Microfluidoc flow system prototype: The University of Leeds has progressed well with the construction of a flow system prototype with the membrane sensor element module. The existing peristaltic pumps have been replaced with syringe pumps which are more easily controlled by automatic system.
Computer software is being developed for automatic control of flow systems and the membrane module has been further miniaturised (Figure 5). The next steps are to introduce an oxygen removal system and to move from single module to multimodular.
Figure 4 : Photograph of automated membrane module prototype with streamlined flow system and flow cell.
Modules for cell cultivation and imaging : Modules have been developed for cell cultivation, imaging and electrical impedance measurement. These modules will be incorporated into a microfluidic system for the initial individual tissue prototype screeners (Figure 6).
Figure 5 : Diagram of first designed tissue screening module.
Incorporation of DNA and miRNA module into the membrane sensor element modular system: Double-stranded 100 base pair long DNA conjugated to lipid tails .have been incorporated into phospholipid vesicles. The vesicles require an incubation time of up to an hour for successful DNA-lipid incorporation.
The DNA-phopholipid doped vesicles as sensor element are then spread on to a phospholipid coated microelectrode. Proof of concept experiments show that the DNA-lipid conjugates sensitise the sensor element to methyl methanesulphonate (MMS) damage by a factor of 100 showing that the methylation of DNA by mms upsets the self-organisation of the phospholipid sensor element which is detected electronically.
Reference NM interaction with biomembranes in the microfluidic module: A series of home synthesised Au and Ag NM of varying shapes and sizes have been screened by the biomembrane sensor element module. Results show that the assay is rapid and indicates a response inversely proportional to particle size and directly proportional to aspect ratio. Ag particles indicate a very clear interaction with the biomembrane sensor element in contrast to soluble silver which shows no significant interaction. A series of JRC standard NM and NM sent out to all partners by ICN Barcelona have also been routinely screened in the microfluidic modular system as part of initial intercalibration experiments within the consortium.
Figure 7: PBPK model for NM biodistribution
Computer program for PBPK model: The PBPK software is being developed. A model system has been built comprising seven separate tissue compartment modules including blood (Figure 6). Results for tissue NM concentrations versus time have been plotted and compared to published data. A good comparison was obtained. These results were extremely encouraging. Subsequent work will focus on comparing the output data from the program with experimental data obtained within the consortium from the membrane and tissue modules Device performance with standard calibrated toxicants: An intercalibration exercise was carried out within the consortium with the aim of aligning and relating together of all the toxicity assays used by each partner. It was planned to use three water soluble toxicant compounds whose physical and toxicological properties were well established. The three toxicants chosen were chlorpromazine, colchicine and methyl
methanesulphonate (MMS). Chlorpromazine is biomembrane active, colchicine is a microtubule inhibitor affecting cell division and MMS methylates the DNA molecule. Results showed for the biomembrane module and cell culture assays a ranking order of toxicity of the toxicant compounds of chlorpromazine
> MMS > colchicine. This indicates that toxicity to cell cultures is of a non-specific biomembrane damage nature. Comet assay showed only MMS to damage the DNA of A549 and HepG2 cell lines which is commensurate with its methylating properties.
Colony forming efficiency chronic exposure tests showed the toxicant ranking order as colchicine > MMS > chlorpromazine.
Colchicine is a microtubule inhibitor and therefore will inhibit cell division to form the cell colony.
7 Expected Impact
The emphasis of this programme is on high level interdisciplinarity which is indispensable for developing a platform technology. This programme brings together the most outstanding workers in nanotechnology, surface and colloid chemistry, toxicology, instrumentation and mathematical modelling to attack the innovative and ambitious problem of developing a global toxicological simulation model and aligning it to a comprehensive and integrated screening platform. The study has a wide impact since it delivers a third generation toxicity screening platform which has a mechanistic and pathway analysis functionality and possesses apparent commercial attractiveness as provides a cheap solution for many valuable practical application such as drug delivery and drug discovery.
The HISENTS project develops a new and comprehensive toxicity testing protocol combining the most up-to-date simulation procedures aligned with a multimodule screening platform where each module carries a specific (bio)barrier and/or (bio)target represented in the simulation. The close reinforcement of dual feedback of simulation with experiment
and experiment with simulation is a disruptive technology and a step change in innovation. This innovation fills an at present unoccupied niche in the toxicity screening area. The new technology improves innovation capacity through its broad interdisciplinarity. The developments in fabrication technology, microfluidics, instrumentation and the interrogation of novel endpoints on a wafer-based platform are each at the cutting edge of 21st century science and can be developed independently for other applications e.g. drug delivery, drug discovery and chemical analysis. The rapid and comprehensive screening ability associated with the simulation model will enable many NM batches to be screened which enables the development of a close understanding of the detailed mechanism of the (bio)nanointeractions. Indeed it was shown in the previous FP7 project ENNSATOX that a close understanding of the toxicity mechanism could only be attained by a rapid screening. This is because NMs change their structure and conformation over time sometimes rapidly.
The HISENTS concept which is the combination of the multimodular screen with the PBPK model is a novel idea. It contains the distinctive features and possesses the positive effects which discriminate the platform from the prior art solutions thereby confirming its high patentability. This screening platform will attract much interest from pharmaceutical and personal care companies and health and safety, water, defence and security industries. There are also several international screening companies such as Cyprotex Ltd who would benefit enormously from adopting this new regime of toxicity testing thus improving their competitiveness.
Personal care companies such as Unilever who are not able to use in vivo testing for the environmental and health screening of their products would find the technology developed during this project highly applicable since its multimodular facility incorporates all tests in one platform and is underwritten by a simulation programme. Pharmaceutical and cosmetic companies will be especially concerned with the structure-activity relationships and also the protocols developed to determine these.
8 Directory
Table 2: Directory of people involved in this project – main contact highlighted in bold.
First Name Last Name Affiliation Address e-mail
Eva Beate Andersen Norsk Institutt For Luftforskning Health Effects Laboratory,
Neus Bastus Fundacio Institut Catala De Nanociencia
I Nanotecnologia Inorganic Nanoparticles Group, Campus De La Uab Edifici Q ICN2, Bellaterra, Barcelona 08193, Spain
neus.bastus@icn.cat
Paul Beales University of Leeds School of Chemistry,
Leeds LS2 9JT, UK p.a.beales@leeds.ac.uk
Rik Brydson University of Leeds School of Chemical &
Process Engineering, Leeds LS2 9JT, UK
r.m.drummond-brydson@leeds.ac.uk
First Name Last Name Affiliation Address e-mail
Tibor Dubaj Slovenska Technicka Univerzita V
Bratislave
Dept Physical Chemistry, Faculty of Chemical and Food Technology, Vazovova 5, Bratislava 81243, Slovakia
tibor.dubaj@stuba.sk
Maria Dusinska Norsk Institutt For Luftforskning Health Effects Laboratory,
Environmental Chemistry Dept, Instituttveien 18, Kjeller 2027, Norway
maria.dusinska@nilu.no
Peter Ertl Technische Universitaet Wien Karlsplatz 13, 1040
Vienna, Austria peter.ertl@tuwien.ac.at
Alena Gabelova Ustav experimentalnej onkologie,
Biomedicinska Centrum SAV
Dubravska Cesta 9 845 05 Bratislava, Slovakia
alena.gabelova@savba.sk
Martin Hanson Blueprint Product Design Ltd Main Office, 38 Market
Street, Keighley BD21 5AD, UK
martin@blueprintproductdesign.com
Nicole Hondow University of Leeds School of Chemical &
Process Engineering, Leeds LS2 9JT, UK
n.hondow@leeds.ac.uk
Nik Kapur University of Leeds School of Mechanical
Engineering, Leeds LS2 9JT, UK
n.kapur@leeds.ac.uk
Mick Karol Blueprint Product Design Ltd Main Office, 38 Market
Street, Keighley BD21 5AD, UK
mick@blueprintproductdesign.com Thorsten Knoll Fraunhofer-Insitut für Biomedizinsiche
Technik IBMT Ensheimer Str. 48, St.
Ingbert 66386, Germany thorsten.knoll@ibmt.fraunhofer.de
Yvonne Kohl Fraunhofer-Insitut für Biomedizinsiche
Technik IBMT Ensheimer Str. 48, St.
Ingbert 66386, Germany yvonne.kohl@ibmt.fraunhofer.de
Rafi Korenstein Tel Aviv University Marian Gertner Institute
for Medical Nanosystems, Dept of Physiology &
Pharmacology, Ramat Aviv 69979, Israel
korens@post.tau.ac.il
Katarina Kozics Ustav experimentalnej onkologie,
Biomedicinska Centrum SAV Dubravska Cesta 9, 845
05 Bratislava, Slovakia katarina.kozics@savba.sk
Nicole Hondow University of Leeds School of Chemical &
Process Engineering, Leeds LS2 9JT, UK
N.Hondow@leeds.ac.uk
Nicole Ludwig Universitaet Des Saarlandes Dept of Human
Molecular Genetics, building 60, Homburg 66421, Germany
n.ludwig@mx.uni-saarland.de
Lea Madi Tel Aviv University Marian Gertner Institute
for Medical
Nanosystems, Dept of Physiology &
Pharmacology, Ramat Aviv 69979, Israel
levana.madi@gmail.com
Eckart Meese Universitaet Des Saarlandes Dept of Human
Molecular Genetics, building 60, Homburg 66421, Germany
eckart.meese@uks.eu
Steve Milne University of Leeds School of Chemical &
Process Engineering, Leeds LS2 9JT, UK
s.j.milne@leeds.ac.uk
Andrew Nelson
(Coordinator) University of Leeds School of Chemistry,
Leeds, LS2 9JT, UK a.l.nelson@leeds.ac.uk
First Name Last Name Affiliation Address e-mail
Vladimir Ogurtsov Tyndall National Institute Dyke Parade, Cork,
Ireland vladimir.ogurtsov@tyndall.ie Victor Puntes Fundacio Institut Catala De Nanociencia
I Nanotecnologia Inorganic Nanoparticles Group, Campus De La Uab Edifici Q ICN2, Bellaterra, Barcelona 08193, Spain
victor.puntes@icn.cat
Mario Rothbauer Technische Universitaet Wien Karlsplatz 13, 1040
Vienna, Austria mario.rothbauer@tuwien.ac.at Elise Rundén Pran Norsk Institutt For Luftforskning Health Effects
Laboratory, Environmental Chemistry Dept, Instituttveien 18, Kjeller 2027, Norway
elise.runden.pran@nilu.no
Peter Simon Slovenska Technicka Univerzita V
Bratislave Dept Physical Chemistry,
Faculty of Chemical and Food Technology, Vazovova 5, Bratislava 81243, Slovakia
peter.simon@stuba.sk
Karen Steenson (Deputy
Coordinator) University of Leeds Engineering Research &
Innovation Service, Faculty of Engineering, Leeds LS2 9JT, UK
k.a.steenson@leeds.ac.uk
Bob Wadey Blueprint Product Design Ltd Main Office, 38 Market
Street, Keighley BD21 5AD, UK
bobw@blueprintproductdesign.com
9 Copyright
© 2017, The University of Leeds, Leeds LS2 9JT, UK, as Coordinator on behalf of the HISENTS Consortium.
HISENTS is a Research & Innovation Action, Collaborative Project, under the European Commission's Horizon 2020 Programme.
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