Grant Agreement: 685817 - H2020-NMP-2015-two-stage Website: http://www.hisents.eu Coordinator: Professor Andrew Nelson, School of Chemistry, University of Leeds, UK Deputy Coordinator: Dr Karen Steenson, Faculty of Engineering, University of Leeds, UK
Table 1 Consortium List.
No. Beneficiary name Short name Country
1 University of Leeds (Coordinator) UNIVLEEDS UK
2 Fraunhofer-Gesellschaft zur Foerderung der Angewandten Forschung e. V. - Institut fuer Biomedizinische Technik
Fraunhofer-IBMT
DE
3 Tel-Aviv University TAU IL
4 Blueprint Product Design Ltd BPDES UK
5 Slovak University of Technology in Bratislava STUBA SK
6 Technische Universitaet Wien TUW AU
7 Ustav Experimentalnej Onkologie Biomedicinska Centrum SAV UEOSAV SK
8 Fundacio Institut Catalá de Nanociencia I Nanotecnologia ICN ES
9 Universitaet des Saarlandes USAAR DE
10 Tyndall National Institute – University College Cork TNI-UCC IRL
11 Norsk Institut For Luftforskning (Norwegian Institute for Air Pollution Research) NILU NO
Contents
1 Summary ... 52 2 Background ... 53 3 Scientific and technological challenges ... 53 4
Objectives ... 54 5
Organisation ... 54
6 Scientific achievements 2016-2017. ... 55 7 Expected Impact ... 56 8 Directory ... 56 9 Copyright ... 58
1 Summary
Project Duration: 36 months (April 2016-March 2019) Project Funding: €6,332,825.00
The HISENTS vision is to address the problem of the dearth of high-quality tools for nanosafety assessment by introducing an innovative multimodular high throughput screening (HTP) platform including a set of individual modules each representing a critical physiological function connected and integrated in a hierarchical vectorial manner by a microfluidic network. The increase of the capacity to perform nanosafety assessment will be realised by innovative instrumentation developments for HTP and high content analysis (HCA) approaches.
Toxicogenomics on chip is also one embedded objective. Our interdisciplinary approach focuses on tools to maximise the
read-across and to assess applicable endpoints for advanced risk assessment of nanomaterials (NM). The main goal is thus to establish individual chip-based microfluidic tools as devices for (nano)toxicity screening which can be combined as an on-line HTP platform. Seven different chip-based sensor elements will be developed and hierarchically combined via a flow system to characterise toxicity pathways of NM. The HISENTS platform allows the grouping and identifying of NM. Parallel to the screening, the pathway and interaction of NM in biological organisms will be simulated using the physiologically based pharmacokinetic (PBPK) model. Using the different sensor modules from the molecular to cell to organ level, HISENTS can input quantitative parameters into the PBPK model resulting in an effective pathway analysis for NM and other critical
compounds. The developed platform is crucial for realistic nano-safety assessment and will also find extensive application in pharmaceutical screening due to the flexible modifications of the HTP platform. The specific objective is the development of a multimodular HTP platform as new a screening tool for enhancing the efficiency of hazard profiling. Currently, no such flexible, easy-to-use screening platform with flexibly combinable chip-based sensors is available on the market.
2 Background
State-of-art toxicology has been an experimental subject whereby the effects of toxicants on living organisms were assessed through in vivo experiments leading to dose-response curves which delivered parameters of median effective concentration (EC50) and/or median lethal concentration (LD50) which are effective and/or lethal respectively to 50 % of a population of animals. These parameters are the standard output of toxicologists. Parallel to the experimental routines, a theoretical predictive enterprise of organism toxicity has been applied for a long time. This is very popular since it avoids the expense and time associated with animal testing and allows for the screening out of putative toxicants. Originally these toxicity predictions were based on partition coefficient data and latterly comparisons with experiment where the hydrophobicity of the toxicant was correlated with its toxicity and/or bioavailability.
These so called structure-activity predictive models have become more refined relating the electronic structure of the molecules to their biological activity. Nonetheless toxicology has always been basically a phenomenon-based science and there has been a dearth of a mechanistic understanding in toxicant biological activity. In addition the in vitro testing has been slow and not robust enough. This together with the emergence of many newly synthesised NM with uncertain biological activity has led to a drive for more rapid toxicity testing using on-line methodology and high throughput testing.
Commonly these tests involve in vitro models but of increasing sophistication than earlier. Procedures are being developed employing artificial tissue on chip-based systems embedded in microfluidic platforms but to date there has been no attempt to integrate these into a global assay system.
This project therefore attempts to advance TWO arms of toxicity testing. The first is to develop these toxicity tests at successive levels of biological organisation and miniaturise them onto a fabricated chip-based platform embedded in a microfluidic network and the second is to integrate them into a global network so a novel systems biological and pathway analysis PBPK type approach can be taken in assessing the biological activity of the NM. The main goal of HISENTS is to establish a comprehensive multimodular screening platform for manufactured NM. An innovative miniaturised HTS tool will be developed with the capability of reliably analysing nano-bio-interactions within a platform of increasing complexity of biological organisation. The smart screening platform will be developed side by side with a PBPK model for predicting the ADME of NMs.
3 Scientific and technological challenges
The project HISENTS focuses on the development and application of a novel technology for nanosafety assessment.
The novelty of this technology is that it allows the development of a multimodular HTS platform where each module represents one particular physiological target/barrier and each target/barrier is monitored separately (see Figure 1). Any kind of monitoring method is possible. All devices will be integrated into one screening platform through innovative instrumentation developments. The design of the multimodular platform will follow the AOP model which accounts for the critical pathway NMs in an organism. At the same time we will simulate the pathway and interaction with targets of a number of NMs in a model biological organism.
Figure 1: The HISENTS experimental/modelling platform.
The modelling procedure we will use is the PBPK model which employs ordinary differential equations to describe transport of compounds/materials from compartment to compartment. A reaction-diffusion approach as well as a MM and QC simulation to describe interaction with targets will be applied to develop the model which qualitatively at first deals with all modules in our proposed sensing platform. The modules will be developed in a hierarchy of organisation. Thus the first module will be the biomembrane which is the primary environment/organism interface, then the lung and intestine which are the routes of intake of NMs, then cells, genetic material and the liver as a site of metabolism of toxicants. Accordingly as a result of the experiments with the different modules, quantitative parameters will be inputted into the model and an effective pathway analysis for the NM as well as for their interaction with critical targets will be extracted. At the same time the simulation development will aid the design of the screening modules. During the project all of the modules will be integrated together according to the PBPK model to realise the concept of a multimodular screening system. The stress throughout the project is to miniaturise modules so that all screening will be chip-based and integrated within a microfluidic network. The project aim is to show by PoC that the multimodular design works which will act as a guiding light or
goal throughout the project. The screening program will be carried out systematically. Initially the screens will be tested and calibrated with the water soluble toxicant tricyclic phenothiazine, chlorpromazine. Once a consensus result has been obtained throughout the consortium, the devices will be tested with related tricyclic pharmaceuticals which have very well characterised ranked biomembrane activity and toxicology to achieve PoC for the toxicity sensing technologies.
Subsequently standard and then novel NMs will be screened.
4 Objectives
The objectives as described below will be followed, and implemented as shown in Figure 2:
Figure 2: Overall strategy of the project HISENTS. Horizontal and vertical arrows indicate activity alignment and programme direction respectively.
Objectives:
1) Synthesise and characterise NM for use as standard test analytes for demonstrating individual device and platform performance.
2) Design and test effective screening devices, each representing a particular physiological function which can be incorporated in a multimodular platform as novel, relevant but realistic devices for sensing nanotoxicity. Individual chip-based tools will be set up as individual devices for (nano)toxicity screening and can be combined as an on-line High Throughput Screening (HTS) and/or High Content Analysis (HCA), as appropriate, multimodular platform. Nine different chip-based sensing modules will be developed each carrying a separate interrogable function/process associated with a biochemical/physiological function. All modules are integrated within a hierarchical flow system to characterise toxicity pathways for the individual toxicants and materials.
3) Configure robust electrochemical and optical techniques to interrogate the individual devices. Develop and innovate instrumentation for interfacing to sensor chips.
4) Incorporate and holistically integrate the screening devices into miniaturised and effective flow systems for HTS of NM along a directional pathway.
5) Develop smart automated signal processing data recognition techniques for qualitative and quantitative analysis of results.
6) Carry out comprehensive performance evaluation of platform initially with respect to each individual device. Calibration of results with corresponding in vivo data using standard invertebrate and mammalian indicator organisms.
7) Employ the concept of adverse outcome pathway (AOP) for deepening knowledge about the progression of toxicity events across scales of biological organisation which lead to adverse outcome (AO) relevant for risk assessment by performing an independent systems biological simulation of mechanistic processes derived from the PBPK model. This will involve the transport and bioactivity of individual NM, mechanistic pathway analysis and, correlation with experimental results.
Figure 3: Detailed WP inter-relationship of HISENTS.
5 Organisation
The scientific (RTD) activities are conducted within six workpackages (WP1-6), with one other work package being specifically concerned with dissemination and exploitation (WP7). Each workpackage is managed by a workpackage manager who is responsible for the timely delivery of deliverables, to budget, to the Coordinator who in turn represents the Consortium to the Commission. WP8 concerns project management, which cuts across all work packages: WP1:
NM synthesis and characterisation (Victor Puntes, ICN); WP2:
HTS smart instrumentation and integration (Vladimir Ogourtsov, TNI-UCC); WP3: HCA toxicogenomics (Eckart Meese, USAAR);
WP4: in vitro sensing modules (Peter Ertl, TUW); WP5:
Modelling mechanisms, pathways and effects (Peter Simon, STUBA); WP6: Calibration, standardisation, performance and validation (Andrew Nelson, UNIVLEEDS); WP7: Dissemination and exploitation (Mick Karol, BPDES); WP8: Management (Andrew Nelson, Coordinator/Scientific Coordinator, UNIVLEEDS). Figure 3 above shows how these WPs are inter-related.
Figure 4: Adverse Outcome Pathway represents the scientific organisation of HISENTS.