 13.

Brodbeck D, Degen M, Kool J, Oesch P. An Investigation of Computer-Generated Visual Feedback for the Support of Low Back Pain Therapy. World Congress on Me-dical Physics an Biomedical Engineering. München, 2009. pp. 214–7.

Brodbeck D, Degen M, Stanimirov M, Kool J, Scheermesser M, Oesch P, Neuhaus C. Augmented Feedback System to Support Physical Therapy of Non-specific Low Back Pain. In: Fred A, Filipe J, Gamboa H, editors. Biomedical Engineering Systems and Technologies: Springer Berlin Heidelberg, 2010. pp. 381–93.

Brodbeck D, Degen M, Stanimirov M, Kool J, Scheermesser M, Oesch P, Neuhaus C. Backtrainer.Computer-aided Therapy System with Augmented Feedback for the Lower Back. In: Azevedo L, Londral AR, editors. Proceedings of the Second International Conference on Health Informatics, HEALTHINF 2009, Porto, Portugal, January 14–17, 2009. pp. 66–73.

Brodbeck D, Mazza R, Lalanne D. Interactive Visualization – A Survey. In: Lalanne D, Kohlas J, edi-tors. Human Machine Interaction: Springer Berlin Heidelberg, 2009. pp. 27–46.

Coste J, Ouchchane L, Sarry L, Derost P, Durif F, Gabrillargues J, Hemm S, Lemaire JJ. New electrophysiological mapping combined with MRI in parkinsonian’s subthalamic region. European Journal of Neu-roscience 2009;29 (8):1627–33.

Daeichin V, Hemm S, Taub E, Schkommodau E. Development of an intraoperative visualisation tool for electro-de placement in deep brain stimulation. In: Ssbe, editor. SSBE Annual Meeting. Bern, 2009.

de Wild M, Leisi D, Schäfer M, Näf M, Schumacher R, Schkommodau E, Bufler M. Investigating the porosity of 3D printed calcium phosphate scaffolds. Meet the Expert. Interlaken: European Cells and Materials, 2010. pp. 5.

de Wild M, Schumacher R, Fabbri S, Yildiz A, Schkommodau E. Analysis and Surface Modification of Rapid Prototyped Titanium Structures. European Cells and Materials 2009;17 (Suppl. 1):6.

de Wild M, Maier K, Schneider M, Tschumi S, Schumacher R, Albrecht H.Surface Modification and In-vitro Investigation of Genera-tively Produced Implants. Biomaterialien 2010;11 (1):157.

Degen M, Luthiger J. Maßgeschneiderte J2ME-Anwendungen. JavaSPEKTRUM 2007 (4):26–8.

Erba P, Wettstein R, Schumacher R, Schwenzer- Zimmerer K, Pierer G, Kalbermatten DF. Silicone moulding for pressure sore debridement. J Plast Re-constr Aesthet Surg 2009.

Hemm S, Coste J, Gabrillargues J, Ouchchane L, Sarry L, Caire F, Vassal F, Nuti C, Derost P, Durif F, Lemaire J-J. Contact position analysis of deep brain stimulation elec-trodes on post-operative CT images. Acta Neurochirurgica 2009;151 (7):823–9.

Hemm S, Schkommodau E, Wild Md. Repositioning precision of EEG-Caps – a preliminary study. World Congress on Medical Physics and Biomedical Enginee-ring. München, 2009.

Hemm S, Wardell K. Stereotactic implantation of deep brain stimulation elec-trodes: a review of technical systems, methods and emerging tools. Med Biol Eng Comput 2010;48 (7):611–24.

Kalbermatten DF, Wettstein R, Erba P, Schumacher R, Dagorov P, Pierer G. Laser scanner analysis in reconstruction of traumatic lacera-tion of the facial nerve. Laser Physics Letters 2007;4 (6):476–9.

Lambrecht J, Berndt D, Schumacher R, Zehnder M. Generation of three-dimensional prototype models based on cone beam computed tomography. International Journal of Computer Assisted Radiology and Surgery 2009;4 (2):175–80.

Molenberg A, Schwarz F, Herten M, Berner S, de Wild M, Wieland M. Improved osseointegration of a novel, hydrophilic Ti surface – a review. Materialwiss. Werkstofftech. 2009;40 (1–2):31–5.

Rieger UM, Erba P, Wettstein R, Schumacher R, Schwenzer-Zimmerer K, Haug M, Pierer G, Kalbermatten DF. Does abdominoplasty with liposuction of the love handles yield a shorter scar? An analysis with abdominal 3D laser scanning. Ann. Plast. Surg. 2008;61 (4):359–63.

Schuler M, Kunzler Tobias P, de Wild M, Sprecher Christoph M, Trentin D, Brunette Donald M, Textor M, Tosatti Samuele GP. Fabrication of TiO2-coated epoxy replicas with identical dual-type surface topographies used in cell culture assays. J Biomed Mater Res A 2009;88 (1):12–22.

Schumacher R, de Wild M, Fabbri S, Yildiz A, Schkommodau E. Rapid Manufacturing of Individualized Ti-6Al-4V Bone Im-plants. European Cells and Materials 2009;17 (Suppl. 1):22.

Schwarz-Wings D, Meyer CA, Frey E, Manz-Steiner H-R, Schumacher R. Mechanical implications of pneumatic neck vertebrae in sau-ropod dinosaurs. Proceedings of the Royal Society B: Biologi-cal Sciences 2010;277 (1678):11–7.

Schwenzer-Zimmerer K, Boerner BI, Müller AA, Jürgens P, Ringenbach A, Schkommodau E. T-Scan: First Experiences with Acquisition of Cleft Morphology. In: Advances in Medical Engineering, 2007. pp. 458–63.

Schwenzer-Zimmerer K, Boerner BI, Schwenzer NF, Muller AA, Jürgens P, Ringenbach A, Schkommodau E, Zeilhofer H-F. Facial acquisition by dynamic optical tracked laser imaging: a new approach. J Plast Reconstr Aesthet Surg 2009;62 (9):1181–6.


Institute for Pharma Technology (IPT)

Arnold Y, Gonzalez RB, Versace H, Kuentz M. Comparison of different in vitro tests to assess oral lipid-based formulations using a poorly soluble acidic drug. J. Drug Delive-ry Sci. Technol. 2010;20 (Copyright (C) 2010 American Chemical Society (ACS). All Rights Reserved.):143–8.

Bendels S, Kansy M, Wagner B, Huwyler J. In silico prediction of brain and CSF permeation of small molecules using PLS regression models. Eur. J. Med. Chem 2008;43:1581–92.

Bittner B, Gonzalez RCB, Bohrmann B, Kuentz M, Huwyler J. Drug-excipient interactions by vitamin E-TPGS: in vitro studies on inhibition of P-glycoprotein and colonic drug absorption. J. Drug Delivery Sci. Technol. 2008;18 (2):145–8.

Ceccarelli SM, Jaeschke G, Buettelmann B, Huwyler J, Kolczewski S, Peters JU, Prinssen E, Porter R, Spooren W, Vieira E Rational design, synthesis, and structure-activity relation-ship of benzoxazolones: New potent mglu5 receptor antago-nists based on the fenobam structure. Bioorg. Med. Chem. Lett 2007;17:1302–6.

Ditner C, Bravo R, Imanidis G, Kuentz M. A Systematic Dilution Study of Self-Microemulsifying Drug Delivery Systems in Artificial Intestinal Fluid Using Dynamic Laser Light Backscattering. Drug Dev. Ind. Pharm. 2009;35 (2):199–208.

Du-Cuny L, Huwyler J, Fischer H, Kansy M. A potentiometric titration method for the crystallization of drug-like organic molecules. Int. J. Pharm 2007;342:161–7.

Du-Cuny L, Huwyler J, Wiese M, Kansy M. Computational aqueous solubility prediction for drug-like com-pounds in congeneric series. Eur. J. Med. Chem 2008;43:501–12.

Huwyler J, Drewe J, Krähenbühl S. Tumour targeting using liposomal antineoplastic drugs. Int. J. Nanomed 2008;3:21–9.

Jaeschke G, Porter R, Buttelmann B, Ceccarelli SM, Guba W, Kuhn B, Kolczewski S, Huwyler J, Mutel V, Peters JU, Ballard T, Prinssen E, Vieira E, Wichmann J, Spooren W. Synthesis and biological evaluation of fenobam analogs as mGlu5 receptor antagonists. Bioorg. Med. Chem. Lett 2007;17:1307–11.

Kapitza SB, Michel BR, van Hoogevest P, Leigh MLS, Imanidis G. Absorption of poorly water soluble drugs subject to apical ef-flux using phospholipids as solubilizers in the Caco-2 cell mo-del. European Journal of Pharmaceutics and Biopharmaceutics 2007;66 (1):146–58.

Kloefer B, Henschel P, Kuentz M. Validity of a power law approach to model tablet strength as a function of compaction pressure. AAPS PharmSciTech 2010;11 (1):467–71.

Kloefer B, Van Hoogevest P, Moloney R, Kuentz M, Leigh MLS, Dressmann J. Study of a Standardized Taurocholate–Lecithin Powder for Pre-paring the Biorelevant Media FeSSIF and FaSSIF. Dissolution Technologies 2010;17 (3):6–13.

Kuentz M. Drug absorption modeling as a tool to define the strategy in clinical formulation development. Aaps J. 2008;10 (3):473–9.

Kuentz MT, Arnold Y. Influence of molecular properties on oral bioavailability of lipo-philic drugs – mapping of bulkiness and different measures of polarity. Pharm. Dev. Technol. 2009;14 (3):312–20.

Kuentz M, Cavegn M. Critical concentrations in the dilution of oral self-microemul-sifying drug delivery systems. Drug Development and Industri-al Pharmacy 2009:1–8.

Kuentz M, Cavegn M. Critical concentrations in the dilution of oral self-microemul-sifying drug delivery systems. Drug Dev Ind Pharm 2010;36 (5):531–8.

Kuentz M, Wyttenbach N, Kuhlmann O. Application of a Statistical Method to the Absorption of a New Model Drug from Micellar and Lipid Formulations – Evaluation of Qualitative Excipient Effects. Pharm. Dev. Technol. 2007;12 (3):275–83.

Malherbe P, Masciadri R, Norcross R, Knoflach F, Kratzeisen C, Zenner M, Kolb Y, Marcuz A, Huwyler J, Nakagawa T, Porter R, Thomas A, Wettstein J, Sleight A, Spooren W, Prinssen E. Characterization of (R,S)-5,7-di-tert-butyl-3-hydroxy-3-triflu-oromethyl-3H-benzofuran-2-one (rac-BHFF) as a positive al lo steric modulator of GABA-B receptor. Br. J. Pharmacol 2008;154:797–811.

Nalluri V, Kuentz M. Advancing Pharmaceutical Dry Milling by Process Analytics and Robustness Testing. Journal of Pharmaceutical Innovation 2010;5 (3):100–8.

Nalluri VR, Kuentz M. Flowability characterisation of drug-excipient blends using a novel powder avalanching method. European Journal of Phar-maceutics and Biopharmaceutics 2010;74 (2):388–96.

Nalluri VR, Schirg P, Gao X, Virdis A, Imanidis G, Kuentz M.Different modes of dynamic image analysis in monitoring of pharmaceutical dry milling process. International Journal of Pharmaceutics 2010;391 (1–2):107–14.

Pellanda C, Strub C, Figueiredo V, Rufli T, Imanidis G, Surber C. Topical bioavailability of triamcinolone acetonide: effect of oc-clusion. Skin Pharmacol Physiol 2007;20 (1):50–6.


Pierson PD, Fettes A, Freichel C, Gatti-McArthur S, Hertel C, Huwyler J, Mohr P, Nakagawa T, Nettekoven M, Plancher J-M, Raab S, Richter H, Roche O, Rodriguez Sarmiento RM, Schmitt M, Schuler F, Takahashi T, Taylor S, Ullmer C, Wiegand R. 5-Hydroxyindole-2-carboxylic Acid Amides: Novel Histamine-3 Receptor Inverse Agonists for the Treatment of Obesity. J. Med. Chem. 2009;52 (13):3855–68.

Poller B, Drewe J, Krähenbühl S, Huwyler J, Gutmann H. Regulation of BCRP (ABCG2) and P-Glycoprotein (ABCB1) by Cytokines in a Model of the Human Blood–Brain Barrier. Cellu-lar and Molecular Neurobiology 2009.

Poller P, Gutmann H, Krähenbühl S, Weksler B, Romero I, Couraud P, Tuffin G, Drewe J, Huwyler J. The human brain endothelial cell line hCMEC/D3 as a human blood-brain barrier model for drug transport studies. J. Neuro-chem. 2008;107:1358–68.

Schwebel HJ, van Hoogevest P, Leigh ML, Kuentz M. The apparent solubilizing capacity of simulated intestinal fluids for poorly water-soluble drugs. Pharm Dev Technol 2010.

Tuffin G, Huwyler J, Waelti E, Hammer C, Marti H. Drug targeting using OX7-immunoliposomes: Correlation bet-ween Thy 1.1 antigen expression and tissue distribution in the rat. J. Drug Targeting 2008;16 (2):156–66.

Vieira E, Huwyler J, Jolidon S, Knoflach F, Mutel V, Wichmann J. Fluorinated 9H-xanthene-9-carboxylic acid oxazol-2-yl-amides as potent, orally available mGlu1 receptor enhancers. Bioorg. Med. Chem. Lett. 2009;19:1666–9.

Woltering TJ, Wichmann J, Goetschi E, Adam G, Kew JN, Knoflach F, Ballard TM, Huwyler J, Mutel V, Gatti S. Synthesis and characterization of 1,3-dihydro-benzo[b][1,4]diazepin-2-one derivatives: Part 3. New potent non-competitive metabotropic glutamate receptor 2/3 antagonists. Bioorg Med Chem Lett 2008;18 (8):2725–9.

Project Portfolio

Since the beginning in the year 2006, the School of Life Sciences FHNW has realized many scientific projects in coope-ration with industrial and academic partners. Within the last five years, the school has established itself as a compe-tent address for cutting edge life sciences.

The following table and figures give an overview of the number of research projects, which have been realized and how they were funded.

total number number number 10'000–100'000 CHF >100'000 CHF projectssince2006 362 265 97

currentprojects 141 77 64

Type of Funding (% of current projects) Research Focus Areas (2009–2010)(% of external Funds)

EU

ThirdParties(directfunding)

SNSF

CTI

SwissFederalOfficesEnvironmental

Management(EM)25%

TherapeuticTechnologies(TT)36%

MolecularTechnologies(MT)

39%


Anders Nättorp, HLS FHNW, MuttenzWie können die Ressourcen im Abwasser genützt werden?

Markus Lenz, HLS FHNW, MuttenzBioremediation of selenium contaminated wastewaters - The solution to pollution?

Thilo Glatzel, Universität Basel, BaselMeasuring Atomic Scale Electrostatic Forces on insulators and Molecules using KPFM

Andreas Hartmann, Novartis AG, BaselGlobaler Umweltschutz bei Novartis: “Trends und Heraus-forderungen“

Ursula Meierhofer, FHNW, BruggGeschlechterspezifische Aspekte in der Arbeitswelt

Monica Dressler, Novartis AG, BaselPharmazeutische Registrierung; vom Molekül im Labor bis zur Apotheke

Michael Schleimer, Basilea Pharmaceutica Ltd, BaselThe Role of Analytics in the Development of Drugs

Gregor Burkhard, HT, FHNWEU-Forschungsprojekt EUPASS “Evolvable Ultra-Precision Assembly Systems”– Resultate und Erfahrungen

Alex Ringenbach, HLS, FHNWHerausforderungen in der Medizinischen Bildverarbeitung - Beispiel Leberchirurgie

Jean-Pierre Locquet, Katholieke Universiteit Leuven (KUL), LeuvenFunctional Oxides for Advanced Electronics Applications

Ralf Schumacher, HLS FHNW, Muttenz Rapid Prototyping und Implantatherstellung – aktueller Stand der Forschung im Bereich Titan und Biokeramiken

Christian Siebenhaar, HLS FHNW, MuttenzHerstellung von Nanopartikel mittels Flammenspraypyrolyse

Andreas Schreiner, Novartis AG, BaselPharmazeutische Entwicklung vom Kristall bis zur Tablette - Herausforderungen in der Prozesstechnik

Verena Christen, HLS FHNW, MuttenzAnalyse der toxikologischen Wirkungen von Pharmazeutika in Fischzellen

Simon Berner, Institut Straumann AG, BaselMaterials und Surface Modifications for Dental Implants

Yves Dudal, HLS FHNW, MuttenzDevelopment of a high-throughput screening for pharmaceu-tical compounds in waters

Peter Biedermann, Medical Cluster Bern, BernMedizintechnikindustrie Schweiz: “Bedeutung, Dynamik und Netzwerke”

Yvonne Arnold, HLS FHNW, MuttenzIn vitro tests as useful tool to predict the solubilization be-havior of poorly soluble drugs incorporated in lipid-based formulations

Jörg Trappe, Novartis AG, BaselInnovative Strategien in der Wirkstoffoptimierung

Klaus Köhler, Endress+Hauser GmbH+Co. KG, Weil am Rhein (D)Moderne Messtechnik und Kalibrierung in Biopharmapro-zessen am Beispiel eines Fermenters

Michael Hoffmann, Universitätsklinikum Freiburg (D)Genetische Marker für kardiovaskuläre Erkrankungen – von der Populationsgenetik zur individuelle Diagnostik

Markus Blümel, Novartis Pharma AG, BaselProtein Analytics: How can proteins be characterized?

Bernd Nowack, Empa, St. GallenWhere to look for the nano-needle in the environmental hay-stack?

Emmanuel Oertlé, HLS FHNW, MuttenzWater loss reduction through intelligent pressure manage-ment

Dirk Benndorf, Max Planck Institut, MagdeburgNutzung von Proteomics für Anwendung in den Bereichen Klärschlamm und Biogas Produktion

Venky Nalluri, HLS FHNW, MuttenzDynamic image analysis of pharmaceutical powders and gra-nules- From on-line analytics to flowability characterization

Sherri Dudal, Novartis AG, BaselTracking biological drugs in the body

Claudio Storck, Universitätsspital Basel, BaselUntersuchung der Biomechanik des Larynx (Kehlkopf) an Dreidimensionalen Bildern

Christoph Minnig, FHNW, Hochschule für Wirtschaft, PMOCURE - Corporate Culture and Regional Embeddedness oder haben Unternehmen eine Heimat?

Konrad Hungerbühler, ETH Zürich, ZürichWhat is green solvent?

Michael Stadler, FMI Basel, BaselComputational Biology

John Zambounis, BASF Schweiz AG, BaselNew Opportunities in Adhesives using Photolatent Bases Technology

Jan Svojitka, HLS FHNW, MuttenzMembrane bioreactors for industrial wastewater treatment and sediment decontamination

Lisseth Sandoval, HLS FHNW, MuttenzEffizienzsteigerung in Landwirtschaftlichen Trocknungsan-lagen

Andreas Hafner, BASF Schweiz AG, BaselTailor-made Micro-, Nano-particles and Systems - the Basis for Novel Effects

Roger Zurbriggen, Akzo Nobel AG, SempachGrenzen der Naturwissenschaften

Georgios Imanidis, HLS FHNW, MuttenzBiorelevante Medien für in vitro Wirkstoffabsorption

Arnulf Bohnacker, HLS FHNW, MuttenzUnternehmensinterne Managementaspekte

Claude Schärer, HLS FHNW, MuttenzAsymmetrische Katalyse in der organischen Synthese

Eric Kübler, HLS FHNW, MuttenzGenetische Diagnostik

Waldemar Hoffmann, HLS FHNW, Muttenz“The osteogenic effect of BMP-2 on freshly isolated human adipose tissue derived stem cells: an in vitro and in vivo study”

Jean-Nicolas Aebischer, HES-SO, FreiburgApplied Physical Chemistry at the HTA-FRIBOURG

Research Seminars at the School of Life Sciences FHNW Autumn 2009–Summer 2011

David Hradetzky, HLS FHNW, MuttenzMikrosystemtechnik zum Einsatz in der Medikamentenab-gabe

Götz Schlotterbeck, HLS FHNW, MuttenzMetabolomics in drug development

Klaus Kümmerer, Universität Freiburg, FreiburgChemikalien, Spurenstoffe, Transformationsprodukte und ihre Bewertung mit Computer basierten Methoden

Daniel Hess, FMI Basel, BaselMass spectrometry based protein analysis and proteomics

Ron Tynes, HLS FHNW, MuttenzIt's a Long Road to the Nucleus: Transient Gene Expression in Modern Protein Biotechnology

Hans Hermann Letzner, Letzner Pharmawasseraufberei-tung GmbH, HückeswagenVom Trinkwasser bis zum Wasser für die Infusionslösung, ohne Krankheitserreger, Rostpartikel und andere Risiken

Wolfgang Riedl, Universität Basel, BaselMembranverfahren in der Biotechnoloige: wo Sizeexclusion zu exklusiven Produkten führt

Christoph Hugi, HLS FHNW, Muttenz“Ressourcenknappheit: Schwellenländer im Wettlauf um Rohstoffe”


Competences

Biochemistry, Bioanalytics, Diagnostic(Bio)-NanotechnologyMolecular Recognition, Organo- and BiocatalysisSynthesis / Sustainable DevelopmentMolecular Diagnostics and Preclinical Development of PharmaceuticalsInstrumental AnalyticsOrganic and Organometallic SynthesisChemical Engineering

Environmental Engineering/Clean TechnologiesEcotoxicologyEnvironmental Biotechnology/MicrobiologyRessource ManagementCleaner Production in Industry (CP)Green Chemistry

Medical Image ProcessingVisual AnalyticsComputer-assisted SurgeryMedical Rapid PrototypingDeep Brain StimulationBiosignal ProcessingTelemedicineData Analysis and ModellingMaterialographyMicrosystem Technology

Intestinal and (Trans) Dermal Active Agent Absorption and Drug DeliveryDrug Targeting and PharmacokinetcsRational Development of Medical Formulations and Dosage FormsProcess Development, Production Planning and Project Management

Research Focus Area

MT, TT

MT, TT, EM

TT

MT, TT

Fields of Research

Biochemistry, Bioanalytics, Diagnostics(Bio)-Nanotechnology, Instrumental AnalysisOrganic Synthesis, Chemical Engineering

Resource Management, Cleaner ProductionEcotoxicologyEnvironmental, Biotechnology and Engineering

Implants and Surgical SystemsBiomedical Information SystemsMedical Image and Signal Processing

Dosage FormsDrug DeliveryProcedures and Production Processes

Institute

Institute for Chemistry and Bioanalytics (ICB)

Institute for Ecopreneurship (IEC)

Institute for Medical and Analytical Technologies (IMA)

Institute for Pharma Technology (IPT)

Research Topics and Competences


Contact

University of Applied Sciences Northwestern SwitzerlandSchool of Life SciencesGründenstrasse 40CH - 4132 Muttenz

Tel +41 61 467 42 [email protected]

Director School of Life SciencesProf. Dr. Gerda Huber, Tel +41 61 467 42 [email protected]

Institute for Ecopreneurship (IEC)Prof. Dr. Philippe Corvini, Tel +41 61 467 43 [email protected]

Institute for Chemistry and Bioanalytics (ICB)Prof. Dr. Gerhard Grundler, Tel +41 61 467 42 [email protected]

Institute for Medical and Analytical Technologies (IMA)Prof. Dr. Erik Schkommodau, Tel +41 61 467 42 [email protected]

Institute for Pharma Technology (IPT)Prof. Dr. Georgios Imanidis, Tel +41 61 467 46 [email protected]

Bachelor of Science (B.Sc.)

– Life Science Technologies Prof. Gianni N. di Pietro, Tel +41 61 467 46 94 [email protected]

– Molecular Life Sciences Prof. Dr. Daniel Gygax, Tel +41 61 467 45 62 [email protected]

Master of Science (M.Sc.)

– In Life Sciences (Molecular Technologies, Therapeutic Technologies) Prof. Dr. Georg Lipps, Tel +41 61 467 43 01 [email protected]

Master of Advanced Studies

– MAS Environment Technology and Management Prof. Dr. Markus Wolf, Tel +41 61 467 43 51 [email protected]

– MAS Nano-Micro-Technology (in cooperation) Prof. Dr. Uwe Pieles, Tel +41 61 467 44 53 [email protected]

PublisherUniversity of Applied Sciences Northwestern SwitzerlandSchool of Life Sciences

EditorsGerda HuberArnulf Bohnacker

DesignBüro für Kommunikationsdesign FHNW

PhotographyUwe Pieles, cover and pages 4, 28, 50 and 62Copyright © Uwe Pieles, all rights reserved

PrintSteudler Press AG, Basel

Circulation800 Copies

No reproduction or publishing is allowed without the prior written consent of the editors

February 2011

Documents

Research Report - FHNW