The FHNW incorporates nine faculties:
Fachhochschule Nordwestschweiz
Hochschule für Life Sciences
Gründenstrasse 40
CH-4132 Muttenz
T+41 61 467 42 42
F+41 61 467 47 01
info.lifesciences @ fhnw.ch
www.fhnw.ch / lifesciences
Research Report
2009/2010
–School of Applied Psychology
–School of Architecture, Civil Engineering and Geomatics
–Academy of Art and Design
–School of Life Sciences
–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
Understanding Biomineralization and its Implication for the Environmental Chemistry of Selenium
Hormonal Activity of Chemicals to which Humans Are Frequently Exposed: Are there Potential Risks?
Hormonal Activity of the UV-Filter Benzophenone-4 Determined by Target Gene Expression Profile in Zebrafish Embryos
Activation of the JAK-STAT Pathway as Potential Molecular Basis for Diagnosis and Therapy of Macular Degeneration
Understanding the Degradation of Hydroquinone, a Key Metabolite in Xenobiotic Degradation
How to Detect Genetically Modified FP967 Flax by Event-Specific Polymerase Chain Reaction
Molecular Identification of a Bacterial Production Strain Used in the Pharmaceutical Industry
MangaCat: Flame Spray Pyrolysis Synthesis of Manganese Oxide Nanocomposite Particles for Catalysis Engineered Supramolecular Surfaces for High-Throughput Polymorphism Screening of APIs
8
10
12
14
16
18
20
22
24
26
Therapeutic Technologies (TT) 31
LANCE: LAccase-Nanoparticle Conjugates for the Elimination of Micropollutants from Wastewater in Bioreactors
Mistletoe-Preparations (Iscador®) Encapsulated in Silica Nanoparticles as a Therapy for Breast Cancer
Porous Shape-Memory-Scaffolds for Bone Implants
Best Environmental Practices in the Healthcare Sector
INOFEA, Innovative Nanomaterials for Environmental Applications, an Ecopreneurship Venture
Absorption Mechanisms of Poorly Water-Soluble Drugs from Self-Emulsifying Formulations in the Caco-2 Cell Model New Technologies for In Vitro Testing of Lipid-Based Drug Formulations
NAPTIS: Nano- and Micro-Scaled Porous Surfaces of Titanium Implants Produced by Spark-Assisted Anodizing
Development of a Functional Model of a Hollow Rotor Axial Flow Pump for Cardiac Assistance
32
34
36
38
40
42
44
46
48
Environmental Management (EM) 53
Waterless Mobile Toilet for Hospital Use
Recovering Phosphorus and Closing the Nutrient Cycle
Water Loss Reduction – A Focus on Pressure Management
Adapting a Membrane Bioreactor to Industrial Wastewater Treatment
54
56
58
60
Appendix 64
Publications
Project Portfolio
Research Seminars
Topics and Competences
Contact
66
75
76
78
80
2 | 3
Editorial
Technologies for the development of innovative healthcare products and
therapy solutions as well as methods and procedures for environmental
sustain­ability 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: “Molecular Technologies” (MT), “Therapeutic Technologies” (TT) and “Environmental Management” (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, bio­medical
products) as well as procedures for sustainable industrial production. In environmental 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 quality 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 relevant foundation work for new consortia like the CTI network for Sustainable Engineering. 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 Huber
Director, School of Life Sciences FHNW
6 | 7
Molecular Technologies (MT)
Technologies for the synthesis and analysis of active compounds
and biological systems
School of Life Sciences
Rapid Development of Stable Cell Lines with Multiple Transgenes
Using Variants of Flp Recombinases and Specific Target Sites
PSV40
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.
PCMV
ATG FRT
GOI
FRT
ZeoR-AcGFP1
Amp
-BR
HygB
PSV40
ATG FRT
-BR
HygB
pUC ori
ori
pUC
PCMV
Amp
expression of hygromycinB resistance gene
Accelerated generation of FlpIn host cells with a tagged
FRT site
The 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 selection (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 continued 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 marker (ZeoR-AcGFP1) (Fig. 1). This modification allowed continued
quality control of selected host cell clones with standard fluorescence microscopy. In addition, this offers the potential to
dramatically accelerate the selection of zeocin resistant host
cells with fluorescence activated cell sorting (FACS). Upon integration of a construct into the FRT site by homologous recombination, the expression of the respective fluorescent protein
was abolished together with the zeocin resistance marker. The
expression of ZeoR-AcGFP1 fusion protein from the integrated FRT site
plasmid to be integrated into the chromosomal FRT site
Flp recombinase
Keywords: Flp recombinase, site directed recombination, cell based assays, High-Throughput Screening (HTS)
Results
pUC ori
Amp
René F. Prétôt, Eleonora Chiri, Hugo Albrecht
School of Life Sciences FHNW
Introduction
The 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 homologous 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 integrated 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 recombinase. Using a fixed chromosomal locus for transgene integration 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 flanking 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 plasmid 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.
8 | 9
Figure 1
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 site
The functional expression of drug targets in mammalian cell
lines often requires coordinated expression of several transgenes. To facilitate these kinds of tasks, we successfully developed 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. Furthermore, the wild type recombinase showed low frequency of recombination 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 functional 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 ZeoRAcGFP1 and DsRed-IRES-PuroR, respectively (Fig. 2). Integration experiments were carried out with cotransfections of a
plasmid construct driving expression of the wt Flp recombinase 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 Outlook
We 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 vectors into the wt FRT and a mutated FRT site, in combination
with the wt recombinase and a mutated recombinase, respectively. In our laboratory we routinely apply a FlpIn compatible target vector with a bidirectional and inducible promoter,
GOI
no promoter
no start codon
FRT
ZeoR-AcGFP1
expression of gene of interest
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 its2promoter as well as its start codon.
Figure
A
B
CHO wt
CHO TetOn FRTZeoAcGFP/FRT*PuroIRESDsRed
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.
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 compatibility 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 development of cellular assays will be most suitable for laboratories
working in the field of pharmaceutical drug development. Relevant applications will be for High-Throughput Screening and
subsequent compound profiling during the lead optimisation
process. In addition, potential uses are possible for recombinant 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 Manipulation 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 Mammalian Expression Cell Lines by Flp Recombinase-mediated Integration. Available under: http://biochem.dental.upenn.edu/GATEWAY/Vector_manual/flpinsystem_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 mammalian cells, thereby reducing development costs.
10 | 11
School of Life Sciences
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 “biomineralized” 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
Indroduction
Selenium 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 observed 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 problems of improper distribution. Within Europe, for example,
Central-Eastern England shows a highly heterogenous selenium soil distribution, with deficient (<0.1 mg/kg) and seleniferous (>0.5 mg Se/kg) soils directly adjacent.
The global selenium cycle is influenced crucially by anthropogenic activities, mainly by combustion of fossil fuels, nonferrous metal melting and agricultural fertilization. When selenium 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 speciation, 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 microorganisms 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 selenium 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 selenium contaminated waters [3]. It has been observed that elemental selenium formed by these microorganisms does not
crystallize to larger particles, yet consists of almost perfectly
spherical nanoparticles (mostly around 300–500 nm in diameter) (Fig. 1), whereas chemical synthesis results in precipitates 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 properties different from particles without such a layer (Fig. 2).
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].
of 3 g/cm³, selenium particles were pelleted (density of 4.8
g/cm³), whereas biomass can be removed from the supernatant (Fig. 3). Subsequently, proteins were subjected to sodium 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 identified. 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 proteins with an anticipated direct functional role in selenium
reduction in spatial association with the bionanominerals
formed. Such proteins include specialized oxyanion reductases and proteins involved in electron transport during microbial 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 conditions. 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 polymers associated with the selenium particles and the extent
of altered environmental behavior of biogenic selenium in
contrast to chemically synthesized selenium.
Figure 1: Scanning Electron Microscopy picture of selenium particles formed by Bacillus selenatarsenatis
interdisciplinary biogeochemical research. It is a future goal
to control selenium biomineralization for (bio)nanotechnology 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 selenium 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 extensionAvailable 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 Ecopreneurship, School of Life Sciences FHNW)
Partner:
Suzette Moes (Biozentrum, University of Basel)
Funding:
Swiss National Science Foundation (SNSF, 200021–126899)
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)
Figure 2: Core-organic polymer layer model of biogenically formed selenium
precipitates
Results
Until recently, it was not known which molecules form the
organic polymer layer around the nanoparticles. We therefore investigated the presence of proteins within the organic
polymer layer by means of Capillary Liquid ChromatographyElectro Spray Ionization-tandem Mass Spectrometry (LCESI-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 nanoparticles potentially bearing a protein layer were isolated from
residual biomass by low speed density based centrifugation.
Using centrifugation in polytungstate solution with a density
Conclusion and Outlook
This study showed that microbially formed selenium particles 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/negative) and their conditions of formation (salt, selenium source,
phototrophic/dark conditions, etc.). These proteins form high
affinity complexes that do not dissociate upon repeated centrifugation/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
Economic efficiency and benefit to society:
Minerals that are produced by living organisms – so called
biominerals – often bear particular physico-chemical characteristics when compared to chemically synthesized analogues. 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 nanomaterials.
12 | 13
School of Life Sciences
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.
tions of these three phthalates. The anti-androgenic activity of
diethylhexyl phthalate (DEHP), dipentyl phthalate (DPP), dimethyl 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].
Karl Fent, Verena Christen
School of Life Sciences FHNW
Keywords: Flame retardants, biocides, phthalates, hormonal activity, androgenic activity, anti-androgenic activity,
cell systems
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 followed 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 reproduction. These data will help in the toxicological and environmental 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.
Indroduction
Flame retardants, antimicrobial agents and phthalates have numerous 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 relevant.They are used in construction materials, furniture, plastics,
electronic equipment, textile, and other products. Until recently,
polybrominated diphenyl ethers (PBDEs) accounted for a large 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 retardants 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 products, 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 contaminants 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 interaction with hormone receptors as agonists or antagonists.
For instance, anti-androgenic compounds can disrupt the action
of steroidal androgens in the foetus, with irreversible consequences (incomplete masculinization and malformations of the
reproductive organs) [4].
The aim of this study was to analyse selected flame retardants,
antimicrobials and phthalates including:
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
–tetrabromobisphenol A (TBBPA),
–hexabromocyclodecane (HBCD)
–penta-bromodiphenylether (BDE) (BDE-100) and hexa-BDE
(BDE-155)
–the antimicrobial compounds TCS and TCC
–eight phthalates
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
Figure 1: Flame retardants HBCD, BDE-100 and BDE-155 enhance androgen
activity
flame retardants. Chemosphere 2008;73 (2): 216–222.
[4] Wilson VS, Blystone CR, Hotchkiss, AK, Rider CV, Gray LE. Diverse mechanisms of anti-androgen action: impact on male rat reproductive tract development.
Int J Androl. 2008;31 (2): 178–187
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 dehydrotestosterone 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 reproducibility 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 toxicological risks are unclear. By using this in vitro test system,
we provide data that may help in the risk assessment of these
chemicals.
Results
No or only weak androgenic activity was observed in all tested 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 dehydrotestosterone-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 activity of flame retardants. In contrast, most phthalates showed
anti-androgenic activity. Butylbenzyl phthalate (BBP), dibutyl
phthalate (DBP) and diethyl phthalate (DEP) showed strong antiandrogenicity (Fig. 3). An up to 80% inhibition of the dehydro­
testosterone response was detected at the highest concentra-
[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.
Research Focus Area:
Molecular Technologies (MT)
Figure 2: Biocides triclosan and triclocarban enhance androgen activity.
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 important for risk assessment and for the safe handling of these
compounds.
Figure 3: Phthalates show antiandrogenic activity
Conclusion and Outlook
Our 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
14 | 15
School of Life Sciences
Hormonal Activity of the UV-Filter Benzophenone-4 Determined by Target Gene Expression Profile in Zebrafish Embryos
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.
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.
[2] Rodil R, Quintana JB, López-Mahía P, Muniategui-Lorenzo S, Prada-Rodríguez D. Multi-residue analytical method for the determination of emerging pollutants 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
Karl Fent, Sara Zucchi, Nancy Blüthgen
School of Life Sciences FHNW
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-
Keywords: Personal care products, benzophenone-4, zebrafish, molecular effects, hormonal activity
phenone-4 alters transcripts of genes involved in hormonal pathways in zebra­
fish (Danio rerio) eleuthero-embryos and adult males. Toxicol. Appl. Pharmacol.
Introduction
Toxicological and ecotoxicological sciences at FHNW are
concerned with the study of potential impacts of chemicals
on human health and the environment. These data will ultimately 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 mechanisms of chemicals in personal care products, pharmaceuticals 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 residues of several UV-filters have been detected. Among UVfilters 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 wastewater 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 vitro 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 endocrinedisrupting activity of BP-4, we evaluate the effects at molecular 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 concentrations 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 continuously incubated at 27±1 °C for approximately 5 days
throughout gastrulation, organogenesis and early larval
development using a static-water renewal procedure by replacing the appropriate BP-4 concentrations in new beakers
every 48 h. At the end of the exposure (120 hpf) eleutheroembryos were anaesthetized and a total of 15 eleutheroembryos 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 transcription PCR (qRT-PCR). We selected oestrogen-related
genes (vitellogenin 1, vitellogenin 3), sex-steroid receptors,
(oestrogen receptor alpha, oestrogen receptor beta 1, androgen receptor) and genes involved in formation of steroid hormones (hydroxysteroid 17-ß dehydrogenase-3, P450aromA,
P450aromB) after exposure during embryogenesis. By applying this approach we aimed to elucidate the toxicological
profile for use in environmental risk assessment.
Results
In 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 biomarker 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 convert testosterone to estradiol. The magnitude of mRNA induction 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.
Figure 1: Induction of vitellogenin 1 (vtg1) transcript by BP-4 exposure in
zebrafish embryos
2011;250 (2):137–46.
Figure 2: Induction of estrogen receptor (esr2b) transcript by BP-4 exposure
in zebrafish embryos
Research Focus Area:
Molecular Technologies (MT)
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, 31003A121829) and Federal Office for the Environment (FOEN)
Figure 3: Induction of P450aromB (cyp19b) transcript by BP-4 exposure in
zebrafish embryos
Conclusion and Outlook
In conclusion, the transcription profile reveal that BP-4 interferes with the expression of genes involved in hormonal
pathways and steroidogenesis [4]. The effects of BP-4 observed 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 environmental risk assessment of this UV-filter. At the same time the
data suggest that a better risk characterization of this compound should be performed with the focus on human health.
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.
16 | 17
School of Life Sciences
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 Gygax
School of Life Sciences FHNW
Keywords: JAK-STAT pathway, ARPE-19, age-related macular degeneration, MHC expression
Introduction
Age-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 characterized 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 underlying 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 constitute a specialized phagocytic system similar to that of macrophages. In addition, the RPE can modulate immune response
through pro-inflammatory cytokine production, including IL1β, IL-6 and tumor necrosis factor alpha (TNF-α). RPE cells
also produce pigment epithelial growth factor, which decreases and increases IL-12 and IL-10 production, respectively.
RPE plays a critical role in photoreceptor renewal, since defects 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 exact 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 phosphorylation. 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 regulation 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 development target for potentially effective AMD therapies.
Methods
Electromobility 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 modulation
We first tested the effects of IFN-α and IFN-γ on HLA-expression 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-regulation 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).
When tested on βCas probe, all nuclear extracts stimulated
with human IL-4 showed activated STAT6. A very strong activation of STAT1:1 homodimer shift was observed after IFN-γ
and IFN-α stimulation on M67 probe. Furthermore, IL-6 activated STAT3 in the ARPE-19 cells. The STAT1:STAT3 complex
after stimulation with IFN-α appeared as a band of intermediate intensity migrating slightly slower than STAT1:1 homodimers. 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 staining
In 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. Representative 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 observed in the cells after treatment with IFN-γ (Fig. 2, red color).
The cytoplasm was stained with anti-cytokeratin 8 antibodies (green color). Strong translocation of STAT6 was observed after incubation with Il-4 and IFN-α induced the nuclear
translocation of both STAT1 and STAT3 (data are not shown).
Figure 2: Nuclear localization of STAT1 observed by confocal laser microscopy after IFN-γ treatment: STAT1 red and Cytokeratin green color
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
Intracellular signaling
We 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 carried out on nuclear extracts and probed with four oligonucleotides 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. Stimulation with IFN-α activated a STAT1-STAT2-p48 complex that
binds to the O15 probe. All positive nuclear extracts of stimulated cells were verified in SuperShift experiments using
specific anti-STAT antibodies.
Conclusion and Outlook
In the present study, JAK-STAT pathway activation in ARPE19 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 cytokine-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 transcriptional 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 macular degeneration. Ophthalmologica. 2005; 219(4):214–221
Research Focus Area:
Molecular Technologies (MT)
Project Team:
Elizaveta Fasler-Kan and Daniel Gygax (Institute for Chemistry and Bioanalytics, School of Life Sciences FHNW)
Partner:
Natasha Barteneva (Immune Diseases Institute and Pro­gram 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.
18 | 19
School of Life Sciences
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 xenobiotics. 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. Corvini
School of Life Sciences FHNW
Keywords: dioxygenases, aromatic ring cleavage
Introduction
Hydroquinone (HQ) is a key compound occurring in the degra­
dation of diverse xenobiotics such as building blocks in polycarbonate production (bisphenol A, BPA) [1], flame retardants
(tetrabromobisphenol A) [2], intermediates of pharmaceuticals,
ammunition and dye production (4-nitrophenol) [3] and degradation products of industrial detergents (branched nonylphenols) [1]. HQ degradation can either proceed via direct ring
cleavage of the benzene ring or ring cleavage of 1,2,4-trihydroxybenzene. The two classes of enzymes that can be involved
in these two pathways, extradiol and intradiol dioxygenases
respectively, represent two evolutionary distinct classes of enzymes. Extradiol dioxygenases are more versatile, as they usually cleave a wider variety of substrates and are also involved
in a wider variety of metabolic pathways, including those degrading 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 enzymes are scarce, as only one member of this family has been
characterized.
Quinonoide compounds derived from HQs are agents of oxidative 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 understanding 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 nonylphenol isomers [1]. It belongs to the genus Sphingomonas (sensu
latu), which comprises strictly aerobic heterotrophic, gram-negative bacteria. Its members are frequently isolated from environmental 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 TTNP3
HQ dioxygenase from strain TTNP3 could be purified to homogeneity by sequential chromatographic purification steps. To
identify enzymatically active fractions, HQ ring cleavage activity 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 determination of its molecular mass by size exclusion chromatography, 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 ferric iron, strongly indicated that the enzyme contained ferrous
iron in its catalytic center. Further analysis by SDS-PAGE produced 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.
Substrate
(200 M)
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
Table 1: Substrate specifity of HQ dioxygenase (relative enzymatic activity
compared to hydroquinone)
Genetic background on HQ dioxygenase from
Sphingomonas sp. strain TTNP3
The respective bands of the enzyme subunits were cut out of the
SDS-PAGE gel and after extraction and subsequent tryptic digestion, analysed via QqTOF. From the mass fingerprints of the
small and the large subunits of the enzyme, four and six oligopeptides 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
Figure 1: Proposed degradation pathway for hydroquinone in strain TTNP3 (gene coding for enzymes whose functionality is proven is boxed)
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 frames coding for putative proteins from Photorhabdus luminescens subsp. laumondii TTO1, a bacterium that can be found in
the gut of entomopathogenic nematodes (51% and 63% amino
acid identity respectively).
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 catabolic pathway in Pseudomonas fluorescens ACB. Journal of Bacteriology
More genes related to the degradation of HQ
In the vicinity of the open reading frames attributed to the HQ
dioxygenase subunits of strain TTNP3, further open reading frames were identified, whose derived amino acid sequences bear
similarities to enzymes probably involved in the further degradation of 4-hydroxymuconic semialdehyde. They revealed 68%,
64%, 56% and 32% amino acid identity to a 4-hydroxymuconic
semialdehyde dehydrogenase (hqdC), a maleylacetate reductase (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 analysis 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 Outlook
HQ dioxygenase is a novel member of the class of HQ ringcleaving 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 metabolites 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 Sphingomonads (such as Photorhabdus, Burkholderia, Pseudomonas and
Ralstonia). Of the six enzymes, only the maleylacetate reductase and the 1,2,4-trihydroxybenzene dioxygenase appeared to
have similar homologs in Sphingomonads. This was an unexpected 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 organization for degradative genes, as these are often scattered
throughout the genome rather than clustered as in this case [5].
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 dioxygenases. Critical Reviews in Biochemistry and Molecular Biology 2006;41
(4):241–67.
[5] Stolz A. Molecular characteristics of xenobiotic-degrading sphingomonads. Applied Microbiology and Biotechnology 2009;81 (5):793–811.
Research Focus Area:
Molecular Technologies (MT)
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 Republic, Centre for Applied Genomics, Prague, Czech Republic)
Frédéric L.P. Gabriel (Institut für Klinische Chemie und Laboratoriumsmedizin, Rostock, Germany) Hans-Peter E. Kohler (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 micropollutants in the environment. It constitutes information for
the development of efficient bioremediation methods.
20 | 21
School of Life Sciences
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 Weston
School of Life Sciences FHNW
Table 1. Amplification data used to determine absolute LOD and LOQ
DNA amount (ng)
No. of positive
signals
Mean Ct
values
SD of observed
Ct values
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
–
–
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.
Table 2. Amplification data used to determine relative LOD and LOQ
GMO % (100 ng total DNA)
No. of positive
signals
Mean Ct values
SD of observed
Ct values
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
–
–
Keywords: Transgenic plant, event-specific assay, FP967 flax
Introduction
Linum usitatissimum L., commonly known as flax, is an important 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 substitution 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 synthase 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 internal 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 linseed form Canada, the European Commission adopted a sampling and testing protocol for Canadian flaxseed exported to
the EU [Sampling and Testing Protocol for the Canadian Flaxseed Exported to the EU]. For detection of the genetic modification 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 specificity at the methods developer’s laboratory [1] and in a verification 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 varieties in the EU event-specific methods have been developed and
made publically available [1].
Haploid genomic
copies
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.
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).
Results
The polymerase chain reaction has revolutionized DNA analytics 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 walking”. The PCR based methods are much faster and less labour
intensive than the traditional genome walking method that requires 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 downstream is generated using specific restriction enzymes. Ligation 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 mediated 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 random 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 flanking 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 “lockednucleic-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 target 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 quantified. 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 quantification assay, a serial dilution containing 1400 to 5 FP967 haploid 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 reactions down to 20 haploid copies (Table 1). From these results
we fixed the absolute LOD and LOQ at about 20 haploid genomic 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 background 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
Conclusion and Outlook
The 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 constructspecific 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.
22 | 23
School of Life Sciences
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 Lipps
School of Life Sciences FHNW
Keywords: Escherichia coli, Laves-Arzneimittel, PCR, identification, quality control
Conclusion and Outlook
The techniques developed in this project now allow the
technically simple, cost-effective and highly-specific identification 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 quality. The work was carried out in cooperation with Laves Arzneimittel 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.
A
Introduction
The company Laves-Arzneimittel produces Colibiogen, a biological drug for patients suffering from inflammatory gut mucosa, e.g. irritable bowel syndome (IBS), infammatory bowel
diseases and radiation colitis. Colibiogen stabilizes the intestinal flora, reduces inflammation and induces relaxation effects in the gut. For the production of Colibiogen, Escherichia
coli strain L1000 (DSM 13792), originally isolated from a healthy human subject by J. Vorschütz in 1931, is used. As Escherichia 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 developed for identifying E. coli L1000 and reliably distinguishing
it from other strains of this genus.
Results
The challenge of the research project consisted in the unambiguous 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 identify 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 based 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 genomic 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 genomic 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 reaction (PCR) assays and optimized for efficiency and specificity 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 detected 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 marker uidA (β-D-glucuronidase) was present in all E. coli strains
tested but the second universal marker lacY (lactose permease) could not be detected in one strain of E. coli. Concerning
the L1000 specific markers, we observed that the five markers 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 reaction uses several primer pairs and therefore generates several 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 distinction 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 marker 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 molecular 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 procedure 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 molecular marker is detected then either the assay failed technically
or a non E. coli strain grew in the culture.
B
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 generated 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.
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-dgalactosidase. Journal of Applied Microbiology 2008;105 (4):970–6.
[2] Horáková K, Mlejnková H, Mlejnek P. Direct detection of bacterial faecal indicators 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.
Research Focus Area:
Molecular Technologies (MT)
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 reliable identification of an industrial E. coli production strain.
24 | 25
School of Life Sciences
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
Introduction
Flame Spray Pyrolysis (FSP) is a versatile technology to produce
inorganic nanoparticulate composite materials in a combustion 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 additive 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 silicasupported catalysts allowing environmentally benign chemical
transformation in chemical production. The process allows the
synthesis of multifunctional catalytic composite materials.
Results
This 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 pyrolysis. The target is the preparation of manganese oxide composite 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. Furthermore an improved nozzle geometry and gas feed were developed
and implemented in the FSP apparatus. This optimisation afforded 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. Different batches of pure manganese oxide nanoparticles were prepared 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 nanocomposite particles prepared (Fig. 3). Oxidation of benzylic alcohols 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 transformation of alcohol to aldehyde using either commercial manganese (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
Figure 2: MnO2 nanoneedles obtained after treatment of Mn3O4 with sulfuric
acid.
Substrates
1
2
3
4
5
6
Conversion
(%)
44
76
52
12
35
35
Crude yield
(%)
NMR analysis
62
74
Quantitative
very low
Quantitative
Quantitative
educt + product
educt + product
educt + product
many side
products
educt + product
educt + product
Table 1: Oxidation reaction performed with 5 eq of nanoparticles (activated)
for 5 days
Figure 1: Structures of Model compounds
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 required sonication or to be warmed up before addition of the oxidant. 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 trans2-methyl-3-phenyl-2-propen-1-ol, we investigated experimental 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 structures (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 represents 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.
Substrates
1
3
5
Conversion
(%)
49
45
45
Crude yield
(%)
NMR analysis
11
44
Quantitative
educt + product
educt + product
educt + product
Table 2: Oxidation reactions performed with 5 eq of nanoparticles (activated) on silica in TBME for 5 days
Conclusion and Outlook
FSP 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 alternative to the precipitation procedure commonly used, which
suffers from bad reproducibility and difficult handling. Reactions carried out with MnO2 produced by FSP are far easier to
work up and therefore give better yields compared to commercially available materials. Furthermore, current FSP technology
will be expanded towards new materials exhibiting multifunctionality 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 Chemical 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 (Copyright (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
Research Focus Area:
Molecular Technologies (MT)
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 materials 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.
26 | 27
School of Life Sciences
Engineered Supramolecular Surfaces for High-Throughput
Polymorphism Screening of APIs
Polymorphism control of crystalline active pharmaceutical ingredients (APIs) is of ongoing interest in the pharmaceutical 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
Introduction
Crystal polymorphism is defined as the ability of a solid substance to exist as more than a single unique crystalline phase.
These different polymorphs have different physical characteristics, 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 legally required for active pharmaceutical ingredients (APIs) to
be allowed as a drug [1].
In this project a new technological approach has been developed, 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 company RPD Tool AG, who are developing automation solutions for
API crystal screening for pharmaceutical industry. This screening 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 direct subsequent crystallisations [3].
The project aims to provide synthetically modified surfaces
to direct crystallisation, achieved by calix[4]arene based amphiphiles, which are synthesised, characterised and appropriately 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.
Results
A series of calix[4]arene amphiphiles was synthesised, following 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 carried four carboxylate groups on the para-positions and dode­cyl
chains at the phenolic rim. It was prepared from the precursor
calix[4]arene 1 by aromatic dealkylation, followed by O-alkylation with an n-dodecyl chain, bromination of the ring, to allow
a copper(I) mediated nitrile exchange, and finally alkaline hydrolysis 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.
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
both aspirin and paracetamol. The behaviour of the aminophenol derivatives 3a–c was then studied, which suggested
that there is a much stronger interaction between the 4-aminophenol derivative 3c and the paracetamol solution when
comparing the 3 derivatives 3a–c.
Having found these interactions, the effect of 2 on actual solution 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
tet­ra-carboxylate 2 added to the air/solution interface. The
amount of amphiphile was varied across the array of experiments, in order to learn what effect the density of calix[4]arene
has on the crystallization. The density is of particular inte­rest,
since the packing of the molecules at the interface depends on
the amount of area available to each molecule, and is therefore 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 interface, 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 subphase.This procedure was deemed necessary, as it is known that
any further crossing of the air/solution interface has a detrimental 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 formation of the thermodynamically more stable form I, which is
expected to be obtained from crystallizations from water.
Figure 3: paracetamol crystals on LB film
Conclusion and Outlook
The 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 parameters 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 model 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
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 behaviour. Its interaction with dissolved APIs was assessed by the
same method, which suggested favourable interactions with
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
Figure 2: paracetamol growing at amphiphile interface
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
Research Focus Area:
Molecular Technologies (MT)
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 ingredients is a major issue in the development of new pharmaceutically 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 crystallization phenomena and thus to contribute to the advancement
of knowledge in this particular field.
30 | 31
Therapeutic Technologies (TT)
Technologies for the development and production of pharmaceutical
and biomedical products taking into consideration sustainable
manufacturing
32 | 33
School of Life Sciences
LANCE: LAccase-Nanoparticle Conjugates for the Elimination
of Micropollutants from Wastewater in Bioreactors
Conclusion and Outlook
Laccase of the white-rot fungi Coriolopsis polyzona was successfully immobilized on fsNP using a novel method. Experiments showed that immobilization to fumed silica is a highly efficient 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 immobilized 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.
Recent advances in nanomaterial sciences have opened new perspectives for environmental biotechnology applications. 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 Corvini
School of Life Sciences FHNW
Keywords: Nanoparticles; Laccase immobilization; Endocrine disrupters; Fixed-bed; Membrane bioreactor
References:
Introduction
Endocrine 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 consequently 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, enzymecatalyzed polymerization (mainly by peroxidases) and precipitation 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 oxidation of a variety of phenols, including BPA [3].
While enzyme-based biocatalytic processes can easily be implemented 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. Furthermore, 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 immobilized enzyme encompass various aspects. For instance, the solid
supports must be biocompatible, while the immobilized enzyme should remain accessible to its cofactors and substrates.
In the past decades, porous inorganic materials, especially mesoporous 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 study
Our final goal is to demonstrate at pilot-scale, at the ARA Birs
wastewater treatment plant, the efficiency of laccase-NP conjugates for the removal of micropollutants. The tests are performed 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 Coriolopsis 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
[1] Greim HA. The Endocrine and Reproductive System: Adverse Effects of Hor-
Conjugate development of nano structured material
and laccase
Laccase-modified NPs were obtained using surface modification, i.e. silanization, followed by a glutaraldehyde cross-linking
procedure. Both, the protein load on fsNP and the binding efficiency (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-Emmett-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 activity and stability were assessed in waste water to evaluate
their applicability for BPA degradation in advanced waste water
treatment systems.
[2] Tsutsumi Y, Haneda T, Nishida T. Removal of estrogenic activities of bisphe-
monally Active Substances? Pediatrics 2004;113 (4):1070–5.
Figure 1: SEM picture of fsNP (left), fsNP after silanization (middle), fsNP
after modification with laccase and glutaraldehyde according to the optimized protocol (right). Samples were taken from a suspension in phosphatebuffer (pH=7), 150’000× magnification, 20 kV.
The stability of the enzymatic activity of laccase- fsNP conjugates (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 experiments
The 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 macropollutants (organic carbon, nitrogen, phosphorous, etc.) while the
UF membrane unit is designed to retain the laccase-NP conjugates as well as biomass from the fixed bed in the system and
produce permeate free of particles.
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.
nol A and nonylphenol by oxidative enzymes from lignin-degrading basidiomycetes. Chemosphere 2001;42 (3):271–6.
[3] Tanaka T, Tonosaki T, Nose M, Tomidokoro N, Kadomura N, Fujii T, Taniguchi
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 successively 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 biological oxygen demand and suspended solids (average effluent concentrations <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 containing 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 backwashed 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 conjugates will be dosed in this UF unit to react with the BPA which is
not degraded in the preceding fixed-bed treatment.
M. Treatment of model soils contaminated with phenolic endocrine-disrupting
chemicals with laccase from Trametes sp. in a rotating reactor. Journal of Bioscience and Bioengineering 2001;92 (4):312–6.
[4] Stanescu M, Fogorasi M, Shaskolskiy B, Gavrilas S, Lozinsky V. New Potential
Bio­catalysts by Laccase Immobilization in PVA Cryogel Type Carrier. Applied Biochemistry 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 partners 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 frame of the Materanet programme initiated by the European Union and the Federal Office for the Environment (FOEN).
Economic efficiency and benefit to society:
Chemical pollution of natural waters has become a major public concern in almost all parts of the world. The LANCE concept is based on the use of biological catalysts to develop an
eco-friendly “de-pollution” technology for the advanced treatment of contaminated water.
Figure 3: The fixed-bed-membrane reactor system
34 | 35
School of Life Sciences
Mistletoe-Preparations (Iscador®) Encapsulated in Silica
Nanoparticles as a Therapy for Breast Cancer
The 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 colorimetric assays.
Introduction
Breast 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 therapies and in the use of adjuvant cytotoxic therapies, the reduction 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 therapies often lead to only partial success, complementary treatment 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 included 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 purified 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 concentrationdependent 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 content, 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 discrepancy 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 nanoparticle (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 extracts 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 encapsulating 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 results
In this work, we demonstrate the successful embedding of
three different mistletoe extracts (Iscador® M, P, Qu) in a coreshell 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 nanoparticles 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.
the different Iscador®-nanoparticles, the data from the MTTassays were used to determine the concentrations which result 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 mistletoe-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 formulations and the naked Iscador 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. Quality 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, Gudmundsdottir G, Hummerston S, Ahmad AM, Platin N, Kearney N, Patiraki E. Use of
complementary and alternative medicine in cancer patients: a European survey,
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 cytotoxic 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 indicative of a wider therapeutic window in the case of Iscador®nanoparticle conjugates.
Conclusion and Outlook
In 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 nanoparticle 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 nanoparticles has increased their cytotoxic effect on the breast
cancer cells tested. Interestingly, this increase can be seen
even at low concentrations on Iscador® nanoparticle conjugates 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 follow 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.
Annals of Oncology 2005;16 (4): 655–663.
[4] Zhao Y, Trewyn BG, Slowing II, Lin VS-Y. Mesoporous Silica NanoparticleBased 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, Frederik PM, de Kruijff B. Nanocapsules: lipid-coated aggregates of cisplatin with high
cytotoxicity. Nat Med 2002;8 (1):81–4.
Research Focus Area:
Therapeutic Technologies (TT)
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 Department, 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.
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
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
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.
36 | 37
School of Life Sciences
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.
tures Ap and Mp, on the energy density applied during the production process was published [5]. Interestingly, the transition
temperatures can be adjusted to the physiological range.
References:
[1] Es-Souni M, Fischer-Brandies H. Assessing the biocompatibility of NiTi shape 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.
Michael de Wild, Uwe Pieles, Therese Bormann, Conradin Döbeli, Waldemar Hoffmann, Uwe Pieles, Erik Schkommodau,
Ralf Schumacher.
School of Life Sciences FHNW
[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-
Keywords: NiTi shape memory alloy, implants, selective laser melting, metallography, differential scanning calorimetry,
micro computed tomography
Indroduction
Selective laser melting (SLM) is an Additive Manufacturing
method that allows the production of complex-shaped metallic structures based on a virtual Computer-Aided Design (CAD)
or a clinical dataset e.g. for a bone scaffold. In the SLM Realizer 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 proficient 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 orthopedic treatment, as an alternative to conventional machining
like turning or milling.
In this project we use nickel-titanium (NiTi) powder, with particle size of around 50 µm provided by SAES MEMRY GmbH,
as a starting material. These nearly equiatomic binary composed nickel titanium alloys are successfully used in biomedical 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-microstructures (DSC 30 from Mettler-Toledo, measurement range
between -100°C and 125°C) and high-resolution micro computed tomography (µCT, SkyScan 1172TM, SkyScan, Kontich, Belgium, 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).
Results
The metallographic analysis of the prepared solids shows an
oriented microstructure with elongated grains in building direction, 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 globular microstructure in the support structure. The pore density
in the material depended on the laser parameters. DSC measurements served for the determination of the transition temperatures. The starting powder, as well as the SLM parts produced, 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 austenite phase change characterized by a change in crystal structure 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-
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]).
nation of strain fields in porous shape memory alloys using micro computed
Figure 2: Metallographic investigations revealed the microstructure in SLM
fabricated samples. Arrows indicate building direction.
tomography. Proc. of SPIE 2010;7804 78041M.
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 pseu­doplastic deformations were quantified using µCT to calculate
the strain field of the entire specimen on the basis of three-dimensional 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 accelerating voltages as high as 180 kV by means of the nanotomTM to
uncover the pore density, pore sizes and pore shapes [4].
perties of NiTi-structures fabricated by selective laser melting. Europ Cells and
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].
Conclusion and Outlook
In collaboration, the University of Applied Sciences Northwestern Switzerland, the University and the University Hospital 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 reversibly 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 fusion test method. Additionally, intense thermo-mechanical characterization including training for intrinsic two-way shape
memory effect will be performed.
[5] Bormann T, Schumacher R, Müller B, Mertmann M, Pieles U, de Wild M. ProMaterials 2010;20 (Suppl 1): 13.
Research Focus Area:
Therapeutic Technologies (TT)
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 Center, 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 collaboration between the University of Applied Sciences Northwestern
Switzerland, the University of Basel and the University Hospital of Basel is leading to symbiotic exchange of knowledge
and technology in the field of biomedical engineering.
38 | 39
School of Life Sciences
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.
Luzmilla private hospital (Fig. 3), both in Amman. These audits 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 financial savings.
Emmanuel Oertlé, Olga Steiger
School of Life Sciences FHNW
Keywords: Best Environmental Practices (BEP), healthcare, sustainable development, cleaner production, environmental audit
Introduction
While environmental management in the healthcare sector in
industrialised countries is common practice due to legislation and standards, it is not the case in developing and transition countries, where hospitals especially often still have
a relevant regional impact on the environment that is neglected or even ignored. Additionally, the costs for energy, water and the disposal of waste can be quite substantial. These
issues therefore offer potential for improvement in efficiency, 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 knowhow to the field of cleaner production as well as to the development of cooperation in the healthcare sector in developing countries. Cleaner production pursues an integrated
strategy, aiming at reducing environmental impacts in a preventive 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 methodology as “Best Environmental Practice” in the healthcare
sector was developed especially for developing and transition countries.
Applied Research
Intensive literature research revealed several fields in the
healthcare sector, with significant potential for the implementation 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 assessment and implementation of measures. For example, hospitals consume large amounts of energy and water. Most of the
energy consumption can be accounted for by heating, ventilation 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.
Figure 2: Prince Hamzah hospital in Amman, Jordan
Simultaneously, two master theses were conducted both in
Jordan and in Switzerland. The thesis evaluating cleaner production 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.
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 under the following URL: http://www.fhnw.ch/lifesciences/iec/
forschungsfelder-und-projekte/download-projekte/projekte/
best-environmental-practices-for-the-healthcare-sector.
Conclusion and Outlook
The research conducted here revealed a substantial potential 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 implementation cases are needed to prove the economic feasibility, real
ecological benefits and the technical and organisational viability 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 settings, 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);
Figure 1: The concept of BEP in hospitals
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 produced every day. Some of these are hazardous for the environment 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 sector in Switzerland. Several field visits were carried out to
learn more about best environmental practice in Swiss hospitals. In addition, numerous presentations of experts from
Switzerland and Germany about energy efficiency, waste management and other subjects were given. All activities conducted 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
Project Team:
Emmanuel Oertlé, Dieter Mutz, Olga Steiger (Institute for
Ecopreneurship, School of Life Sciences FHNW)
Partner:
Sustainable Business Associates (SBA) Lausanne, Royal Scientific Society (RSS) Jordan
Figure 3: Conduction of an environmental audit in Luzmilla hospital in
Amman, Jordan
Results
One of the main results was the development of the “Best Environmental Practices in the Healthcare Sector” guide. [2] The
aim of this guide is first of all to raise awareness of the necessity and the benefits of implementing BEP. The guide provides a methodology to overview, understand and characterize 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 implementation progress. Following the introduction to the hospital 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-
Funding:
Swiss Secretariat for Economic Affairs (SECO)
Economic efficiency and benefit to society
The 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 patients and employees.
40 | 41
School of Life Sciences
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 Dudal
School of Life Sciences FHNW
Keywords: Spin-off Company – nanomaterials – environmental applications – Basel Incubator
Introduction
Research 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 target 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-efficient. It enables the production of kilograms of this virus binding nanomaterial in a few hours from simple, easily available
and non-expensive raw material.
Results
The 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 submission,
–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 INOFEA, 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 Federal Institute of Intellectual Property (MAREG sca/602193);
the domain names corresponding to INOFEA (www.inofea.
ch and www.inofea.com) and corresponding email addresses are reserved;
Furthermore new products based on the patent will be developed either by INOFEA itself or with the financial help of the
CTI and acacemic institutions, such as the School of Life Sciences 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 Venture 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.
–An exclusive license pre-agreement for environmental applications has been reached with the School of Life Sciences
FHNW regarding use of the patent.
A preliminary survey of the potential market has been performed 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, bioanalytical laboratories, etc. Finally, the chemical industry and
chemical retailers could also be a potential market.
In numerous studies, a fast growing market for these applications 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 represent $21.5 million.
The INOFEA business model is based on the B to B marketing
of virus binding nanomaterial, which is easy, fast and costefficient 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 nanomaterial-embedded products together with our customers.
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.
Conclusion and Outlook
We have identified the critical milestones leading to the foundation 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 quantifying the market potential are crucial next steps to be continued.
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.
Figure 1: Inofea Team
Research Focus Area:
Therapeutic Technologies (TT)
42 | 43
School of Life Sciences
Absorption Mechanisms of Poorly Water-Soluble Drugs from
Self-Emulsifying Formulations in the Caco-2 Cell Model Using
Biorelevant Media
Drug development encompasses the formulation and manufacture of a delivery system which provides adequate bioavailability. 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
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.
Keywords: Drug absorption, intestinal, formulation, Caco-2, biorelevant media, self-emulsifying drug delivery system
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 understood and the rate of success is still variable. In the present work, the absorption kinetics of model poorly water-soluble drug danazol formulated as a lipid based SMEDDS was
investigated in the Caco-2 cell culture model. This is an established in vitro model for intestinal absorption. To improve 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 biore­levant 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.
Methods
Danazol was formulated as SMEDDS consisting of tri-, diand monoglyceride, Cremophor EL as surfactant and ethanol as cosolvent. This formulation spontaneously produces
a microemulsion upon mixture with aqueous media. Caco-2
cells were cultured on Transwell inserts according to standard 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 experiments 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.
Results
Permeability 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 microemulsion and the biorelevant media increased drug solubility
in water (not shown). The model-deduced media-to-cell partition 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
Figure 2: Deduced permeability coefficient of danazol for different lipid
concentrations of SMEDDS in aqueous transport medium and biorelevant
media FaSSIFCaco and FeSSIFCaco.
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 contributing factors.
(Eq. 1)
1 =
1
P Pdbl,d (qz+ 1– zV
f
q
+
(
) z Pm,d +
1
Pm,L
z
Pm,LVf
)
where,
P is the measured permeability coefficient
z is the free fraction of drug in the colloidal dispersion
Pdbl,d is the permeability coefficient of the diffusion boundary
layer (unstirred water layer) for the free drug
Pm,d is the permeability coefficient of the free drug for the
cell membrane
Pm,L is the permeability coefficient of lipid particle-associated drug for the cell membrane (due to direct transfer upon
impact)
Vf is the volume fraction of water (=1-φ), φ being volume fraction of the lipid phase
q accounts for the difference in diffusion boundary permeability between the free drug and the colloidal particle-associated drug and is given by
q=
²/ 3
( )
D w,d
D w,L
with D being the diffusion coefficient of the drug molecule
and of the colloidal particle in water, respectively. Vf is commonly >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/s
Pm,L = 1.1E-3 ± 3.6E-4 cm/s
Pdbl,d = 1.05E-3 ± 2.4E-4 cm/s
Hence, permeation by direct drug transfer from the lipid particle to the cell membrane must be taking place in parallel
with permeation of free drug.
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.
Conclusions
Incorporation of the lipophilic drug in the colloidal structures of SMEDDS was found to increase its overall drug
solubility in the medium. The propensity of the drug, however, to permeate the cell membrane was generally reduced.
This effect of SMEDDS was evident in fasted and fed state
bio­relevant 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 permeability coefficient of the drug for the cell monolayer compared
to the fasted state fluid. The increased drug solubility elicited by SMEDDS may, however, compensate or even overcompensate 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. Development of self-microemulsifying drug delivery systems (SMEDDS) for oral bioavailability 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 Proximal Human Gastrointestinal Tract: An Update. Dissolut Technol. 2009;16(3):21–5.
Research Focus Area:
Therapeutic Technologies (TT)
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 Technology, 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 expedite drug development. Understanding the mode of action
of drug delivery systems under these conditions will allow
the introduction of potentially life saving drugs.
44 | 45
School of Life Sciences
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
Introduction
New drugs often exhibit low aqueous solubility, which can
result in biopharmaceutical issues of incomplete drug absorption as well as of high intra- and intersubject variability.
These biopharmaceutical issues are major concerns for the
development of novel drug products. Pharmaceutical technology can here provide an important contribution to drug products 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-emulsifying systems [2]. The complex mixtures are named according to their dilution performance as self-emulsifying or selfmicroemulsifying 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 dispersion 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 formulation 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.
Figure 1: Fate of a lipid-based drug formulation in the gastro-intestinal tract.
A formulation should therefore keep the drug in a solubilized form throughout the intestinal transit. This solubilization ability can be tested in vitro by using sophisticated
dispersion/precipitation tests. It is further possible to analyze the formulation digestion [4, 5]. These tests are currently 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.
Results
A 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 Gattefossé as main sponsors. Further partners come from academia including eight universities. As one of the academic partners, 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 formulations. Some tests are targeted for an early development
phase so that the experimental set-up as well as the analytical 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 formulation dispersion at a later development stage.
The main objective of the Institute of Pharma Technology
(School of Life Sciences FHNW) is to evaluate new technologies for in vitro dispersion testing of lipid-based formulations. Simple dilution tests were studied to learn about experimental 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 phase, we studied more sophisticated biopharmaceutical precipitation tests. Fig. 2 illustrates such an assay including
simulated gastric as well as simulated intestinal fluid. This
biopharmaceutical method has the advantage that the transit from the stomach to the intestinal environment can be
simulated and it is therefore called a transfer test. For initial
Figure 2: Scheme of a biopharmaceutical transfer test and installed monitoring tools. Initial results are shown using dipyridamole as model drug.
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 technologies. 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 analyzed 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 online 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 planned to assess the suitability of the new in vitro test for a
series of lipid-based drug formulations.
Conclusion and Outlook
The 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 provide 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 formulations. Based on this characteristic behavior the existing
categorization of lipid-based formulations will be reconsidered. 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 administration: 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 watersoluble 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.
46 | 47
School of Life Sciences
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
Introduction
The 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 development of infections around the implant. Generating roughness
and porosity on the implant surface at the nano- and micrometer 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 implant 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 reducing infection risk as well [3]. The goal of the project NAPTIS is to develop surfaces of titanium implants which fulfill
both requirements focussing on the development of new porous surfaces of titanium implants by spark assisted anodizing. 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.
Results
The surface treatments applied in the course of this project
include precleaning, spark-assisted anodizing in proprietary 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
Figure 1: Layers produced by the spark-assisted anodizing process
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 discovered 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 demonstrated that the mechanical pretreatment for deburring
has no significant effect on the overall anodization process.
Although similar in chemical composition and morphological 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).
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 demonstrate the long-term efficacy of the antimicrobial active
sample (solid assay). First results of the antimicrobial activity 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 elements in the course of the anodizing process, as well as with
regard to other microorganisms.
To determine the biocompatibility and incorporation potential of the titanium implant surfaces developed, several
bioassays were performed. Bone marrow derived mesenchymal stem cells were plated on titanium sample discs and
cultured for 20 days under conditions either favouring (OM)
or preventing (CM) differentiation towards osteoblastic lineage 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 differentiation inducing medium (Fig. 3b).
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.
elemental conditions will be studied for their antimicrobial
activity and effects on stem cell differentiation. The most promising 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: material 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
Research Focus Area:
Therapeutic Technologies (TT)
Figure 2: Scanning electron micrographs of different surface modified
samples (sample 13A, 13B, 13C)* (buttom: results of the Calo- test)
Samples treated according to the protocol developed* have
been subjected to test methods for the further characterization of the modified titanium surfaces e.g. confocal microscopy to determine the surface roughness (SRa), biocompatibility 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 active copper deposits were prepared and tested. The samples
containing copper, which were introduced during the electrochemical process in the presence of the proprietary electrolyte* 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 certain cases bacterial growth is inhibited by extracts from metallic copper surfaces whereas in other cases direct contact
of the microorganism with the copper surface seems to be required. Using copper discs we found that E.coli bacteria die
Project Team:
Uwe Pieles, Joachim Köser, Michael de Wild, Waldemar Hoffmann (Institute of Chemistry and Bioanalytics, Institute of
Medicinal and Analytical Technology, School for Life Sciences
FHNW)
Ivan Martin (Department of Biomedicine, University of Basel)
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.
Conclusion and Outlook
In 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 experiments 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 electrochemical treatment processes utilizing electrolytes with different
Partner:
Falko Schlottig (Thommen Medical AG)
Christiane Jung (KKS Ultraschall AG)
Funding:
Swiss Nanoscience Institute (SNI)
Economic efficiency and benefit to society
Medical 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.
48 | 49
School of Life Sciences
Development of a Functional Model of a Hollow Rotor Axial
Flow Pump for Cardiac Assistance
References:
[1] Marseille O. Entwicklungs- und Bewertungsverfahren für Rotationsblutpumpen. Dissertation. RWTH Aachen, 2001.
[2] Stepanoff AJ. Centrifugal and Axial Flow Pumps: Theory, Designs and
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.
Application. 2nd ed., Florida: Krieger Publishing Company, 1992.
[3] Behbahani M, Behr M, Hormes M, Steinseifer U, Arora D, Coronado O, Pasquali M. A Review of Computational Fluid Dynamics. Euro J Applied Mathematics 2009;20 363–397
Erik Schkommodaua, Sandro Fabbria, Matthias Jekera, Doan Baykutb
aInstitute for Medical and Analytical Technologies, School of Life Sciences FHNW, bCorasMedical AG
Research Focus Area:
Therapeutic Technologies (TT)
Keywords: Silica core-shell nanoparticles, 14C-labelling, fluorescent, polyethylene glycol
Method
Since 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 abovementioned 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 VeroBlue™/ 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 dematerialized and degassed water and a bypass- pump was installed 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 parameters 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 Instruments analogue to digital signal converter was used; measurement data were detected in real time and processed with a
measuring program based on LabView®.
Figure 2a: Drive unit
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 transplantation problems including their waiting procedures
would become less severe.
Figure 1: Electromechanical pump test stand
Results
An axial-flow pump with circumferentially positioned rotor
blades was designed. This unique “hollow-rotor” design eliminates the central body without reducing pumping perfor­m­
ance [fig. 2]. By diminishing blood friction surfaces, a re­duc­
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 geometries 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 developed [fig. 1]. The functionality and hydraulic performance of
the different designs were tested and analysed. The most appropriate 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 Outlook
The experiments showed that even in this early phase of development, 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 efficiency factor in comparison to volume is promising for intracorporal blood conveyance.
Figure 2b: Hollow-rotor
Based on the verified functionality of the hollow-rotor concept,
an extracorporeal operating VAD for in vitro tests will be developed. The next steps comprise active/passive magnetic levitation, electrical drive, hydrodynamic design and haemocompatibility. For an accurate prediction of flow field and shear
stress Computational Fluid Dynamics (CFD) will be used.
differential pressure [mmHg]
Introduction
In 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 transplantation (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 downsizing of the devices for convenient implantation.
Project Team:
Erik Schkommodau, Sandro Fabbri, Mathias Jeker (Institute
for Medical and Analytical Technologies, School of Life
Sciences FHNW)
190
180
170
160
150
140
130
120
110
100
90
80
70
60
50
40
30
20
10
0
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
volume flow rate [l/min]
7000 rpm
8000 rpm
9000 rpm
10000 rpm
11000 rpm
12000 rpm
Figure 3: Head-capacity curve of rotor #26
5.5
6
6.5
7
7.5
8
8.5
52 | 53
Environmental Management (EM)
Holistic environmental management for the sustainable use of
resources and the preservation of an environment worth living in.
54 | 55
School of Life Sciences
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 Furstos
School of Life Sciences FHNW
Keywords: Waterless toilet, water saving, micro-pollutant free sewage water
Introduction
Until now, handling human waste in all developed countries
was based on flush toilets, using water as transporting, diluting 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 because 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
chem­ical 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 former High School of Applied Sciences FHBB [2].
In the area of waste management, preventive resource protection 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.
Results
The 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 better 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 industrial 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 Outlook
The 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 position 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 human 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 technological 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 waterless 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 Erfahrungen 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
Figure 1: Waterless toilet ready for move (left); ready for use (right)
Research Focus Area:
Environmental Management (EM)
Project Team:
Claude Lüscher, Julien Furstos (Institute for Ecopreneurship,
School of Life Sciences FHNW) and Henri Zinsli, Münchenstein
Partner:
FOLAG AG Sempach; Zim-Clip GmbH Neuchâtel; espro AG
Stallikon
Funding:
Federal Office for the Environment (FOEN), Förderfonds Aargau, 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.
56 | 57
School of Life Sciences
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
Process chain 0 is the reference chain without phosphorus recovery. In process chain 1 the phosphorus is precipitated from
the liquor from the sludge dewatering; the sludge is incinerated 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.
Anders Nättorp, Claude Lüscher
School of Life Sciences FHNW
Keywords: Phosphorus, Sustainable, Resource Management, Recovery, Incineration, Technology
Introduction
Phosphorus 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 replenished. 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 phosphate 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 supply 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 consumption. 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].
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 agriculture or further processing and a solid suitable for landfill.
Sewage sludge is in general an important source of phosphorus 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 elimination of phosphorus increases. The most recent statistics indicate 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 phosphorus 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 information necessary for implementing a new sewage sludge
treatment in 2015.
Results
15 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 (mineralization). 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 processes of every type were chosen: for recovery from water,
Only some types of mineralization allow recovery of phosphorus 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 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 triplesuperphosphate. 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 process is not sufficiently soluble for plants; further development is needed.
Conclusion and Outlook
Switzerland imports 16'000 t of phosphorus every year, mainly 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 Canton of Zürich a monoincineration plant is planned. The Federal
Office for the Environment is revising waste disposal regulations and is considering obligatory recovery of the phosphorus
in wastewater.
References:
[1] U.S. Geological Survey, Mineral Commodity Summaries, 2010. Available un-
The recovery potential, specific cost and specific energy demand 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 available phosphorus can be recovered.
–Recovery from sludge or extraction from the mineral residue (process chains 2, 4 and 6) yields three quarters of the
available phosphorus. However, those processes are expensive because of the high chemicals demand.
der: http://minerals.usgs.gov/minerals/pubs/commodity/phosphate_rock/mcs2010-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 Umwelt, 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
250
1
WWTP
WWTP
Stabilization
Stabilization
Dewatering
Dewatering
Drying
Drying
Monoincineration
MSWI
Landfill
Landfill
200
0
1
P-Recovery
sludge liquor
2
WWTP
Stabilization
Dewatering
P-Recovery
sludge
Drying
3
WWTP
Stabilization
Dewatering
Drying
Monoincineration
4
5
WWTP
6
Stabilization
Dewatering
Drying
Wetoxidation
MSWI
P-Recovery
purify
residue
Landfill
3
P-Recovery
extraction
from residue
P-Recovery
purify
residue
P-Recovery
extraction
from residue
2
Energy demand compared to reference process chain 0
(kWh/kg phosphorus)
0
68%
150
100
83%
5
50
87%
X
-2
-4
0
0
87%
7
2
4
83%
3
6
-50
-100
25% 1
4
-200
Cost compared to reference process chain 0 (CHF/kg phosphorus)
1
2
3
4
5
Landfill
Figure 1: The evaluated process chains: mineralization processes in orange and processes for phosphorus recovery in white.
6
From process water
5 Purification of residue of wet oxidation
From sludge
6 Extration from residue of wet oxidation
Purification of mono-incineration ash 7 Mining of phosphorus and processing to
Extraction from mono-incineration ash Triple-superphosphate (TSP)
%
Project Team:
Anders Nättorp, Claude Lüscher, Thomas Wintgens (Institute
for Ecopreneurship, School of Life Sciences FHNW)
6
-150
Landfill
Research Focus Area:
Environmental Management (EM)
2
Recovered phosphorus-percentage (% from inflow)
Figure 2: Evaluation of the process chains.
8
4
10
12
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 enterprises with a good basis for sustainable decisions. The study
shows that phosphorus recovery is, or will soon become, economically and ecologically advantageous.
58 | 59
School of Life Sciences
Water Loss Reduction – A Focus on Pressure Management
Customers worldwide expect to be supplied with affordable drinking water in sufficient quantity, quality and continuity, but water losses often represent a major challenge to achieving these aims. Thus, we are involved in a partnership to introduce, promote and support Pressure Management (PM) as a best practice instrument for water loss
reduction, especially for developing countries and emerging economies.
Demonstration of improved management in pilot areas
The technical implementation of the guidelines will be examined together with the implementation of the required technical 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.
Emmanuel Oertlé
School of Life Sciences FHNW
Furthermore, new pipe breaks have been reduced by approximately 50% with a project payback time of 4–5 months.
Conclusion and Outlook
Knowledge 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. Offering the guidelines with working materials in combination
with training measures and pilot projects for pressure man­
Keywords: Water Loss Reduction, Pressure Management, Guidelines, Technical Cooperation.
Technology
Period
Background
Sustainable 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 water resources and on the infrastructure that supplies drinking 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 politicians, 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 distributed without being invoiced, called ‘apparent’ water losses. The
sum of real and apparent water losses and unbilled authorised 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, nonrevenue water accounts on average for 40–50% of the water
produced.
Figure 1: Standard terminology for the water balance according to IWA [2]
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 developing 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 supply 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 Approach
From 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 international partners has been created. Within this project, FHNW is
responsible for project management and coordination, organisation of stakeholder dialogues, the project homepage and
parts of the developed technical manual.
The main objective of the development partnership is the reduction of water losses through improved management of existing water supply networks at different levels of intervention:
Development of guidelines for water loss reduction –
a focus on pressure management
These 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 instruments for water loss reduction. This ambitious task is
based on intensive dialogues, literature research, field experience 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 complementary 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. implementation, operation and maintenance), tailor-made training
plans and materials have been developed for the design, installation and management of pressure management areas
including valve design, maintenance and repair. The content
of the guidelines provides the framework for a comprehensive training programme for technical directors and technicians 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.
Water losses Water savings
(%)
(m3/month)
Without PM
Jan. 2005–Sep. 2005
301.7
–
Time based modulation
Oct. 2005–Jan. 2006
203.9
-33%
Remote node based modulation Feb. 2006–Jun. 2006
178.0
-41%
Figure 3: Results obtained in the district of Santo Amaro in Brazil between
January 2005 and June 2006
agement implementation is seen as an effective approach for
a long-term reduction of leakage from water distribution networks and more sustainable water resources management.
References:
[1] World Water Council, Istanbul Water Consensus for Local and Regional
Figure 2: Implementation of pressure management component in Ain Al
Basha, Jordan, where water losses were reduced by up to 40%
Authorities. Proceedings of the 5th World Water Forum, Istanbul, Turkey, 2009.
[2] Lambert AO, Hirner W. Losses from Water Supply Systems: Standard Terminology and Recommended Performance Measures. International Water As-
The Pressure Management Homepage –
www.waterloss-reduction.com
In order to exchange information, facilitate contacts and allow downloading the guidelines free of charge, the project
homepage is available at the following URL: www.waterlossreduction.com.
sociation (IWA), 2000.
[3] Knobloch A, Klingel P, Oertlé E, Mutz D, Fallis P, Sorg F, Ziegler D. Guidelines 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.
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.
Proceedings of the IWA Specialised Conference 'Leakage 2005', Halifax, Nova
The Example of Santo Amaro, São Paulo, Brazil
In the metropolitan area of São Paulo, with continuously increasing 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 effectively, 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 reduction system that includes pressure regulating valves, control
panels, telemetry installations and the supporting software
[5]. Two technologies were implemented: in a first stage, timebased modulation and in a second stage, remote node based
modulation, which resulted in the benefits shown in Fig. 3.
Partner:
Deutsche Gesellschaft für Internationale Zusammenarbeit
(GIZ) GmbH, VAG-Armaturen GmbH (VAG), Karlsruhe Institute of Technology (KIT).
Scotia, Canada, 2005.
[5] Oppinger P. Reducing Water Losses by Intelligent Pressure Management,
Water & Wastewater International, 2009.
Research Focus Area:
Environmental Management (EM)
Project Team:
Emmanuel Oertlé, Dieter Mutz (Institute for Ecopreneurship
IEC, School of Life Sciences FHNW)
Funding:
German Ministry for Economic Cooperation (BMZ) and VAGArmaturen 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 million people in the developing countries.” (World Bank, 2006)
60 | 61
School of Life Sciences
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.
fact can be explained by the possible presence of microorganisms which have similar metabolism compared to nitrifying
bacteria. It means that their activity could be inhibited by allylthiourea although they were able to oxidize some organic
compounds of industrial wastewater. Thus their respiration
Lukas Dvorak, Jan Svojitka, Thomas Wintgens
School of Life Sciences FHNW
Keywords: membrane bioreactor, industrial wastewater, activated sludge, nitrification, adaptation
Introduction
Nitrification (biological oxidation of ammonia nitrogen to nitrate) is carried out by autotrophic bacteria which are more
sensitive to different inhibitory effects compared to heterotrophic bacteria. Inhibitory effects of various compounds on
nitrification can occur with some industrial wastewaters, especially 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 biodegradable 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 required [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 ordinance (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 municipal 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 chemical 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 tests
The respirometric tests served for the evaluation of nitrification 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 industrial wastewater ranging from 0 to 100%. Results of respirometric 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 another wastewater treatment plant and were not exposed to the
Figure 3: AUR for tested shares of IWW - reference activated sludge
activity will be increased after the addition of a small amount
of industrial wastewater; due to the respiration test methodology, their activity will be measured together with the nitrifiers. 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.
Conclusion and Outlook
From 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 nitrification 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 concentration 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 increasing 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 proportion 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 identification 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
Figure 1: Photo of adaptation membrane bioreactor
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 nitrification respiration rate in the test with 100% industrial wastewater compared to the test with 0% industrial wastewater. Before
the start-up of the adaptation reactor the inhibition rate was
Figure 2: AUR for tested shares of industrial wastewater (IWW) under different IWW concentrations in the MBR influent
about 95%; after 164 days of adaptation and stepped increase
of industrial wastewater in the influent into the MBR, the inhibition was only 61%. In contrast, the inhibition effect on the
samples of reference activated sludge did not change substantially 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
Results of chemical analyses
The results of regular chemical analyses showed that the increase in the content of industrial wastewater in the MBR
influent had almost no influence on the concentration of ammonia nitrogen in the effluent from the MBR. The average concentration 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 contributed to the overall high efficiency of ammonia nitrogen removal – on average 98%.
In terms of nitrite nitrogen concentration, its very high concentration (up to 10 mg/L­nitrite 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 stabilization of MBR operation, the nitrite nitrogen concentration
decreased to 0.6 mg/L­and 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 concentration 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 efficiency was above 88%.
From the results mentioned above it is obvious that the application of membrane technology is promising for industrial wastewater treatment, particularly if good effluent quality
and high efficiency of nitrification should be achieved.
[3] Qin JJ, Wai MN, Tao G, Kekre KA, Seah H. Membrane bioreactor study for
reclamation of mixed sewage mostly from industrial sources. Sep. Purif. Technol. 2007; 53 (3): 296–300
[4] Swiss Federal Council. Gewässerschutzverordnung des Schweizerischen
Bundesrates vom 28. Oktober 1998.
Research Focus Area:
Environmental Management (EM)
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, Switzerland
Institute of Chemical Technology Prague, Czech Republic
Funding:
Sciex-NMSch (Scientific Exchange Programme between Switzerland 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.
64 | 65
Appendix
66 | 67
School of Life Sciences
Publications
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 acting 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
nano­mechanical cantilever sensors. J. Phys.: Conf. Ser.
2007;61:612–7.
Institute for Chemistry and Bioanalytics (ICB)
Barblan M, Mollet D.
Key performance indicators for chemical plants. Chimia
2009;63 (7–8):523–5.
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 applications. 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 macroscopic 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-Assembly 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 quantification 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 diagnosis 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 nanoparticles 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 resorcinarenes in water. New J. Chem. 2010;34 (12):2704–14.
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, Housecroft 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 Nanopowders 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 islandicus. 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, inhibits Nuclear Factor-kB and reduces myocardial infarct size in
rats. Eur. J. Pharmacol. 2008;586 (1–3):251–8.
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
mo­di­fication 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 immunosorbent 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 enzymelinked 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 Environmental 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 potential bioreactor applications. Biochem. Eng. J. 2008;39 (3):586–9.
Wagner M, Berkner S, Ajon M, Driessen AJM, Lipps G,
Albers S-V.
Nault L, Cumbo A, Pretot RF, Sciotti MA, Shahgaldian P.
Cell transfection using layer-by-layer (LbL) coated calixarene-­ Expanding and understanding the genetic toolbox of the
based solid lipid nanoparticles (SLNs). Chemical Communi- hyperthermophilic genus Sulfolobus. Biochem. Soc. Trans.
cations 2010;46 (30):5581–3.
2009;37 (1):97–101.
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.
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-absorbing compounds (UV filters) in environmental samples. J.
Chromatogr., A 2008;1202 (1):64–74.
68 | 69
School of Life Sciences
Institute for Ecopreneurship (IEC)
Bayer C, Follmann F, Melin T, Wintgens T, Larsson K,
Almemark M.
The ecological impact of membrane-based extraction of phenolic compounds – a life cycle assessment study. Water Science & 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 microporous hollow-fibre membranes. Desalination and Water Treatment 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 CYP3A 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. Chemosphere 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. Applied 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 -ethinylestradiols 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.
Fent K, Zenker A, Rapp M.
Widespread occurrence of estrogenic UV-filters in aquatic
ecosystems in Switzerland. Environmental Pollution 2010;158
(5):1817–24.
Kochan J, Wintgens T, Wong JE, Melin T.
Polyelectrolyte-modified polyethersulfone ultrafiltration
membranes for wastewater treatment applications. Desalination and Water Treatment 2009 (9):175–80.
Geranio L, Hommes G, Shahgaldian P, Wirth-Heller A,
Pieles U, Corvini P.
Radio (14C)- and fluorescent-doubly labeled silica nanoparticles for biological and environmental toxicity assessment.
Environmental Chemistry Letters 2010 (8):247–51.
Kochan J, Wintgens T, Wong JE, Melin T.
Properties of polyethersulfone ultrafiltration membranes modified by polyelectrolytes. Desalination 2010;250 (3):1008–10.
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 Levels. Environ. Sci. Technol. 2009;43 (13):4783–90.
Heim T, Hengevoss D.
Mit Cleaner Production zu mehr Ressourceneffizienz. io new
management 2010 (1004).
Evangelou M, Ebel M, Hommes G, Schaeffer A.
Biodegradation: The Reason for the Inefficiency of Small Organic Acids in Chelant-Assisted Phytoextraction. Water, Air, &
Soil Pollution 2008.
Hengevoss D.
Umweltverträgliche Abfallentsorgung. Umwelt Perspektiven
2007 (1).
Fent K.
Ecotoxicology of engineered nanoparticles. In: Frimmel FH,
Niessner R, editors. Nanoparticles in the Water Cycle: Springer, Berlin Heidelberg, 2010.
Hengevoss D.
Umweltverträgliche Entsorgung von Abfällen. Blickwechsel –
das Magazin des Lichtensteinischen Entwicklungsdienstes
(LED) 2007 (2):13–4.
Fent K.
Effects of Pharmaceuticals on Aquatic Organisms. In: Kümmerer K, editor. Pharmaceuticals in the Environment: Springer, Berlin Heidelberg, 2008. pp. 175–203.
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 & Technology-WST 2010;62 (6):1265–73.
Fent K.
Ökotoxikologie: Georg Thieme, Stuttgart, 2007.
Fent K.
Permanent Fisch Cell Cultures as Important Tools in Ecotoxicology. ALTEX 2007;24 (Special Issue):26–8.
Fent K, Kunz PY, Gomez E.
UV Filters in the Aquatic Environment Induce Hormonal Effects 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-trimethoxycinnamate, benzophenone-3, benzophenone-4 and 3-benzylidene 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-trimethoxycinnamate, benzophenone-3, benzophenone-4 and 3-benzylidene camphor. Mar Environ Res 2009;10:10.
Fent K, Weisbrod CJ, Wirth-Heller A, Pieles U.
Assessment of uptake and toxicity of fluorescent silica nanoparticles in zebrafish (Danio rerio) early life stages. Aquatic
Toxicology 2010;100 (2):218–28.
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 understanding 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-modified polymeric membranes used in membrane bioreactors.
Chemical Papers 2009;63 (2):152–7
Kolvenbach B, Schlaich N, Raoui Z, Prell J, Zuehlke S,
Schaeffer 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.
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 (Pimephales 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 Sulfurospirillum barnesii for simultaneous selenate and nitrate removal. 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 Complicated. 19th Annual V.M. Goldschmidt Conference. Davos: Geochimica 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 Spectroscopy of Sequentially Extracted Anaerobic Biofilms. Environ.
Sci. Technol. 2008;42 (20):7587–93.
70 | 71
School of Life Sciences
Lenz M, van Hullebusch ED, Farges Fo, Nikitenko S,
Corvini PFX, Lens PNL.
Combined Speciation Analysis by X-ray Absorption NearEdge Structure Spectroscopy, Ion Chromatography, and Solid-Phase Microextraction Gas Chromatography−Mass Spectrometry 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 acetoclastic 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 single 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 paddy 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 biotransformation 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 – Erfahrungen 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.
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 fractionation. Biogeosciences 2010;7 (1):333–41.
Svoijtka J, Wintgens T, Melin T.
Treatment of landfill leachate in a bench scale MBR.Desalination and Water Treatment 2009 (9):136–41.
Niewersch C, Meier K, Wintgens T, Melin T.
Selectivity of polyamide nanofiltration membranes for cations and phosphoric acid. Desalination 2010;250 (3):1021–4.
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.
Nowak KM, Kouloumbos VN, Schaeffer A, Corvini PFX.
Effect of sludge treatment on the bioaccumulation of nonylphenol in grass grown on sludge-amended soil. Environ.
Chem. Lett. 2008;6 (1):53–8.
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.
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 Conference "Water Loss 2010". Sao Paulo, 2010.
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.
Oggier DM, Weisbrod CJ, Stoller AM, Zenker AK, Fent K.
Effects of diazepam on gene expression and link to physiological effects in different life stages in zebrafish Danio rerio.
Environ Sci Technol 2010;44 (19):7685–91.
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.
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.
Weston A, Caminada D, Galicia HF, Fent K.
Effects of lipid-lowering pharmaceuticals bezafibrate and
clofibric Acid on lipid metabolism in fathead minnow (pimephales promelas). Environ Toxicol Chem 2009;28 (12):2648–55.
Riefer P, Klausmeyer T, Schwarzbauer J, Schäffer A,
Schmidt B, Corvini P.
Rapid incorporation and short-term distribution of a nonylphenol isomer and the herbicide MCPA in soil-derived organoclay complexes. Environmental Chemistry Letters 2010:1–5.
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.
Sandoval Soto L, Jutz M, Langbein A, Wintgens T.
Gras effizient trocknen. Swiss engineering STZ 2009 (6):30–1.
Wintgens T, Hochstrat R, Kazner C.
Water recycling in Europe and the Mediterranean: framework
conditions and technologies for future development. WORKSHOP Membrane technologies for alternative water resources.
Thessaloniki, 2009. pp. 7–10.
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 manure of young pigs treated with a veterinary antibiotic. J. Environ. Sci. Health, Part B 2008;43 (1):8–20.
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.
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 exposure through breast milk. Chimia 2008;62 (5):345–51.
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.
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 fractionation. Biogeosciences Discuss. 2009;6 (5):9279–300.
Zenker A, Schmutz H, Fent K.
Simultaneous trace determination of nine organic UV-absorbing 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 Engineering 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 doublecut 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 Medical 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, editors. Human Machine Interaction: Springer Berlin Heidelberg,
2009. pp. 27–46.
72 | 73
School of Life Sciences
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 Neuroscience 2009;29 (8):1627–33.
Daeichin V, Hemm S, Taub E, Schkommodau E.
Development of an intraoperative visualisation tool for electrode 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 Generatively 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, SchwenzerZimmerer K, Pierer G, Kalbermatten DF.
Silicone moulding for pressure sore debridement. J Plast Reconstr 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 electrodes 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 Engineering. München, 2009.
Hemm S, Wardell K.
Stereotactic implantation of deep brain stimulation electrodes: 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 laceration 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 Implants. 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 sauropod dinosaurs. Proceedings of the Royal Society B: Biological 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 Delivery 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 relationship of benzoxazolones: New potent mglu5 receptor antagonists 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 compounds 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 efflux using phospholipids as solubilizers in the Caco-2 cell model. 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 Preparing 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 lipophilic 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-microemulsifying drug delivery systems. Drug Development and Industrial Pharmacy 2009:1–8.
Kuentz M, Cavegn M.
Critical concentrations in the dilution of oral self-microemulsifying 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-trifluoromethyl-3H-benzofuran-2-one (rac-BHFF) as a positive
al­lo­steric 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 Pharmaceutics 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 occlusion. Skin Pharmacol Physiol 2007;20 (1):50–6.
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School of Life Sciences
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.
Project Portfolio
Since the beginning in the year 2006, the School of Life Sciences FHNW has realized many scientific projects in cooperation with industrial and academic partners. Within the last five years, the school has established itself as a competent 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.
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. Cellular 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. Neurochem. 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.
total number
projects since 2006
362
current projects
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.
number
>100'000 CHF
77
64
141
Type of Funding (% of current projects)
Tuffin G, Huwyler J, Waelti E, Hammer C, Marti H.
Drug targeting using OX7-immunoliposomes: Correlation between 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.
number
10'000–100'000 CHF
265
97
Research Focus Areas (2009–2010)
(% of external Funds)
EU
Swiss Federal Offices
Third Parties
(direct funding)
Environmental Management (EM)
25%
Molecular Technologies (MT)
39%
CTI
Therapeutic Technologies (TT)
36%
SNSF
76 | 77
School of Life Sciences
Research Seminars at the
School of Life Sciences FHNW Autumn 2009–Summer 2011
Dirk Benndorf, Max Planck Institut, Magdeburg
Nutzung von Proteomics für Anwendung in den Bereichen
Klärschlamm und Biogas Produktion
David Hradetzky, HLS FHNW, Muttenz
Mikrosystemtechnik zum Einsatz in der Medikamentenabgabe
Venky Nalluri, HLS FHNW, Muttenz
Dynamic image analysis of pharmaceutical powders and granules- From on-line analytics to flowability characterization
Götz Schlotterbeck, HLS FHNW, Muttenz
Metabolomics in drug development
Sherri Dudal, Novartis AG, Basel
Tracking biological drugs in the body
Claudio Storck, Universitätsspital Basel, Basel
Untersuchung der Biomechanik des Larynx (Kehlkopf) an
Dreidimensionalen Bildern
Anders Nättorp, HLS FHNW, Muttenz
Wie können die Ressourcen im Abwasser genützt werden?
Christian Siebenhaar, HLS FHNW, Muttenz
Herstellung von Nanopartikel mittels Flammenspraypyrolyse
Markus Lenz, HLS FHNW, Muttenz
Bioremediation of selenium contaminated wastewaters - The
solution to pollution?
Andreas Schreiner, Novartis AG, Basel
Pharmazeutische Entwicklung vom Kristall bis zur Tablette Herausforderungen in der Prozesstechnik
Thilo Glatzel, Universität Basel, Basel
Measuring Atomic Scale Electrostatic Forces on insulators
and Molecules using KPFM
Verena Christen, HLS FHNW, Muttenz
Analyse der toxikologischen Wirkungen von Pharmazeutika
in Fischzellen
Andreas Hartmann, Novartis AG, Basel
Globaler Umweltschutz bei Novartis: “Trends und Herausforderungen“
Simon Berner, Institut Straumann AG, Basel
Materials und Surface Modifications for Dental Implants
Christoph Minnig, FHNW, Hochschule für Wirtschaft, PMO
CURE - Corporate Culture and Regional Embeddedness oder
haben Unternehmen eine Heimat?
Konrad Hungerbühler, ETH Zürich, Zürich
What is green solvent?
Michael Stadler, FMI Basel, Basel
Computational Biology
John Zambounis, BASF Schweiz AG, Basel
New Opportunities in Adhesives using Photolatent Bases
Technology
Ursula Meierhofer, FHNW, Brugg
Geschlechterspezifische Aspekte in der Arbeitswelt
Yves Dudal, HLS FHNW, Muttenz
Development of a high-throughput screening for pharmaceutical compounds in waters
Monica Dressler, Novartis AG, Basel
Pharmazeutische Registrierung; vom Molekül im Labor bis
zur Apotheke
Peter Biedermann, Medical Cluster Bern, Bern
Medizintechnikindustrie Schweiz: “Bedeutung, Dynamik und
Netzwerke”
Michael Schleimer, Basilea Pharmaceutica Ltd, Basel
The Role of Analytics in the Development of Drugs
Yvonne Arnold, HLS FHNW, Muttenz
In vitro tests as useful tool to predict the solubilization behavior of poorly soluble drugs incorporated in lipid-based
formulations
Andreas Hafner, BASF Schweiz AG, Basel
Tailor-made Micro-, Nano-particles and Systems - the Basis
for Novel Effects
Jörg Trappe, Novartis AG, Basel
Innovative Strategien in der Wirkstoffoptimierung
Roger Zurbriggen, Akzo Nobel AG, Sempach
Grenzen der Naturwissenschaften
Klaus Köhler, Endress+Hauser GmbH+Co. KG, Weil am Rhein (D)
Moderne Messtechnik und Kalibrierung in Biopharmaprozessen am Beispiel eines Fermenters
Georgios Imanidis, HLS FHNW, Muttenz
Biorelevante Medien für in vitro Wirkstoffabsorption
Gregor Burkhard, HT, FHNW
EU-Forschungsprojekt EUPASS “Evolvable Ultra-Precision
Assembly Systems”– Resultate und Erfahrungen
Alex Ringenbach, HLS, FHNW
Herausforderungen in der Medizinischen Bildverarbeitung Beispiel Leberchirurgie
Jean-Pierre Locquet, Katholieke Universiteit Leuven (KUL),
Leuven
Functional Oxides for Advanced Electronics Applications
Ralf Schumacher, HLS FHNW, Muttenz
Rapid Prototyping und Implantatherstellung – aktueller
Stand der Forschung im Bereich Titan und Biokeramiken
Michael Hoffmann, Universitätsklinikum Freiburg (D)
Genetische Marker für kardiovaskuläre Erkrankungen – von
der Populationsgenetik zur individuelle Diagnostik
Markus Blümel, Novartis Pharma AG, Basel
Protein Analytics: How can proteins be characterized?
Bernd Nowack, Empa, St. Gallen
Where to look for the nano-needle in the environmental haystack?
Emmanuel Oertlé, HLS FHNW, Muttenz
Water loss reduction through intelligent pressure management
Jan Svojitka, HLS FHNW, Muttenz
Membrane bioreactors for industrial wastewater treatment
and sediment decontamination
Lisseth Sandoval, HLS FHNW, Muttenz
Effizienzsteigerung in Landwirtschaftlichen Trocknungsanlagen
Arnulf Bohnacker, HLS FHNW, Muttenz
Unternehmensinterne Managementaspekte
Claude Schärer, HLS FHNW, Muttenz
Asymmetrische Katalyse in der organischen Synthese
Eric Kübler, HLS FHNW, Muttenz
Genetische 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, Freiburg
Applied Physical Chemistry at the HTA-FRIBOURG
Klaus Kümmerer, Universität Freiburg, Freiburg
Chemikalien, Spurenstoffe, Transformationsprodukte und
ihre Bewertung mit Computer basierten Methoden
Daniel Hess, FMI Basel, Basel
Mass spectrometry based protein analysis and proteomics
Ron Tynes, HLS FHNW, Muttenz
It's a Long Road to the Nucleus: Transient Gene Expression
in Modern Protein Biotechnology
Hans Hermann Letzner, Letzner Pharmawasseraufbereitung GmbH, Hückeswagen
Vom Trinkwasser bis zum Wasser für die Infusionslösung,
ohne Krankheitserreger, Rostpartikel und andere Risiken
Wolfgang Riedl, Universität Basel, Basel
Membranverfahren in der Biotechnoloige: wo Sizeexclusion
zu exklusiven Produkten führt
Christoph Hugi, HLS FHNW, Muttenz
“Ressourcenknappheit: Schwellenländer im Wettlauf um
Rohstoffe”
78 | 79
School of Life Sciences
Research Topics and Competences
Institute
Fields of Research
Competences
Research
Focus Area
Institute for Chemistry and Bioanalytics (ICB)
Biochemistry, Bioanalytics, Diagnostics
(Bio)-Nanotechnology, Instrumental Analysis
Organic Synthesis, Chemical Engineering
Biochemistry, Bioanalytics, Diagnostic
(Bio)-Nanotechnology
Molecular Recognition, Organo- and Biocatalysis
Synthesis / Sustainable Development
Molecular Diagnostics and Preclinical Development of Pharmaceuticals
Instrumental Analytics
Organic and Organometallic Synthesis
Chemical Engineering
MT, TT
Institute for Ecopreneurship (IEC)
Resource Management, Cleaner Production
Ecotoxicology
Environmental, Biotechnology and Engineering
Environmental Engineering/Clean Technologies
Ecotoxicology
Environmental Biotechnology/Microbiology
Ressource Management
Cleaner Production in Industry (CP)
Green Chemistry
MT, TT, EM
Institute for Medical and Analytical Technologies (IMA)
Implants and Surgical Systems
Biomedical Information Systems
Medical Image and Signal Processing
Medical Image Processing
Visual Analytics
Computer-assisted Surgery
Medical Rapid Prototyping
Deep Brain Stimulation
Biosignal Processing
Telemedicine
Data Analysis and Modelling
Materialography
Microsystem Technology
TT
Institute for Pharma Technology (IPT)
Dosage Forms
Drug Delivery
Procedures and Production Processes
Intestinal and (Trans) Dermal Active Agent Absorption and Drug Delivery
Drug Targeting and Pharmacokinetcs
Rational Development of Medical Formulations and Dosage Forms
Process Development, Production Planning and Project Management
MT, TT
80 | 81
School of Life Sciences
Contact
University of Applied Sciences
Northwestern Switzerland
School of Life Sciences
Gründenstrasse 40
CH - 4132 Muttenz
Bachelor of Science (B.Sc.)
–Life Science Technologies
Prof. Gianni N. di Pietro, Tel +41 61 467 46 94
[email protected]
Tel +41 61 467 42 42
[email protected]
–Molecular Life Sciences
Prof. Dr. Daniel Gygax, Tel +41 61 467 45 62
[email protected]
Director School of Life Sciences
Prof. Dr. Gerda Huber, Tel +41 61 467 42 42
[email protected]
Master of Science (M.Sc.)
Institute for Ecopreneurship (IEC)
Prof. Dr. Philippe Corvini, Tel +41 61 467 43 44
[email protected]
Institute for Chemistry and Bioanalytics (ICB)
Prof. Dr. Gerhard Grundler, Tel +41 61 467 42 27
[email protected]
Institute for Medical and Analytical Technologies (IMA)
Prof. Dr. Erik Schkommodau, Tel +41 61 467 42 46
[email protected]
Institute for Pharma Technology (IPT)
Prof. Dr. Georgios Imanidis, Tel +41 61 467 46 80
[email protected]
–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]
Publisher
University of Applied Sciences Northwestern Switzerland
School of Life Sciences
Editors
Gerda Huber
Arnulf Bohnacker
Design
Büro für Kommunikationsdesign FHNW
Photography
Uwe Pieles, cover and pages 4, 28, 50 and 62
Copyright © Uwe Pieles, all rights reserved
Print
Steudler Press AG, Basel
Circulation
800 Copies
No reproduction or publishing is allowed without the prior
written consent of the editors
February 2011