Final Report Summary - FUTURENANONEEDS (Framework to respond to regulatory needs of future nanomaterials and markets)
Executive Summary:
FutureNanoNeeds was designed to develop a novel framework to enable the naming, classification, hazard, and environmental impact assessment of the next generation nanomaterials (NM’s) prior to their widespread industrial use. The goal was to develop a suite of integrated tools and concepts which will form the basis of a “value chain” regulatory process which allows each new nanomaterial to be assessed for different applications on the basis of available data and the specific exposure and life cycle concerns for that application. The project has succeeded in doing so.
Roadmaps for future nanomaterial value chains were developed by promoting multi-stakeholder dialogues between researchers from industry, SME’s and other advisors. Eight value chains (VCs) were identified and prioritised by a Value Chain Advisory Committee (VCAC) and subjected to a detailed analysis to determine value chains with a high social and economic impact in the medium to long term, and with a focus on nanomaterials which have a significant research interest and/or synthesis and manufacturing capacities. Policy recommendations for future European cofounded innovation and research actions, or coordination and support actions, were released and validated through an online survey open to external stakeholders. A framework was developed to forecast the potential release of the selected VCs during different life cycle stages. For this purpose, an iterative tiered approach was developed by using mass flow analysis and Bayesian Belief Network methodologies to predict NM release, in each instance using case studies of different VCs. These methods considered the associated data gaps and uncertainty to account for data rich/poor scenarios. An important outcome was that these methods identified the End-of-Life (EoL) mechanical recycling processes as important hotspots in the future. Several measurement studies were conducted to identify and characterize next-generation nanomaterials in the workplace setting. To keep pace with the development of novel structures and functionalities, two new innovative, easy-to-calibrate and transportable devices were developed. In addition, a literature review was conducted on new concepts of exposure metrics and their applicability through the OECD (2015) measurement strategy. Based on real-world case studies, a practical manual on best practice for safe nanotechnology action plan was completed.
Responding to the VCAC, the consortium developed and produced a variety of homologous series, each one varying in a controlled manner to explore the impact of structural details on biology. It has involved the synthesis of nanocrystals with radically different shapes, geometries, compositions, and surface treatments. Special attention has been paid to the purity, monodispersity, and shape control (production of different geometries of the same chemical substance) to avoid confounding the biological outcomes with different active species in the same sample. Each particle type was made by at least two different laboratories. Nanocrystals sizes ranged from few atoms to hundreds of nanometres, and they presented with different dimensionalities (0D, 1D, 2D, 2D and intermediate dimensions as stars (2.5 D). Attention was paid to the control of synthetic environments and processes to obtain ‘reproducible’ particles, monodisperse in size and shape, free from chemical and biological (endotoxin) contamination. Remarkably, internal and small informal round robins and quality assurance checks were made to provide a complete audit of the processes underpinning quality and material supply. The success in these processes was considered a milestone of the program. Samples produced were analysed with state-of-the art characterization techniques. Beyond synthesis, partners carefully studied and analysed the evolution of nanocrystals during time and storage conditions, and in different working media. Nanoconstructs were distributed across the FutureNanoNeeds consortium for subsequent biological eco-tox evaluation. Significant effort has been spent in the proper labelling of nanoparticles as appropriate for biological studies.
The project carried out a series of experiments on different types of polymer nanoparticles (NPs) and studied their bioavailability and targeting. PCL3 NPs are deemed an interesting tool for the development of nanodrugs or to predict the fate of NPs in organs and cells. Studies were carried out on the dispersion states of NMs in biofluids and found that in general the materials were stable. The project also studied the effect of the protein corona on the stability of NMs. In situ and in-line monitoring of the fate of the particles has been studied and the results show NP stability in biofluids containing proteins only at certain concentrations. The physical size and shape of NMs was found to influence the orientation of protein adsorption. Finally, the degradation of NMs in a variety of environmental fluids has been studied.
Intentional (drugs, foods, cosmetics) and unintentional (pollution, workplaces) exposure to NMs has been enormously increased over the last decades. The perception of risk is controversial and the correlation between the main physico-chemical parameters characterizing NMs and their effect on cells and organs is still far from being elucidated. To answer to this pressing need, an innovative nanosafety platform was built within the project. Three main biological matrices with an increasing level of complexity (biological fluids, cells, healthy rodents) were considered to achieve reliable and transferable results for human health. Briefly, we confirmed that independent to the material, other parameters such as size, shape, and surface, can strongly influence the biological response. Overall, the results from this project provide useful suggestions to drive the production of safer industrial NMs and also to predict the potential impact of nano-pollutants. The approach could help develop new way of considering and critically evaluating the risks emerging from nanotechnology, in order to create a more responsible and easily transferable strategy of validation.
The project, in its investigation of environmental impact, found that the nanoparticle dispersion state in the aqueous phase was related to nanoparticle toxicity. Lead ion release was found to be the cause of toxicity of perovskite nanomaterials in adult zebrafish through a dietary exposure. Organ uptake and internal distribution should be monitored more closely to provide more in depth information of the toxicity of particles. In soil, the low mobility of perovskite nanomaterials may lead to an accumulation in the upper soil layers. The consortium found that toxicity study exposure must be expressed on the basis of time weighted average particle concentrations. This must explicitly take particle dissolution as well as sedimentation into account. Particle shape and size were found to be the key properties affecting the toxicity of nanoparticles. The coating of nanoparticles may determine toxicity, while the toxicity of metal nanoparticles may be via a ‘trojan horse effect’ whereby dissolution is achieved following nanoparticle-organism interaction or nanoparticle internalisation.
Durable links to other key EU projects, stakeholders, and regulatory bodies, were created during the course of FutureNanoNeeds, ensuring the communication and sharing of new understanding and findings beyond the project end. The project had significant success in the organization of several workshops to disseminate key results and open discussions on topics such as the standardization, classification, and categorization for present and future nanomaterials. In particular, the Conference on the Categorization of Next Generation Nanomaterials in Brussels, was successfully organized in 2017, preceded by several satellite meetings and the involvement of worldwide experts.
Project Context and Objectives:
Work Package 2 Scientific Coordination
The main objectives of Work Package 2 were the coordination and leadership of the overall program and the in-depth management and administration of workflows, and the administration of materials flow and QA. Throughout the project, regular communication between the work packages was facilitated through teleconferences, face to face meetings, emails, phone calls, and the FNN website (https://www.futurenanoneeds.eu). This work package was responsible for the initial round-robin process, compiling and updating SOP’s, and compiling the annual, periodic, and final reports, gathering and submitting deliverables, organising reviews etc.
In terms of materials flow, teleconferences and discussions were held between relevant partners to make sure the flow of materials in the project was effective, including the implementation of a spreadsheet to help predict Nanoparticle requirements (type and quantity) in the medium term. While there was concern regarding the quantities of nanoparticles available for some experiments, through effective communication and cooperation between partners, this was resolved in the final half of the project allowing the research to progress to a successful conclusion.
Work Package 3: Roadmapping of value chain materials platforms, and consequently their exposure scenarios
Work in WP3 contributes to the development of a novel framework to enable naming, classification, hazard and environmental impact assessment of the next generation nanomaterials prior to their widespread industrial use. To achieve this, new and existing concepts have to be integrated to produce a robust, versatile and an adaptable framework and methods to forecast release of next generation NMs and to ensure its safe use. Different life cycle stages and the characterization of NM release in each instance are important issues to consider for this purpose. The scope within WP3 includes occupational and environmental exposures as a starting point, although the focus for measurement studies and the best practices action plan relates to occupational exposure.
In terms of objectives, the central objective that relates to WP3 and all other WPs within the FutureNanoNeeds projects was to develop roadmaps for future nanomaterial value chains by promoting multi-stakeholder dialogues between
researchers from industry, SME’s, and other advisors. A Value Chain Advisory Committee (VCAC) was appointed to maintain, develop and update those decisions made within the VCAC, and to translate them into action, and therefore encourages an intensive engagement involving many specialists from different stakeholders, advisors, extending from industry to innovators and venture capitalists and others. The main objective was to prioritise value chains for FutureNanoNeeds within the Value Chain Advisory Committee (VCAC), and to analyse them in detail for range of nanomaterials likely to be used, thereby proposing homologous series of representative nanomaterials that exemplify the value chain chosen.
Another crucial objective relevant for the entire work package was to develop a framework that allows for the forecasting of potential release during production and other stages of these chosen next-generation nanomaterials/product value chain. For this purpose Bayesian networks are proposed to model complex systems with many related variables, while considering associated uncertainties and to model complex systems with many related variables. It was of importance to keep pace with development of novel structures and functionalities by exploring concepts of new methods
and techniques to monitor release or exposure. Also, the identification of potential for (occupational) exposure to the next generation of nanomaterials and development of (innovative) methods/tools to identify and control possible exposure in the workplace and in ambient air along the value chain should be considered. And lastly, the development of a manual on best practice for safe nanotechnology action plan should ensure that control measures are implemented and tested to ensure optimal performance.
Work Package 4: Synthesis and exhaustive characterization of materials of the future
Responding to the Value Chain Advisory Committee (VCAC) the main activity of WP4 has been the development and production of a variety of nanoparticle homologous series corresponding to value chains (see table). Taken as a basic starting point the available nanomaterials structures routinely made in the laboratories of the Partners in WP4 and considering the inputs from VCAC, series of NCs were developed. In detail, high quality homologous series of NCs have been prepared, each series varying selected parameters in a controlled manner to explore the impact of structural detail on biological and other outcomes. It has involved the synthesis of different series with radically different shapes, geometries, compositions, and surface treatments. A full list is available in the attached report.
Within WP4, Task 4.1 focused on the synthesis of novel geometries and shapes of nanomaterials, particularly those structures plus variants around them that allowed the study of biological and environmental interactions with a wide variety of different geometries. A variety of techniques were applied allowing the kinetic control of specific crystal planes to make different geometries of the same chemical substance. Task 4.2 was intended to explore variations going far beyond those envisaged in Task 4.1. In this regard, hybrid nanoparticles composed of a combination of different materials in core-shell, hollow or heteromer-like configurations were produced. These new hybrids represented a powerful strategy for modifying nanoparticle properties, combined the properties of the individual components, or even exhibit novel and unique properties that originate from collective interactions between constituents. Task 4.3 evolved the nanomaterials formed in Task 4.1 by (post-) modifying their surface, particularly with different charge and hydrophilicity/hydrophobicity. Samples were characterized both dry (where appropriate) and dispersed in various simple media within Task 4.4. The most basic characterization of particles involved (average) size/dimensions, ‘charge’ (averaged mobility) and surface area. In some cases new instrumental developments (or evolution of existing ones) were required to meaningfully characterize these novel shapes and geometries.
During the lifetime of the project special attention has been paid to the control of synthetic environments and processes to obtain ‘reproducible’ particles, monodisperse in size and shape control, free from chemical and biological (endotoxin) contamination to avoid confounding the biological outcomes with different shapes in the same sample. Each particle type was made by at least two different laboratories, in order to provide a comparative study of the synthetic processes and expecting to provide a significant achievement in the refinement of synthetic methodology of shape control synthesis. Remarkably, internal and small informal round robins and quality assurance checks were made throughout the lifetime of WP4 activity aiming to provide a complete audit of the processes underpinning quality and materials supply. The success in these process was considered a milestone of the program. Samples produced were analysed with state-of-the art characterization using established methods, and introduction of novel approaches appropriate for new material.
Beyond synthesis, WP4 partners carefully studied and analysed the evolution of the physicochemical properties along the time and storage conditions and/or in different media.
NPs were distributed across the FutureNanoNeeds consortium for subsequent biological (WP5, WP6) and eco-tox (WP7) evaluation. Full list of samples prepared and delivered can be found in D4.5 D4.6 D4.7 D4.8 and D4.9. Significant effort has been spent in the proper labelling of nanoparticles as appropriate for biological studies.
Work Package 5: Biological and environmental interactions of future nanomaterials
In this workpackage we successfully created high quality, characterized (and realistic) dispersions of the nanoparticles decided by the VCAC in different biological media, as appropriate for WP6, We also investigated and created examples of high quality, characterized (and realistic) dispersions of nanomaterials in different environmental media. To make sure these scenarios were realistic we applied a balanced portfolio of strategies (high content screening, uncovering biological pathways and mechanisms, hypothesis driven detailed study, including in vivo). We also advanced the understanding of the state of organization of non-standard-geometry (shape, topology) nanoparticles in biological media both in relation to dispersion, and in relation to the biomolecules adsorbed to their surfaces (‘ biomolecular corona’). This was done by altering the kinetics of internalization and sub-cellular localization. We also modified the activation of the main systems of metabolism and degradation. This work package had multiple objectives (early alert to hazard, understanding of connection between structure and action, future design principles) which we feel we have addressed fully.
Work Package 6: Biological identity, processes and human health hazards of future nanomaterials
The main goal of this WP was to understand how physico-chemical parameters of novel NMs (e.g. shape, size, curvature) could influence their interaction with biological targets at different levels of complexity (biological fluids, cells, animals). Specific hallmarks, such as the protein corona formation, the cell penetration, the kinetics and accumulation in cells and organs, and, finally, the potential triggering of detrimental effects, have been carefully evaluated along the 6 tasks of the WP. We were mainly interested to understand if and how parameters such as shape, size, curvature, surface might influence the interaction with biological fluids. This is relevant for both nanotoxicology and nanomedicine. The interaction with proteins, sugars, lipids and other components may indeed strongly influence the behaviours of NMs once penetrated in the host tissues. To this end a deep analysis with biochemical, bio-molecular and morphological assays was carried out before moving to single cell investigations.
The in vitro studies were the second, and most extensive part of this WP. They concern at least four different tasks, devoted to the determination of the influence of specific physico-chemical features of NMs on different cell types. The comprehension of the potential alterations of morpho-functional parameters (not exclusively associated with death or high toxicity) after exogenous compounds exposure is always important. However, it becomes even more important in nanotoxicological studies, due to the peculiar ability of nanoparticles (NPs) to be efficiently internalized inside the cells through an endocytotic mechanism commonly defined as “Trojan horse effect”. The first part was therefore aimed at measuring the NP uptake and localization inside the cells depending on the dose, the material, the shape, the size, the curvature. Afterwards, a deep investigation of the “fate” of internalized NPs was carried out. To this end we planned a series of experiments matching different approaches, such as EM, fluorescence imaging, ICPMS and cytoViva, to investigate the presence and dynamics of processes of intracellular dissolution, degradation kinetics and exocytosis of different NMs in different cell types. The rate of penetration into the cell and the consequent accumulation, cannot be automatically translated to a toxic effect. In fact, there may be materials that, although penetrate and accumulate inside the cell, are easily isolated from cytoplasmic structures or are chemically inert. The studies of uptake and accumulation, are therefore necessary but not sufficient to define the potential hazard. The following part of the study was therefore devoted to the effect of NPs after internalization. However, we did not exclusively investigated the survival, but also looked for upstream and subtler alterations, such as immunomodulation, inflammation, oxidative stress and other sub-lethal processes. Finally, for a restricted type of materials (gold and TiO2) we evaluated the effect of size and shape on organ biodistribution, accumulation, clearance and potential detrimental effects (inflammation, immune response, histopathology) after single administration in healthy immunocompetent mice. This last part is crucial to understand if the results coming from in vitro studies can be somehow transferred to more complex systems and to complete the development of this integrated platform devoted to nanotoxicology.
Work Package 7: Exposure and environmental interactions of future nanomaterials
Project FutureNanoNeeds (FNN) studied the fate and effects of novel nanomaterials (NMs) along selected value chains in close collaboration with key stakeholders. Within FNN Workpackage 7 (WP7) addressed “Exposure and environmental interactions of future nanomaterials” and its main aim was to study environmental fate and effects of the so-called new generation NMs. This aim was addressed via these key objectives:
1. To translate the general exposure scenarios (WP3) considered most important along selected value chains and life cycle of future NMs into an understanding of the in situ exposure conditions;
2. To investigate the nature of surface adsorbed molecules derived from product formulation modifications to the NMs (e.g. surface modification derived from the embedding matrix of the product) for exemplary value chains as decided by the VCAC (WP4);
3. To characterize the nature and structure of adsorbed molecules, and other surface modifications, derived from the natural aquatic environment (freshwaters), from solids as well as from wastes or effluents (characterisation of for instance the specific interactions with Dissolved Organic Matter, but also the specific interactions with selected contaminants present in the natural medium (like metals, PAHs, etc.);
4. To develop suitable exposure models for environmentally relevant species, and sentinel species, and to investigate the interactions of novel NMs with them.
The above objectives were fulfilled in this WP by research structured around the following tasks:
• Task 7.1 Environmental binding and ecoidentity of future NMs;
• Task 7.2 Development and application of environmentally relevant organism exposure protocols;
• Task 7.3 Material transformation (including degradation) in the environment;
• Task 7.4 Interaction with environmentally relevant species, including ‘sentinel’ species.
Research carried out within WP7 was undertaken by 7 partners, namely, NUID-UCD (P1); IUTA (P5); HWU (P6); LIST (P7); UoB (P8); MNI (P11); RIVM (P12) TUL (P15), with different and complementary expertise and techniques. The practical approach adopted followed the conceptual model presented in Figure 1 of the attached report.
Results from the work undertaken in WP7 was comprehensively reported in the following deliverables:
D7.1 Identification of the composition of the ‘eco-corona’ formed in specific environmental media on next-generation NMs;
D7.2 Report on the propensity of next-generation NMs to provide a ‘Trojan horse’ effect of environmental pollutants;
D7.3 Report on proposed standardized exposure conditions and protocols for environmental species, building upon the general exposure scenarios identified in WP3;
D7.4 Identification of possible ‘hot-spots’, where material transformations are most likely to occur, along several value chain platforms;
D7.5 Material transformations along several value chains and their consequences identified;
D7.6 Report on methods and protocols for measuring NMs interactions with environmental compartments;
D7.7 Preliminary results on NMs interactions with environmental compartments for selected value chain platforms;
D7.8 Report highlighting any novel toxicity or toxicity mechanisms derived from standardized exposure conditions.
Work Package 8: Key elements of dissemination, potential changes to risk assessment approach for future nanomaterials
The FutureNanoNeeds project comes into existence at a time when research, industry and society (through, among others, regulatory bodies and consumers) are beginning to come in contact with what we here have termed next generation nanomaterials. More complex, versatile, engineered nanomaterials (ENMs) are characterized by parameters beyond their size and core composition, such as shape, surface chemistry, biological and environmental identity. These materials hold great promise for innovation, but they also present unique challenges for their safe use and commercialization of the products that incorporate them. The FutureNanoNeeds project aims to address these emerging issues by, among others, creating the framework for a fruitful conversation among all of the stakeholders involved in the research, commercialization, use and regulation of next generation nanomaterials. WP8 focuses on the dissemination aspects related to the many facets of this conversation.
Tasks 8.1 through 8.5 in WP8 were carried out by a unique mixture of partners. They brought into discussion nanosafety and the development of novel nanomaterials in a very broad and inclusive context, aiming for i) the development of classifications systems for the next generations of engineered nanomaterials (Task 8.1) ii) assessing the impacts of uncertainties surrounding nanosafety and facilitating innovation and commercialisation on the basis of safety assessment, with the collaborative efforts of researchers, regulators and industry (Tasks 8.2 8.3 and 8.4) and iii) the development of cost-cutting tools for SMEs in the context of safety in innovation (Task 8.5).
Work Package 9: Liaison with NMP projects, national and other international projects
Nanotechnology and engineered nanomaterials hold the potential to make qualitative improvements in rapidly developing markets such as energy harvesting and storage, construction, advanced materials for electronics and biomedical devices. FutureNanoNeeds project has produced a totally new workflow for the classification of nanomaterials and complex nanomaterials of the future by reading across physical-chemical properties, biological hazard and environmental impact assessment. This complex framework for the next generation nanomaterials will allow to better regulate and therefore facilitate the introduction of engineered nanomaterials for a widespread industrial use.
Two main goals, strictly connected each other have emerged along the project:
• To ensure a smooth benchtop-to-market transition for nanomaterials in future industries by predicting the nature of future nanomaterials and by providing reliable tools to assess potential health and environmental hazards ahead of time.
• To promote a standardized system of classification and regulation across EU and worldwide for nanomaterials.
The data generated along the process will form the basis of a “value chain” regulatory process. The nanomaterials will be assessed for different applications (and for each step of the production value chain) on the basis of FNN findings and the specific exposure scenarios and life cycle concerns for that application. In this context it is of paramount importance to manage the data and the findings emerged from the project, making them accessible and available for the scientific community, the stakeholders and regulators, beyond FNN. In this sense, the role of WP9 has been to collect, summarize and disseminate the main results obtained for the other WPs as well to create the basis for a broader discussion involving key players in the EU and international regulation in the world stage. The main urgency now is to ensure a future to all the project findings and discussion on promoting strategies to reach consensus. This can be done beyond FNN, with the creation of databases, bridging together FNN and other relevant EU projects and the EU regulation organs.
Project Results:
Work Package 3: Roadmapping of value chain materials platforms, and consequently their exposure scenarios
NfA (RINA-C, supported by MBN Nanomaterialia and Prodintec) ensured the integration of the outcomes of a VCAC web meeting on FNN policy recommendations, the link between FNN deliverables and selected value chains, a revision of selected patent and market analyses and finally the integration of the results of an online survey open to external stakeholders aimed at validating key findings of the deliverable. A list of 8 Value Chains centred on key nanomaterials has been identified and analysed though a vigorous discussion by all consortium partners (especially the material researchers in WP4) and by selected external experts. General descriptions of nanomaterials in terms of key properties and of the target applications and final products were provided. Advantages with respect to state-of-the art materials or competing nanomaterials were summarized. Current Technology Readiness Level (TRL) of the value chain was also estimated. Market analysis was described in detail for each value chain, estimating market size, trends, geographical distribution, and segmentation by technology/material and by application and top companies. Patent and literature analyses were provided, aimed to enhance the development phase of the value chains and the innovation potential. A summary of the “hot spots” was also provided for each value chain: these are likely points of exposure to nanomaterials during the life cycle. Finally, policy recommendations for future European cofounded innovation and research actions or coordination and support actions were released and validated through an online survey, open to external stakeholders. The survey showed that for the majority of value chains, the most critical VC step was considered the recycling and disposal step, followed by nanomaterial synthesis.
To provide input for the roadmapping activities, TUL was involved with UCD/ /CRF in a conjoint project: the biological and environmental impact of reduced graphene oxide (rGO) nanomaterials (gemmate s.r.l.) intended to be used as motorcar lubricant. CRF was active in the definition and updating of value chains for relevant nanomaterials and was responsible for keeping up to date the nano-lubricant value chain. That activity was based on the two types of reduced graphene oxide procured by CRF and supplied by Gemmate Technologies. The two materials were thermally and chemically reduced graphene oxides (T-rGO and C-rGO). These results demonstrate the potential of the applications of r-GO as friction modifier in industrial lubricants and enforce the industrial interest in the nano-lubricant value chain. For this reason UCD, TUL and CRF studied the biological and environmental impact r-GO nanomaterials intended to be used as an automotive lubricant. IUTA had been involved into the VCAC actions, for example initiating two major VCs including Li-Ion Battery Technology and the thermoelectric value chain. IUTA played a major role to develop a powerpoint template that provides a brief overview of the value chains and the corresponding results. NANOGAP has provided useful information for the inclusion of one of our products (AgNW) as value chain material, including market and production process information.
A two-tiered iterative approach is proposed to prioritize the focal point and to forecast the potential release and emissions at different levels of detail. The framework assesses material flows along different life cycle stages. The developed Tier 1 approach (an adapted EUSES-based methodology) was expanded to include a more in-depth assessment (Tier 2). The Tier 2 includes two levels to assess release of nanomaterials. The level 1 assessment applies a Material Flow Analysis (MFA) intended for a selected ‘hotspot’ Life Cycle stage. The methodology for the analysis is based on previous work conducted by IUTA within the FP7 Project BUONAPRT-E and was extended to close the mass balance during the possibly different end-of-life steps. Three case studies were worked out to illustrate its application, i.e. (i) Silicon nanoparticles for battery applications (VC2), (ii) Silicon-germanium powder to be used in thermoelectric generators (VC3) and (iii) MoS2 fullerenes used as lubricant additive for automotive applications (VC 4). IUTA received input from various partners incl. CEA and TNO for these cases. The developed simple spreadsheet software allows an easy identification of possible release hotspots and the final compartments in which the nanomaterials end up after the life cycle is closed. It was revealed that currently a lot of work has been conducted and data complied with regard to possible release and exposure during the synthesis, compounding and assembly of nano-enhanced products (occupational settings) but that tremendous knowledge gaps exists for release during the use and especially also for the recycling phase.
The overall mass flow analysis showed that approximately 20% of the nanoparticles will end up in environmental compartments with the majority going into air and soil, which is mainly due to the use phase within the motor oil. About 15% end up in landfills and another approximately 65% are being fed into recycling processes. Overall, the detailed mass flow analyses revealed, that during the production/assembly phase a major release of the nanomaterials is generally unlikely. Uncontrolled release occurs during the use phase and for the end-of-life phase there are still many unknowns with regard to recycling pathways. Also, possible transformations of the materials are not covered by this kind of analysis.
The level 2 involves the application of a Bayesian Belief Network (BBN) to forecast release of NMs at process level. The process “shredding” was selected because mechanical recycling during End-of-Life was found to be an important ‘hotspot’ (from Tier 1) for next generation NMs. The following value chains were selected for this development: (i) perovskites in solar panels, (ii) graphene/Si in lithium-ion batteries and (iii) quantum dots in display technologies. TNO lead the development of this model and invited a BBN expert from TNO to facilitate the development process. IUTA, CEA and external experts actively participated in the development of the Bayesian Belief Network during two intensive (two day) F2F workshops and five (half-day) follow-up teleconferences over a period of 9 months. The model is presented in the form of a graphical representation of the BBN and is underpinned with probability tables based on the expert elicitation procedure obtained during the workshops and follow-up meetings. Different influencing factors on release with regard to the specific activity were identified, showing the general applicability and versatility of the approach also with regard to future nanomaterials not yet covered. The model was tested with shredding data from CEA (i.e. shredding electrodes of batteries).
To define ‘hotspots’ or focal points, the developed Tier 1 approach (an adapted EUSES-based methodology) was applied. The main objective of these assessments are to identify hotspots, that flag potential specific areas of concern in the nanomaterial life cycle and may relate to substantial release, emission or exposure, or where unknown changes and alterations of the material are expected. The following NM Value Chains were identified by the Value Chain Advisory Committee (VCAC) as reported in Deliverable 3.3 and subsequently subjected to a Tier 1 assessment and the identification of focal points (as presented in Deliverable 3.7): (i) VC Energy harvesting: focal points Perovskites, (ii) VC Energy harvesting: focal points Boron doped silicon, (iii) VC Energy storage: focal points Silicon Nanowires, (iv) VC Lubricants: focal points fullerene and graphene flakes, (v) VC Thermoelectrics: focal points of nanocomposites in thermoelectric generators and (vi) VC Display screens: focal points Quantum Dots (CdSe). For each VC, a flow chart was developed with mass flows, incl. information sources and assumptions are indicated. Based on the outcome this flow and the criteria, the focal points of concern were identified and summarized. The tier 1 assessment indicates semi-quantitatively the emission to the environmental compartments, which could be useful for benchmarking worst-case concentration assumptions for toxicological studies. TNO conducted the material flow analysis for the VCs, VC owners, i.e. EPFL, PUM, IUTA, CFR and task partners, i.e. IUTA and CEA, reviewed the assessments. Various partners were involved, incl. TUL who provided information on the biological and environmental impact of reduced graphene oxide (rGO) nanomaterials intended to be used as motorcar lubricant (FNN value chain 4 Transportation/ Industry). The resulting toxicological assessment is intended to help the translation to the market for the novel nano-lubricant, providing specific information for the risk assessment and regulation of a novel nanomaterial.
For methods and devices to detect, characterise and quantify exposure to next-generation nanomaterials, a review was conducted of new methods. IUTA provided valuable input for the overall Deliverable. Of all the detection techniques available, a common drawback identified is that most of the approaches are particle-dependent and therefore a versatile tool would require a multi-sensor approach to be effective and efficient. TUL and TNO worked on a literature review on new exposure metrics and involved in state of the art on “new concepts of exposure metrics” for next generation nanomaterials. It is currently not possible to obtain the size distribution and the chemical composition of the released particles into the air using affordable portable devices. Personal monitors provide the lung-deposited surface area (LDSA) of the nanoparticles in few seconds while personal samplers provide the quantitative chemical composition of the collected particles over a certain period of time using among other X-ray fluorescence spectroscopy. Measuring the chemical composition of the collected particles allows dealing with the nanoparticle background which is known to be variable in time and space. However, it is very difficult and time consuming to correlate size and chemical composition.concepts of exposure metrics are explored in this task through a literature review.
CEA and IUTA was involved in the selection of methods and devices to detect, quantify and characterize exposure to next-generation nanomaterials. IUTA also actively participated in the discussions on best practices to ensure the safety of these nanomaterials. To disseminate the results with regard to release and exposure several oral presentations at international conferences on a framework of release (based on work conducted within the FP7 Project MARINA and jointly extended by TNO, CEA and IUTA) had been given. CEA continued the development of its electrostatic precipitator which is an innovative device, easy-to-calibrate and transportable. It provides a size in real time and the chemical composition for several size ranges off-line in order to give a response for ENMs that could be easily differentiated from the atmospheric background or when there is several sources of nanoparticle that can lead to exposure. IUTA participated in the discussion on test methods and devices able to detect next generation nanomaterials. The work included the further development of the OECD tiered approach for exposure assessment in occupational settings in view of possibly unknown particle properties. TUL worked on a literature review on new exposure metrics methods devices to detect, characterise and quantify exposure to next-generation nanomaterials. Alternatives to current exposure metrics were explored and evaluated under the frame of the OECD general tiered approach for exposure assessment. The possible parameters to be used in the exposure assessment scheme were defined along with a list of available methods, tools and devices for their measurement. Moreover, innovative methods and devices that could overcome some of the current difficulties observed during field measurement were developed during the project. In particular, CEA developed and evaluated the concept of a novel device, easy-to-calibrate and transportable which could provide a size distribution in real time and the chemical composition for each size bin off-line. Several prototypes were designed, built and evaluated using both simulation tools (Finite Elements Analysis - COMSOL Multiphysics) and experiments performed in laboratory with controlled aerosols. The devices were improved iteratively and are currently reaching TRL 4. Two patent applications were filled in July 2015 and were extended in 2017. The innovation is to sort and collect charged particles in specific places of a circular substrate based on their electrical mobility. The concept developed through this task allowing to correlate size and chemical composition of airborne particles is promising since it would allow the discrimination of secondary sources of airborne particles in occupational settings.
CEA performed several field measurements campaigns along with Tier1, Tier 2 and Tier 3 evaluations were conducted during the project to generate new information on the operative conditions and levels of exposure for several case studies on next-generation nanomaterials:
•Production of nanoenabled electrodes for Li-ion batteries
•End-of-life of nanoenabled electrodes for Li-ion batteries
•Production of thermoelectric generators based on nanostructured silicides
•Additive manufacturing
Those visits and campaigns also allowed the identification of experimental challenges related to the measurement of emissions in occupational settings and contributed to confirm that research activity on the development of personal devices was required. This is particularly the case since those tools could be useful for Tier 2 evaluation in SMEs and small scale production lines.
CEA developed a manual on best practice for safe nanotechnology action plan. The best practices identified throughout the technical activities conducted during the project were regrouped in a manual along with generic guidelines for ensuring exposure-driven safety of the next generation nanomaterials being developed for industrial applications. It is also based on extensive literature review and considers the life cycle stage and hierarchy of control (source to worker). Best practices were identified using inputs from other WP3 outcomes (VC roadmapping, field study data, etc). It presents an approach on best practices for ensuring safety of the next generations of nanomaterials being developed for industrial applications. Those selected value chains are representative of the 3rd and 4th generation nanomaterials. The action plan will be beneficial to the scientific community, regulators and stakeholders outside of the project since there is a strong need to reach consensus on methods and devices to assess exposure in practice along the whole value chain (workplace, consumer and environment). This manual will also allow workplace hygienist, workers and executives to make appropriate decisions to tackle efficiently risks associated to manufactured nanomaterials.
The work was directed towards a safer and more responsible development of nanotechnologies. It allows workplace hygienist, workers and executives to make appropriate choices to efficiently address risks associated to manufactured nanomaterials. A user-friendly action plan table was developed that summarizes the best practices for each risk management controls and life cycle stages. CEA, IUTA, NANOGAP and TNO participated in discussions and review of the work.
Work Package 4: Synthesis and exhaustive characterization of materials of the future
The main activity of WP4 has been the synthesis of novel materials relevant in future value chains as agreed with the VCAC. The work has been structured in 4 different tasks. The main science and technological results are detailed below. At the end of the document a summary of nanomaterials structures made in the laboratories of the Partners in WP4 classified by specificity (size, dimension, composition, shape, and application) and partner production is provided.
Task 4.1 Synthesis of novel materials as analogues of those relevant (or likely to be) in future value chains
Within this task WP4 has synthesized nanoparticle classes as agreed with the VCAC. We focused on the synthesis of novel geometries and shapes of nanomaterials, particularly those structures plus variants around them that allowed the study of biological and environmental interactions with a wide variety of differential geometries. For the first time synthetic laboratories were asked to produce a relatively uniform distribution in shape to avoid confounding the biological outcomes with different shapes in the same sample. Within this task we achieved significant advance in the refinement of synthetic methodology of shape control synthesis. A variety of techniques were applied allowing the kinetic control of specific crystal planes to make different geometries of the same chemical substance.
Task 4.2 (VHIR, TCD, NUID-UCD) Combinatorial nanomaterial libraries and collections
This task was intended to explore variations going far beyond those envisaged in Task 4.1. Assembling several materials into a single nanostructure was the strategy to design and produce systems possessing diverse physical and chemical properties. In particular, WP4 partners succeeded in producing hybrid nanoparticles composed of a combination of different materials in core-shell or heteromer-like configurations. These new hybrids represented a powerful strategy for modifying nanoparticle properties, combined the properties of the individual components, or even exhibit novel and unique properties that originate from collective interactions between constituents.
Among the different hybrid structures, WP4 partners succeeded in producing magneto-plasmonic and metal-semiconductor nanoparticles with more complex function and shapes. Magneto-plasmonic particles have been proposed as bi-functional systems for a variety of applications in theranostics, in which the metal shell acts as a protecting agent for the magnetic core and as a highly functionalizable surface. Similarly, metal-semiconductor nanoparticles are ideal candidates for solar energy harvesting applications, in particular photocatalysis, in which light energy is stored into chemical bonds. Thus, the semiconductor part of the particle can be tailored for optimal light absorption while the combination with the metal component facilitates charge separation and the reactive metal surface with a large surface area can serve as the proper catalytic substrate for the intended reactions. This combination leads to novel electronic properties which may ultimately be expressed in biological or other interactions.
CBNI-UCD in WP4 mainly focus on the synthesis and advanced characterisation of gold nanoparticles with complex nanostructures for biological applications (see gold library in Figure 2). Despite the almost limitless possibility in the synthesis of “gold shapes”, most of the methodology reported in literature are not well reproducible or not well described often leading to low quality products. Aiming to understand the effect of different shapes in biology CBNI-UCD invested a great effort in order to carefully select and make reproducible SOPs and high-quality NPs. For this reason, round robins on different synthetic methods and advanced physico-chemical characterisation have been performed on several batches. To perform a deeper characterisation on shaped GNPs CBNI-UCD developed an advanced TEM analysis able to establish a kind of “shape distribution” (more discussed in D8.2) and (in collaboration with FILARETE, Nanoscale, 2017, 9, 2778–2784) SPES analysis was also adopted. However, these techniques concern mainly the study of the core structures in water suspension of the NPs but this is not enough toward biological applications. Several batches of shaped NPs with different synthetic surface chemistry were prepared to be tested for their stability in relevant biological media (as discussed in WP5). The new surface chemistry (biomolecular corona) of the NPs exhibiting good stability in biological fluid were investigated by mass spectrometry (collaboration with Prof. Gabriella Tedeschi UNIMI) and also by QCM mapping technique. Significant differences in the biomolecular corona were found in both composition and orientation.
Figure 2: Gold shape library synthesized by CBNI-UCD
We believe the biomolecular corona can represent the biological identity of the NPs in vivo influencing their cell trafficking, biodistribution and immunological effects (Nanoscale Horiz., 2, 187-198 (2017). For this reason, it is fundamental to exclude biocorona contaminations. It is now well known that endotoxins (as LPS) can adsorb on the surface of the NPs and can be recognised by specific cell receptor (for example in the liver) possibly modifying the original biodistribution or activating certain specific immuno responses. In CBNI-UCD a special effort has been dedicated to build up an endotoxin-free synthesis laboratory (see Figure 3). In this laboratory (with restricted access) strict SOPs are followed to guarantee an almost 100 % rate of LPS-free sample production. All the sample produced are tested for the LPS content by LAL and western-blot (LPS testing protocols are described in WP2).
Figure 3: Endotoxin-free synthesis laboratory in CBNI-UCD
The LPS-free samples were characterised as previously described and sent to several partners for biological studies, including in vivo studies (ACS Nano, 2017, 11 (6), pp 5519–5529) in collaboration with Istituto Mario Negri (Milano) leading to significant biological outcomes (see WP6).
Task 4.3 (PUM, EPFL, TCD, NUID-UCD, Filarete, IUTA, SolarPrint) Surface treatment and modification of novel materials
This task evolved the nanomaterials formed in Task 4.1 by (post-) modifying their surface. Series of nanoparticles with different charge (electrophoretic mobility) and hydrophilicity/hydrophobicity (including superhydrophobic and superhydrophilic modifications) were created using standard surface chemistries.
Task 4.4 (NUID-UCD, IUTA, EPFL, TCD, ICN, USC, PUM, Filarete, LIST/CRP-GL, SolarPrint) Dispersion and characterization of novel materials.
Samples were characterized both dry (where appropriate) and dispersed in various simple media. The most basic characterization of particles involved (average) size/dimensions, ‘charge’ (averaged mobility) and surface area. In some cases new instrumental developments (or evolution of existing ones) were required to meaningfully characterize these novel shapes and geometries. Full characterization of the materials was provided with respect to elemental/chemical composition, morphology and structure. Obtained nanomaterials were characterized for their elemental/chemical composition (UV-Vis-, emission- infrared-, Raman-spectroscopy, EDX, EELS, ICPMS) and as well as for their colloidal properties such as hydrodynamic and geometric size and size distribution (dynamic light scattering (DLS), nanoparticle tracking analysis (NTA)), surface area (gas adsorption - BET method), shape (TEM, STEM, HAAF-STEM, HRSEM, and WDX), crystallinity (x-ray diffraction), porosity (BET/SEM), surface charge (Zeta-potential), isoelectric point (Zeta potential) as well as their dispersability and stability. All characterization protocols will be carefully prepared following OECD, NIST and ISO standards in order to ensure the comparability of experimental data and therefore fulfill information standards. The quality/stability of the NCs was monitored throughout the project to identify potential variations in the material properties. Optical properties of NC-based photocatalysts, in particular the emission, was a central focus of attention. We systematically measured the quantum yield of NPs and hybrid systems using organic dyes such as Rhodamine B and Rhodamine G6 as a standard probe as well as NC life-time. Besides, modeling tools (NanoHub platform) were used to correlate and model the properties of the NPs. Due to the large set of different types of nanoparticles prepared in this project, appropriate high-throughput screening methods for surface characterization was of interest. The use of high throughput methods for studying surface interactions significantly aided in the identification of the basic interaction mechanisms between nanostructures and biological systems.
Summary of nanomaterials structures classified by composition and shape.
-Composition: A wide range of different nanomaterial types were produced: inorganic, metals, inorganic-metal composites, polymeric, metal-polymer, inorganic polymer particles.
-Shapes: Nanoparticles with different sizes and shapes, and different geometry families were prepared from metals and semiconductors. Some of these series of particles corresponded to series of conventional regular geometries (spherical) where only one feature was varied within the series (e.g. a size dimension). Others corresponded to radically different geometries. For example, particles with precisely tuned cavities/pores were prepared. This was the case of hollow shapes (hollow cubes, hollow spheres, hollow wires -tubes-, hollow triangles and hollow rods) and semiconductor-like subnanometric clusters of metals with different sizes, displaying very different physicochemical properties than the corresponding metal nanoparticles. Some materials (Au/Cu/Se/Te/S) have permitted the fabrication of spiky nanoparticles which were of interest because of their different optical and electronic transport properties, suggesting possible future use in optoelectronics, solar cells and light emitting devices. Spiky metal nanoparticles have improved plasmonic properties, which is very promising for surface enhanced Raman scattering (SERS) and thus for applications as molecular sensors.
-Doping of particles: Doped semiconductor systems have been explored due to their broader access to visible light and enhancing photocatalytic activity. Crucially doping changed not just the optical or electronic properties of nanoparticles (e.g. fluorescent semiconductor quantum dots), but also tuned dissolution of the nanoparticles and degradation of the particles, allowing such aspects as the rate of degradation to be controlled as part of a series.
Summary of nanomaterials structures classified by Partner.
-NUID-UCD Contribution. NUID-UCD developed a shape reference library of anisotropic gold nanoparticles (GNPs) with monodisperse distributions of particle sizes and shapes. The library included the following geometries: spherical, rod-shaped, nanocubes, nanostars (and other branched GNPs), nanotrisoctahedra and gold nanoprisms. The particles were produced in large scale and were applied in subsequent cross-collaborative studies of biocompatibility involving other partners of the FNN project. To render the particles biocompatible, and minimise surfactant-induced cytotoxicity surface of the particles several of these were custom functionalised. Successful surface modification was succeeded in the case of spherical, rod shaped and star shaped gold nanoparticles, where the surface chemistry was normalised using a biocompatible carboxy-PEG-thiol ligands. These ligands provided exceptional stability and biocomplatibility of gold particles of the reported size range in aqueous and biological media. A great effort was also done in order to produce endotoxin-free NPs by developing a platform/laboratory specific for the production of “clean nanomaterials”.
-ICN2/VHIR Contribution. ICN2/VHIR developed a comprehensive reference library of nanomaterials with monodisperse distributions of particle sizes and shapes. The library included the following nanomaterials: i) Shape reference library of TiO2 NPs with narrow size distributions of particle sizes and shapes: spherical, rods, bipyramids and platelets; ii) Homologous series of NPs with different compositions and surface chemistries for co-exposure experiments: TiO2, ZnO, CeO2, FexOy, CdSe, Ag and Au; iii) Shape reference library of hollow Au NPs with narrow size distributions of particles sizes and shapes: spherical, rods, cubes, triangles and wires; iv) Inorganic fullerene-like (IF) MoS2 nanoparticles and V) metallic Ni nanoparticles. In all cases, the surface chemistry of NPS was precisely adjusted in order to ensure their colloidal stability and dispersability.
-PUM Contribution. PUM has synthetized different homologous series of NPs libraries including water soluble gold shapes, Fe2O3, CdS and CdS doped with Mn, ZnS and ZnS dopped with Mn NPs. Some of these NPs were firstly synthesized in organic solvents. Afterwards, all the NPs were transferred to water using an amphiphilic polymer. This approach is based on the use a polymer which contains a hydrophilic backbone and a hydrophobic domain formed by aliphatic chains. This hydrophobic domain interacts with the aliphatic chains that are stabilizing the NPs after their synthesis in organic solvent. Finally, raising the pH, the rings of the hydrophilic domain of the polymer are opened. This process creates a surface rich on carboxylic groups on the NP that provides colloidal stability.
Figure 4. TEM micrographs reporting some example form the GNPs library prepared by UCD and further functionalized by PUM from up to down and left to right: FNN_PUM_Au_#3; FNN_PUM_Au_#4; FNN_PUM_Au_#5; FNN_PUM_Au_#6; FNN_PUM_Au_#7; FNN_PUM_Au_#8; FNN_PUM_Au_#9; FNN_PUM_Au_#10 and FNN_PUM_Au_#11. NPs were distributed across the FutureNanoNeeds consortium for subsequent biological (WP5, WP6) or eco-tox (WP7) evaluation.
-IUTA Contribution. IUTA has worked on the synthesis of materials on the pilot-plant scale along several value chains. Within WP 4 IUTA has used its pilot plant including three different reactor concepts (hot-wall reactor, flame reactor, microwave assisted plasma reactor) to generate materials.
-LIST/CRP-GL Contribution. CRP-GL has worked on the synthesis of Fe3O4, ZnO and PEG-coated Ag Nanoparticles. Fe3O4 nanoparticles were synthesized by a co-precipitation method and stabilized by an oleic acid coating and further dispersed in hexane. ZnO nanorods were synthesized by hydrothermal synthesis. Polymers were used during the synthesis to control the aspect ratio. Citrate-stabilized Ag nanoparticles (20, 40, 80 nm) were functionalized by using the well-known thiol-silver chemistry PEG-SH (MW 3000).
-TCD Contribution: TCD has worked on the synthesis and characterization of CdS, CdS doped with Mn2+ and TiO2 NPs. CdS nanotetrapods of approximately 15 nm size were produced through the use of an aqueous reflux in the presence of the enantiomerically pure stabilising ligand D or L penicillamine. CdS nanotetrapods doped with Mn2+ ions were synthesized in a similar fashion to the synthesis of pristine CdS nanotetrapods above with a number of changes. TiO2 nanoparticles were produced through a non-aqueous reflux.
-FILARETE Contribution: TCD has worked on SPES (Single Particle Extinction & Scattering) technology which is capable of characterizing nano and micro particles in complex fluids with unprecedented high-quality results.
Figure 5- TEM/STEM images TiO2 NCs synthesized by ICN2/VHIR from up to down and left to right: FNN_ICN2_TiO2_#2; FNN_ICN2_TiO2_#3; FNN_ICN2_TiO2_#4; FNN_ICN2_TiO2_#7; FNN_ICN2_TiO2_#8¸ FNN_ICN2_TiO2_#9. NPs were distributed across the FutureNanoNeeds consortium for subsequent biological (WP5, WP6) or eco-tox (WP7) evaluation.
-NANOGAP Contribution: NANOGAP has synthesized and sent to partners both, fluorescent Au clusters with enhanced Stokes shift, and surfactant-free ultrasmall Ag clusters, according with Task 4.1. Results from partners have shown that clusters synthesized in NANOGAP were endotoxin-free, as required for toxicology testing. IN detail, they succeeded in the development of synthetic and purification techniques for the production of Ag AQCs with a specific number of atoms with a concentration of different species low enough to avoid interference in the observed properties. The species produced by NANOGAP in an almost pure form are Ag3 and Ag5, with very different biological effects one from the other. Besides, a much simpler synthetic approach has been developed for the production of complexes of Au AQCs which, due to their radically new luminescent properties, have enormous potential in any application concerning fluorescence. This fact makes very interesting the study of their biological interactions and the potential toxicity of the product.
Figure 6 - TEM images homologous of series of NPs with different compositions, and surface chemistries. The library includes the following compositions: TiO2, ZnO, CeO2, FexOy, CdSe, Ag and Au. NPs were distributed across the FutureNanoNeeds consortium for subsequent biological (WP5, WP6) or eco-tox (WP7) evaluation
-USC Contribution: USC has worked on the preparation of silver nanoclusters with sizes slightly larger (6-10 atoms) than those prepared by Nanogap (2-5 atoms) and with the highest monodispersity in size. They used different methods for achieving this objective: i) Direct reduction of Ag(I) salt by UV irradiation (254nm wavelength);ii) Chemical reduction of Ag(I) salt with strong reducing agents (e.g. sodium borohydride); iii) Electrochemical synthesis using two Ag foils; iv) Two-step synthesis: electrochemical synthesis of small Ag clusters followed by chemical reduction with strong reducing agents; and v) Two-step synthesis: electrochemical synthesis of small Ag clusters followed by UV irradiation (254nm wavelength). From these previous methods, the one-step methods showed long-term stability problems (aggregation and settling) and some polydispersity in sizes. The results were much better with the two-step methods, being the last method the best one.
Figure 7. TEM images of Au-Ag hollow nanoparticles after reaction of Ag templates of different shapes with different volumes of HAuCl4: (A) Au-Ag hollow spheres (FNN_ICN2_AuHollow_#1, 53nm in diameter), (B) Au-Ag hollow cubes (FNN_ICN2_AuHollow_#2, boxes) (36 nm side) (C) Au-Ag hollow rods (FNN_ICN2_AuHollow_#3, 60x27x27 nm), (D) Au-Ag hollow prims (FNN_ICN2_AuHollow_#4, 45x45x10 nm), and (E) Au-Ag hollow wires (FNN_ICN2_AuHollow_#5, tubes) (> 1000 x 50 x 50 nm).
Work Package 5: Biological and environmental interactions of future nanomaterials
Within WP5 TNO performed an analysis of the protein corona formation around nanoparticles and investigated the implications for safe design. In order to control the activation of specific biological pathways, it is therefore essential to understand the formation of the biocorona in terms of composition and amount of adsorbed proteins and its relation to the physicochemical properties of nanomaterials and proteins. In this way, it would be possible to develop structure-activity relationships, allowing the engineering of nanomaterials in order to develop nanostructures with controlled fate and bioactivity/toxicity. It was found that the profile of corona composition around different nanoparticles is basically the same, and that this depends on the plasma concentration and molecular weight of the proteins. For the most abundant proteins, we also find that electrostatic interactions play a role in determining the amount of adsorbed proteins. For surface modification of nanomaterials, in order to selectively bind or repel specific proteins, may not be functional to control specific biological effects or avoid biological pathways.
Partner CBNI-UCD: The stability of several nanomaterials (including, but to not limited to different gold nanoparticles with different size and shapes (as spheres, rod-like, urchin-like and star-like GNPs) and different carbon based nanoparticles (as graphene and nanodiamonds) dispersed in relevant biological media have been investigated. To this purpose several biofluids and biological media have been selected, ranging from full human plasma or serum (to mimic in vivo scenarios) to complete growth medium (RPMI and MEM supplemented with different concentrations of faetal bovine and human serum, in order to mimic in vitro conditions). The nanoparticles have been therefore suspended in these milieu and incubated at 37 °C for different time points (up to 24 hours). Optimised suspension protocols have been developed. Generally, the NP dispersions in biological media of the analysed nanoparticles turned out to be stable in “realistic biological conditions”.
A further work was performed in collaboration with Filarete. DCS and SPES were both employed to successfully analyse in situ and in line the stability of different GNPs shapes in biofluids. This is an important achievement allowingthe study of NPs suspension in real conditions without washing/isolation of the NPs. Several NPs at certain concentration are well stabilised by the large excess of proteins present in biological media but lack of stability below a certain protein concentration (i.e. after washing/centrifugation).
CBNI also investigated the biomolecular corona composition of different shaped nanomaterials (synthetized in the group / WP4) exposed to relevant biological fluids. Some of these shaped nanoparticles were also sent to FILARETE for investigation of the corona by SPES and shotgun mass spectrometry (in collaboration with UNIMI).
CBNI-UCD also investigated how the shape can influence the orientation of the protein during the adsorption on the NPs surface. The study was performed by QCM (quartz microbalance) using nanospheres and nanostars of same size with transferrin corona. The analysis showed a significantly higher number of Tf with the “right orientation” for nanostars compared to nanospheres. These results confirmed an effect of the shape not simply in the biocorona composition but also in the epitope presentation and therefore potentially different biological recognition. Also the prepared surface chemistry was shown to parametrize the NP-protein complexation. Indeed, for gold nanostars functionalised with BSPP the recognition was much higher than the one observed for the very same nanostars functionalised with PEG. This is probably due to the reduced interaction of the proteins with the PEGylated NPs surface.
Moreover, the influence of the shape (and related biomolecular corona) have been tested in vitro. Briefly, CBNI developed a cell platform to assess the role of specific receptors (mostly express in the liver) in the uptake of NPs. Human Embryonic Kidney 293 T cells (HEK-293T cells) have been used as a model for the transfection of the specific receptors of interest, due to its high efficiency of transfection and protein production.
Furthermore, in collaboration with TUL, LIST and FILARETE, CBNI-UCD investigated the stability/degradation of different NPs suspensions in different environmental fluids as peatbog water, sea water and reservoir water. Preliminary data suggest that again combining SPES, DCS (as complementary techniques) nad UV/Vis it is possible to follow the overtime evolution of the NPs degradation.
TUL leaded D5.2 Report on novel methods to assess degradation, which included examples on assessment of nanomaterial degradation in most important biological and environmental media, particularly in FBS, hepes buffer, PBS buffer, artificial lysosomal fluid, simulated body fluid, bacterial growth media, algal growth media, in soil, contaminated groundwater, and freshwaters. This deliverable served as a basis for WP6 and WP7 partners performing toxicity and ecotoxicity studies in terms for providing recent advances in methodology for characterisation of nanomaterials. Moreover, TUL developed a new standard (nAu) for differential centrifugal sedimentation analysis, because commercial PVC standard was found to be unsuitable for all types of nanomaterials. A scientific article is now under preparation (Mikšíček. et al. Quality control of differential centrifugal sedimentation analysis and its use for determining the stability of synthesized with a gold and iron oxide nanoparticles).
Further we performed research on transformation of new FNN nanomaterials in biological media, particularly in algal and bacterial growth media and in activated sludge from wastewater treatment plant. Results are present in D5.3 Data inventory of solubility and degradation kinetics of novel nanomaterials in biological milieu. Generally, this study helped us to understand behaviour of the materials is different exposure media and thus better understanding of ecotoxicological data (within WP7). Several scientific articles are now under preparation in collaboration with RIVM, UoB, and UCD.
Dispersion, aggregation and stability of FNN nanomaterials (VHIR_AgNP_15 nm_060516, mesoporous silica NPs coated by APTES with magnetic cores, and Si@Gra01) in reservoir water, media of low and high pH and media of different ionic strength were tested at TUL. Results were described in D5.6: Characterisation of dispersions in several environmental media and in conference extended abstract (Wacławek et al. 2016). Finally, TUL leaded a study on FNN ZnO materials obtained from LIST (nanoscale ZnO-HP1, ZnO-HP1-Mirj S40, LNP ZnO-HP1, ZnO nanowires) in natural waters (reservoir representing normal pH and ionic strength conditions, peatbog representing acidic environment, and seawater representing higher ionic strength environment) in collaboration with MNI (EOS Instruments) and NUID-UCD. Results are being analysed and an article intended for journal with high IF will be prepared.
Contribution from MNI:A limited series of experiments has been performed to evaluate the influence of the material on cell internalization mechanism. To this aim NPs with similar size, shape, z-potential and surface functionalization (PEG) but differentiating among them for the chemical nature of the Polymer (PMMA, PCL3 and PLA8 respectively) were incubated in breast cancer cells MDA-MB231.1833. Rhodamine-B was added to the monomer and the process of polymerization was undertaken in order to have a strong signal and to avoid the biological elution of the dye. Representative images showing the pattern of RhB distribution of NPs are shown in figure 8.
Figure 8: Representative images showing the accumulation of red signal (associated with NPs) in MDA-MB231.1833 cells (identified by the presence of nuclei in blue). All field of views were acquired by exciting the sample with a laser at 405 nm and immediately after with a laser at 546 of λ. The merge between the two signals is reported in these images. Scale bar: 75 µm. Scatterograms showing the merge of events selected by TissueQuest in each experimental group. For each group nine different fields of view, obtained by two different experiments, were processed. In the X-axis the fluorescence intensity for each single spot is reported. In the Y-axis the intensity of fluorescence for each single spot is reported.
The quantification of fluorescence is reported in figure 9. Our data showed that the NP accumulation significantly increase from at each time-point for each type of materials but the uptake was faster and stronger in MDA-MB231.1833 cells incubated with PCL3.
Figure 9.A: Histograms showing the fluorescent area for each experimental group (n = 20 fields of views). The same fields of view processed for the analysis of RhB were analyzed to determine if the time of incubation and/or the type of nanomaterial may influence the number of cells.
In figure 10 A depicts the cell internalization of the three materials is shown.
Figure 10A: Representative pictures showing the staining associated with the nuclei (left column), the staining for RhB (central column) and the merge between the two signals (left column) in MDA-MB231.1833 cells incubated for 24 hours without NP (upper line), PMMA-NP (second line)PLA8-NP (third line) and PCL3-NP (lower line).
B: High magnified pictures showing the different localization of NPs (red) 24 after incubation in MDA-MB231.1833 cells. PMMA-NP incubation (LEFT panel) led to a massive internalization of NP (see green arrow) and a weak staining surrounding the peripheral rig of the cell (yellow arrow). PLA8-NP (central panel) mainly remained confined on the external surface of NP (yellow arrow) with a weak penetration inside the cell (green arrow). A homogenous distribution of red spots was instead observed in cells exposed to PCL3-NP (right panel).
The vehicle-treated cells (upper panels) were added to demonstrate that, by this procedure, the red signal associated with was almost completely broken down (the black arrow show a signal apparently unrelated to the nuclei). Both PMMA-NP and PCL3-NP incubation was characterized by separated dots and mainly concentrated close to the perinuclear region. On the other hand, the staining for RhB after 24 hours of PLA8 was almost exclusively confined to the external part of the cells.
Experiments with blockers of endocytosis figured out that, whilst both PMMA and PCL3 staining was greatly reduced after treatment, no significant alterations were found in cells treated with PLA8. This result furthermore confirmed the lack of endocytosis by PLA8-NP.
Overall, the present results furthermore confirm the dramatic changes in bio-nano interaction after modification of a single parameter of NPs (in the case the softness of the material). This is an extremely important point to be considered to both evaluate the efficacy of a treatment or, opposite, to predict its potential toxic effect.
Work Package 6: Biological identity, processes and human health hazards of future nanomaterials
As described above, in this WP the biological impact of several NMs having different physico-chemical properties has been carefully evaluated by a multi-step platform starting from the basic interaction with molecules or fluids and, passing through analyses in cells, reaching the in vivo characterization in rodents.
A first period was devoted to set-up and standardize the experimental procedures required to this platform development. This approach is in line with the whole philosophy of the project, in which there has been extensive effort to make reliable and reproducible the data generated throughout the lifetime of the study. This has included a centrally managed workflow of materials produced and distributed to partners for testing. The existence of a wide variety of data from previous FP programs already suggested guidelines and rules to be followed along the whole duration of the project among all partners involved in this WP. To this end standard operating procedures (SOP) have been tested in a number of Round Robin and interlaboratory comparisons and compared to those tested with the reference materials. In detail, viability assays, mainly through MTS assay experiments, were carried out using the same stock of cells representative of the alveolar epithelium (A549 cells). The results, that were then uploaded to Teamlab to be easily shared with other partners, unveiled a high level of reproducibility among the different labs involved in this WP. Further to this, the results are in good agreement with previous interlaboratory comparisons performed using the same SOP and materials within the QualityNano Infrastructure. This indicates a high level of confidence in the reproducibility of results within WP6 and the potential for read-across of a number of biologically relevant endpoints across numerous cell lines. Another important point to be assessed before the different effect of NPs in biological fluids, cells and animals is the level of contamination. To this end TUL an accurate analysis of endotoxins in new NM samples obtained from FNN project partners was carried out. This was important for WP6 partners performing toxicity studies with mammal cell lines, because some toxic or inflammatory effects, which might be attributed to NPs, can be in fact caused by the presence of endotoxins. Therefore, we newly established chromogenic Limulus amoebocyte lysate (LAL) assay and fluorogenic EndoLisa assay for detection of endotoxins. Both methods were developed for medical purpose and had to be adjusted for NMs. For example, presence of NPs can cause false positive and false negative results. Hence, a control displaying possible inhibition or enhancement of the signal was included. During screening of FNN NMs for endotoxins, we found out that considerable number of samples was contaminated by endotoxins and we promptly communicated these results to the other WP partners involved on the toxicological studies. Guidelines for FNN WP4 partners on how to minimize the introduction of endotoxins during NM synthesis were then prepared and circulated. A study to compare four methods for detection of endotoxins was therefore performed. Experiments with E. coli lipopolysascharide (LPS) spiked samples of ultraclean Au-NPs and SiO2-NPs LPS were done using LAL assay and EndoLISA to find out the limits of the methods and possibilities for further optimization. Another important aspect that had to be considered before biological tests was the behaviour of NMs over time. Particle storage and aging, dissolution in short and long term and in relevant media may lead to a change in the physico-chemical properties of materials and, consequently, modify their biological identity. In this WP we focused on the attention on the selection of the best procedures to follow the fate of the different NMs after their synthesis. This was pivotal to control the quality of the products and to remove impurities in a broad range of material types. This allowed us to undertake the next phases of the project with an inner control based on the purity and the maintenance of the basal characteristics of the different NP before the next part of the WP, devoted to the in vitro studies.
The first feature we evaluated in cells was the ability to uptake NPs and the consequent intracellular accumulation. The mechanism of uptake and localization of novel materials, graphene and graphene oxide (of interest for VC4), long-tipped gold nanostars (due to unorthodox shape parameters), gold nanorods (possible shape of interest for some applications) and spheres (reference material) was investigated in detail in this WP. The study of NP-cell membrane interactions is the more realistic way but is a really complex system. To get results we performed these experiments with high speed spinning Disk confocal LASER microscopy. Utilising spinning disc confocal LASER microscopy it is possible to create ‘movies’ of NPs as they interact with the cell membrane. Our results highlight that interaction between a NP and the cell include several processes such as, the diffusion near the cell membrane, the diffusion to the cell membrane and the endocytosis. It is difficult to completely separate those phenomena as some of them might be time based, e.g. particles which stick to the membrane could be internalized at different time periods. We found that the time a particle spends on the membrane prior to internalization can range across a large period of time, internalization events could be seen to range from time scales of seconds to upwards of 30 minutes and that that the rarity of combination between NP and receptor is not directly dependent on its physico-chemical details, such as shape and material, but rather depend on particle/cell concentration and conditions of the study. It is known the uptake can be influenced by the percentage of serum conditions. The achieved results indicate that there are a higher number of events under the lower serum condition for the same time period per unit length of membrane. Hence it is more likely to engage in an interaction event with the membrane, meaning a higher probability to potentially undergo endocytosis compared to the same type of NPs incubated in a higher concentration of serum.
To further investigate the involvement of the receptor receptor-dependent uptake of NPs, we focused our attention on the action played by scavenger receptors. Macrophage scavenger receptors are a family of trimeric membrane glycoproteins responsible for the endocytosis of a large range of macromolecules, likely proteins linked to NPs. To this end, the human embryonic kidney cell line (HEK293) was induced to express specific scavenger receptors on their surface to evaluate the importance of each individual member of these scavenger receptors. In this study graphene, silica and gold NPs were extensively used to test the efficiency of uptake. In a receptor, a transmembrane protein devoted to recognition of bacteria fragments or other non-self-agents circulating in the bloodstream, a progressive reduction of the overall uptake was observed when the serum concentration increased. However, the 33% of NPs were still uptake by the scavenger receptor 1 even at high level of serum in the medium. These results suggest that despite the low interaction due to the presence of human serum proteins the receptor is still able to recognize NPs in the medium and internalized them. Using another scavenger receptor, we found that both star and spherical gold NPs behave in the same way and that the complexity of the cell surface increase when the cells are exposed to gold NPs. This suggests that NPs can adhere to the cell membrane inducing relevant alterations in the homeostasis of the cell surface.
Once determined the most critical parameters influencing the cell surface-NP interaction and the consequent uptake, the attention was pointed at to their localization inside the cell. We used TEM to evaluate the subcellular localization in macrophages of gold NPs incubated after 24 hours. As expected, our study revealed that NPs mainly localized into the cytoplasm, in organelles probably related with the endocytic pathways and vesicles. After NP uptake by endocytosis the primary location is in acidic organelles with different levels of maturation; this can go from primary endosomes, late endosomes, lysosomes and lamellar bodies. To combine observational to quantitative results, ICP-MS experiments were carried out. Results showed a different level of uptake by the two cell types which varied between different materials, sizes and shapes. Very interestingly, two different sizes of the gold nanospheres were mixed (at 5% v/v of the respective stock suspensions) the uptake by A549 cells appeared to be determined by the uptake rate of the largest NPs, whereas in Caco-2 cells uptake remained similarly low for both sizes.
Once internalized, NPs can be either dissolved or put out from the cells (exocytosis) or remain inside the cells leading to a progressive accumulation. It is clear that both NP characteristics and cell type may greatly influence their fate after penetration. In this multivalent process, lysosomes play a pivotal role in processing because of their ability in digesting and controlling the release of materials out of the cells. Using A549 cells, exposed to different types of NPs for two hours and then maintained for a longer window of time (weeks), we found that the isolated lysosomal fraction showed a trend of decrease of NPs over the time. Obviously, this decrease was tightly associated with the nature of the NM and their main physic-chemical features. In order to understand if this reduction was due either a real dissolution of the NM or related to a clearance by exocytosis, the NPs were separated from isolated lysosomes and TEM analyses were carried out. Quite interestingly we found that gold and silica NPs did not show any sign of dissolution whereas magnetite core NPs had a significant change on particle structure. This is a very important finding to predict what could happen once ingested or inhaled by humans and penetrated in the cells of digestive or respiratory tract. The interaction between NPs and lysosomes was also evaluated by super resolution microscopy using fluorescent polymeric NPs in human breast cancer cells. This study showed that 2 hours after incubation NPs are already in contact with mature lysosomes, that hey actively enter inside the lysosome passing through its membrane to be degraded. Very interestingly several agglomerates of lysosomes containing NPs, resembling a multivescicualr bodies, were found at the periphery of the cells starting from 24 hours of NPs. One week after NPs injection the signal associated with them almost completely disappeared and lysosome organization was similar to the resting state. This strongly suggested that a complete clearance of polymeric NPs from the cells was occurred. To mimic the in vivo condition, spheroids form A459 were added to this platform to check the processes of NP degradation in a 3D model. Our results revealed a mild but progressive decrease of silica NPs over time. In contrast, the rate of decrease for non-degradable polystyrene showed a significant reduction within the first week but a maintenance of these levels at the latter times, indicating that there was no further dilution of the particles. This is a crucial point in case of repeated exposure, accumulated NPs could indeed lead to relevant functional alteration, first a lower efficiency of lysosomes but not only. To better evaluate this relationship between uptake-accumulation and downstream effects, a careful investigation on the systematic toxicity of NMs was carried in the next step of this integrated platform for the nanosafety assessment. The combination of MTS assay and High Content Imaging high-throughput method was applied to test the viability and other parameters of toxicity after exposure to 30 different NMs at the three different concentrations in three different cell lines. This analysis generated a very large number of data. However, it is possible summarize that many parameters, such as the geometry of NPs, their concentration, the nature of the material and the cell type may lead to different results. In addition, it is also important to underline that relevant hallmarks of toxicity (e.g. ROS production, genotoxicity, mitochondrial impairment) found with specific NMs were not always strictly associated with the lethal effect of them. This is a very important point because it underlines that the mere evaluation of cell survival is not such a reliable approach to deeply evaluate their potential effect to the human health.
Another important question is whether NP accumulation can be safe for the target but, at the same time can induce on it some physiological alterations that can be detrimental for neighbouring cells. This always happens in pathological condition such as automminune disorders or reactive gliosis in neuroinflammatory diseases. To this aim NP-dependent immunomodulation was investigated by inflammasome activation. Inflammasomes comprise a group of intracellular multiprotein complexes readily triggered by exogenous stimuli. They mainly lead to cytokine expression and release and activation of a pro-inflammatory phenotype from a resting to an activated macrophage. Among the several results achieved we interestingly found that CeO2 nanospheres had a dose-dependent activation of Il-1β without changes in cell viability (in blood monocytes THP-1), whereas CeO2 stamps sharply decreased the viability but did not increase the cytokine release. Other NMs, such SiO2 or gold, showed a shape dependent infammasome activation. All this data is extremely important both in nanotxicology but, above all, in nanomedicine. To know that an inflammatory activation of circulating cells can be somehow associated with a specific physic-chemical feature of injected nanocarrier would be extremely important to develop NPs for pharmacological purposes.
Although this in vitro screening is very important to maximally reduce preclinical studies, they cannot completely exclude the use of rodents to predict crucial bio-nano parameters such as organ distribution, the clearance and the occurrence of detrimental multy-systemic effects. To this end, on a very restricted number of mice, a comparative evaluation of the effect of intravenously injected NPs was carried out. In a first study the role of the size and the shape of endotoxin-free gold NPs was evaluated. We selected this kind of pure NPs to be close to the clinical practice. The animals were sacrificed 1 hour, 1 day and 5 days after the treatment because these three time points are representative of three different situations, filter organ penetration, clearance and long-term accumulation respectively. Both shape and size strongly influenced the filter organ accumulation and the excretion, no relevant improvement of biological barrier passage was instead observed for any kind of geometry tested. Very interestingly, the injection of endotoxin-free gold NP did not lead to either any evident histopathological alternation or signs of inflammatory activation. A similar approach was used to define the shape-dependent behaviour of TiO2 NPs. In this case we found a lower ability of accumulation, and the shape did not lead to significant difference, but a marked but transient response of blood, liver and lungs (the three organs with the higher presence of NPs) accompanied to a pro-inflammatory activation. In particular rods, gave a better effect in spite of a lower persistence and bio-accumulation thus indicating these two latter factors are not always tightly correlated.
Although the present platform has been mainly exploited to evaluate the behaviour of NPs with a well-defined shape, it can be easily transferable to the evaluation of the effect of NMs with unusual geometry. In the past three decades, nanostructures with a variety of morphologies, components, chemistries, and functional molecules have been extensively developed, and the tuning of these physicochemical properties can be directly used to influence nano-bio interactions. Given the significant nano-immune interactions for all types of nanostructures and all delivery routes, we have decided to focus our attention on understanding the impact of NP with irregular shapes on the innate immunity. First, we examined the role of surface geometry on the gold (spheres or stars) NP uptake in human macrophages. We noticed that both spherical and star-shaped gold nanoparticle could be internalized by macrophages, however, it was observed for different concentrations that the uptake of gold nanospheres (GNP) is more efficient than that of gold nanostars (GNS). Compared to GNP, the uptake rate of GNS was much lower at the initial exposure and remained steady until 16 h, where it showed a trend of reaching a plateau. In order to identify the signalling pathways and mechanisms that contribute to the immune responses to different shapes of gold nanoparticles, a dual-colour transcriptome microarray was performed in THP-1 cells 24 hours after incubation. Gold NPs exposure triggered to a significant alteration in gene expression. Overall, we observed that gene expression regulation was more significantly affected by GNP, compared to GNS in the macrophage model.
The immunomodulant effect of the shape was also seen by a chronic repeated subcutaneous administration of endotoxin free GNP and GNS in healthy rats. An increase in the circulating IgG levels was indeed observed in certain animals treated with GNS suggesting again that there might be a differential processing in vivo compared to GNP. Moving from the stars and spheres, we decided to further synthesize more complex branched nanostructures and screen the adjuvant effects of the geometry in mice, previously immunized by Ovalbumin and then treated with spheres, urchin, long-trip urchin and flowers respectively. Mice were sacrificed at 42th day, axillary lymph nodes, Bone marrow from hind limbs and spleen were harvested and homogenized in a single cell suspension, PBMCs were extracted from blood samples. Mice treated with spheres showed a very weak production of antibodies almost comparable to the ovalbumin alone whereas urchin-like NPs did not show any production of antibodies. Long-urchin like NPs seemed to be more efficient in stimulating antigen-specific antibody response than free ovalbumin and an increase of IgG production after second booster comparable to free ovalbumin. Although other experiments should be carried out to better define the immunogenic power of these different shapes, including the ability to migrate into the sentinel lymph-node (axillary) an evidence of a shape-dependent influence is emerging from this study. These preliminary results suggest that GN shape convey the adjuvanticity, which is consistent with previous observations with nanostars and nanospheres. It is important to understand the mechanism underlying the shape-dependent adjuvanticity. The ongoing work by NUID-UCD is focusing on further phenotyping and analysis of different antigen-presenting cells to understand the quality of these adjuvanticity.
Work Package 7: Exposure and environmental interactions of future nanomaterials
Task 7.1 Environmental binding and ecoidentity of future NMs
Work carried out within WP7 pioneered the investigation of the role of organism-secreted biomolecules in modulating the acquired biomolecule corona and thus the implications and impacts of NMs on living systems. Figure 11 shows schematically the approach used to collect the secreted biomolecules (conditioning of the medium), in which NMs are incubated prior to their presentation to organisms for assessment of uptake, retention and impacts.
Figure 11. Schematic illustration of the process of conditioning medium, incubation of NMs in the conditioned medium, and subsequent presentation of the eco-corona coated NMs to organisms, in this case the aquatic crustacean, Daphnia magna.
Thus, the secreted corona arises from the fact that organisms secrete biomolecules such as proteins, carbohydrates and lipids, which adsorb selectively to the NMs to create an eco-corona or layer of biomolecules around the NM which changes the identity of the NM (i.e. confers and environmental identity) and alters their interactions, stability, uptake by, and toxicity to organisms.
Notably, in cellular systems the reduction of surface energy through binding of biomolecules and corona formation always leads to reduced NM uptake and cytotoxicity, while in the case of environmental organism, with their myriad of uptake modalities, this is not always so straightforward. The impacts from secreted coronas observed within FNN include:
1. Biomolecules can have destabilising or stabilising effects on NMs, which can cause them to agglomerate and become a more or less attractively sized food source for D. magna which in turn can drastically affect their toxicity. It was observed that the secreted proteins caused agglomeration of polystyrene NMs, which lead to increased retention of the NMs in the D. magna gut, and thus our working hypothesis is that bigger particles (closer to the algae size) are more recognisable and potentially more attractive as a food source, though NM dissolution also needs to be taken into account for many NM types. The addition of natural biomolecules during NM toxicity studies should be considered in NM exposure protocols. Furthermore, whole organisms such as D. magna are themselves dynamic systems and therefore potential changes to the NMs may occur once consumed in, for example, the gut, which can be considered as a sub-environment where NMs may be altered.
2. It is a well-established fact that D. magna decrease their grazing if they are already fed. It has been found that NMs may become lodged within the gut, in proximity to the bush border, which appears to be causing the D. magna to feel full, resulting in reduced uptake over the subsequent period. A wide range of NMs, or different sizes and compositions have been found to be retained by D. magna, over at least 6 hours post-exposure to NMs, in the absence of feeding. It is well-known that D. magna require a food source to push out previously ingested material so that the amount remaining in the gut may be cleared in real environmental scenarios where food is present. At the same time, NMs can bind to a food source incidentally increasing their uptake so that a food source acts as a modulator of both uptake and excretion. Thus, more realistic exposure scenarios need to be developed, that account for the physiological features of the test organisms and the unique considerations of NMs. Assays to assess the fate of the NMs following uptake also need to be developed.
3. The presence of the corona and secreted proteins in the medium can have a profound effect on the dissolution of NMs, and specifically on the mobilisation of specific metals contained within some NMs. Extensive work with lead iodide perovskite particles, comparing their toxicity in standard media versus conditioned media again found enhanced toxicity in the case of the conditioned media. However, the mode of toxicity in this case was related primarily to the ability of the secreted proteins to sequester lead and thus drive the release of lead from the perovskite NMs and thus enhance its bioavailability to the organisms. Design of the NM surface to minimise contact with the specific lead-binding proteins may be one potential route forward to reduce the toxicity of these materials during use and end-of-life. Work is ongoing to confirm whether the mobilisation of lead occurs outside the organisms or is enhanced at the low pH of the D. magna gut.
Limited work on the development of a workflow and methodology for assessment of the other constituents of the eco-corona, beyond the proteins, was also undertaken. Assays for total carbohydrate concentration and for identification of the functional groups in the carbohydrates were developed, given the strong prevalence of polysaccharides in the environment. Additionally, a workflow to identify the small-molecule corona, also called the metabolite corona, have been developed based on mass spectrometry again utilising the conditioned medium as the starting point, with limited success. However, this is a very promising area of future research, and several different avenues are being pursued in parallel to optimise the small-molecule corona workflow.
In summary, research undertaken to address task 7.1 focussed on characterisation of the secreted corona (proteins, polysaccharides, small molecules, including method development for small molecule (metabolite corona) and evaluation of the impact of the route of exposure to NMs (with/without food before, during and after exposure) on the resulting effects of the NMs. Specific features of NMs linked with toxicity included the release of toxic ions (e.g. lead from perovskites) or effects from cationic charges and rod shaped particles that lead to excessive reactive oxygen species production, which required the organisms to divert energy usually required for moulting to fighting the oxidative stress, resulting in delayed development through delayed shedding of the carapace. In the absence of the conditioning step, D. magna were able to mitigate the oxidative stress induced by cationic particles within 24 hours. By contrast, the same NMs exposed at the same concentrations but following incubation in the D. magna conditioned medium, were found to be unable to recover with >90% dead within the 8 hours of exposure (at the EC40 concentration for the bare NMs). The role of the corona in enhancing oxidative stress, or preventing the functioning of anti-oxidant molecules is an important aspect worthy of additional research.
Task 7.3 Material transformation (including degradation) in the environment
In WP7 partners conducted leaching experiments in soils with different perovskites as well as nanoscale MoS2 and non nanoscale MoS2, as for these materials a release into soils are suggested. The primary particle size and the hydrodynamic diameter, zeta potential as well as the solubility of the materials was tested before the leaching experiments. For the solubility experiments the NM mixture was shaken for 24 h (overhead shaker). After this the suspension was centrifuged (5000 g) for 30 min and filtered by using a 0.02 µm syringe filter. The filtrate was analysed with ICP MS (Mo for MoS2 and Pb for the perovskites).
For the soil experiments, glass columns with an inner diameter of 4 cm were filled with soil (dystric cambisol) to a height of 10 cm. Afterwards the soil was pre-wetted from the bottom to the top with 0.01 M CaCl2 solution and drained again before the NM suspension was applied on the top of the soil column as suspension. After this "artificial rain" 0.01 M CaCl2 solution was applied on the top for 48 h. The eluate was sampled after 3 h, 6 h, 24 h and 48 h and analysed to the Mo and Pb concentration. After the experiments different segments of the soil column were analysed (NM conc.) with ICP MS.
No breakthrough was observed for the three tested perovskites (FALi, MAFA, MALB), these materials show a low mobility. FALi shows a slightly higher mobility, followed by MAFA and MALB. The MoS2 materials show a breakthrough with a significant higher mobility of the nanoscale material. No correlation was observed with the agglomerate size or solubility. For the agglomerate size the material with the highest size the highest mobility was observed and no ranking between the perovskite materials. As both MoS2 materials show a slightly higher agglomerate size, compared to the perovskites we can conclude that the agglomerate size did not significantly affect the mobility. For the zeta potential a trend was indicated showing the highest mobility for the materials with the highest zeta potential and vice versa.
WP7 was able to develop mobility experiments of the next generation of NMs in soil columns. The work conducted leads to a better understanding of the mobility of novel NMs in soil columns, helping to understand the expected possible behaviour in the environment as well as to identify exposure hot spots in soils relevant for hazard assessment for soil organisms, plants and assess groundwater threats.
Task 7.2 Development and application of environmentally relevant organism exposure protocols and Task 7.4 Interaction with environmentally relevant species, including ‘sentinel’ species.
When assessing the impacts of new generations of NMs, it is essential to combine quantitative exposure assessment with effect assessment, independent of the type of toxicity assessment. Amongst others, time weighted average (TWA) particle concentrations are well suited to express the effective exposure. Although TWA concentrations are commonly based in mass-units, it is also possible to express TWA in alternative dose expressions like number of particles and total surface area to which the biota are exposed. When assessing TWA concentrations, it was found to be essential to explicitly take particle dissolution as well as sedimentation into account as both processes can (dependent on the composition of the medium in terms of especially ionic strength and intrinsic particle properties like chemical composition of particle core and particle coating) be extremely fast with half-live as low as just a few minutes.
Homologies series of NMs allowed to assess that particle shape and particle size are the key properties affecting acute toxicity of NMs. In most cases, the impact of shape and size is related to the kinetics of ion release of the particles. Studies using homologues series of Pb-based perovskites, Ag, Cu and ZnO NMs indicated that in most cases, toxicity is dictated by the release of toxic metal ions whereas in general the contribution to acute of the NMs is limited. No acute effects were found for stable NMs like TiO2.
An ex vivo exposure of embryos of the waterflea Daphnia magna to polystyrene NMs (PSNMs) demonstrated a similar accumulation of PSNMs in or on lipophilic cells, illustrating the likelihood of brood pouch-mediated PSNMs uptake by embryos. By demonstrating embryo PSNM uptake via the brood pouch, novel insights in bioaccumulation of NMs and likely other lipophilic contaminants, were obtained. Since this uptake route can occur within a diverse array of aquatic organisms, it is clear that brood pouch-mediated accumulation needs to be considered in studies of the hazards and risks of NMs contamination. Up till now, this exposure route is ignored in such kind of studies despite the fact that the embryos thus affected may well be inherently sensitive to NMs.
Particle uptake of PSNMs in zebrafish embryos was found to be restricted to oral exposure, whereas the dermal route resulted in adsorption to the epidermis and gills only. Ingestion followed by biodistribution was observed to be size-dependent with uptake possible only for relative small particles of 25 and 50 nm. The particles spread through the body and eventually accumulated in specific organs and tissues such as the eyes. Particles larger than 50 nm were predominantly adsorbed onto the intestinal tract and outer epidermis of zebrafish embryos. Embryos exposed to particles via both epidermis and intestine showed highest uptake and eventually accumulated particles in the eye, whereas uptake of particles via the chorion and epidermis resulted in marginal uptake. Organ uptake and internal distribution should be monitored more closely to provide more in depth information of the toxicity of NMs.
One important focus of WP7 was to investigate relevant exposure conditions. One way of assessing this was to evaluate hazard via aqueous and dietary exposures on endpoints at various levels of biological organization. Another important area of investigation was to assess interactions between NMs and possible co-contaminants.
The main findings were: 1) sorption of environmental contaminants on NMs can change the bioavailability of the contaminant in the aqueous phase. Specifically, sorption of copper and benzo(a)pyrene (under fluorescent light) on NMs reduced the adsorbent bioavailability. In contrast, benzo(a)pyrene and anthracene, when adsorbed on TiO2 or Si NMs, were photo-catalysed under UVA and in the case of benzo(a)pyrene, highly toxic photo-by-products showed increased bioavailability in the presence of the NMs; 2) higher toxicity was found when NMs were in dispersion, indicating toxicity is related to the dispersion state of NMs; 3) lead-halide perovskite acute toxicity was attributed to lead ion dissolution based on induction of metallothionein 2 gene expression through aqueous and dietary exposure, and 4) the perovskite-spiked diets did not disrupt zebrafish gut microbiome after a 14-d exposure while disruption of gut microbiota by equivalent Pb(NO3)2 diets was observed.
As a general conclusion of the co-contaminant study (‘trojan-horse’), sorption of copper was confirmed on negatively charged NMs with sorption being closely related to surface area. Sorption of photo-labile organic compounds occurred on semi-conductor NMs which under UVA promoted photo-catalysis of the organic compounds. Toxicity of adsorbed contaminants in aquatic organisms strongly related to sorption efficiency and while copper sorption on NMs showed a protective mechanism reducing copper bioavailability in larval zebrafish and C. vulgaris, the NMs under UVA can catalyse organic compounds to toxic photo-by-products and obtained in this study indicate that benzo(a)pyrene photo-by-product bioavailability was increased in the presence of NMs. This study also indicated that controlled conditions of NM exposure in the aqueous phase lead to consistent results and higher toxicity of NM in zebrafish larvae indicating that NM toxicity depends on NM aqueous dispersion. Ag nano-prisms and CeO2 NMs showed highest toxicity in larval zebrafish and C. vulgaris compared to other FNN NMs used. Regarding possible modified assay conditions for improved exposure, the designed exposure chamber can provide the start for a standardised methodology that would limit variation in results among laboratories and will enable to critically compare data and conduct correct risk assessment. It would be interesting to further investigate the physicochemistry of NM during the controlled conditions of the fish larvae toxicity test using the exposure chamber, hence link toxicity with NM physicochemistry.
The perovskite studies indicate that lead dissolution is responsible for lead-halide perovskite NM toxicity observed in all studies, using different test models. The perovskite NMs dissolved lead in the aqueous phase and via dietary exposure in zebrafish inducing a metal bioavailability specific biomarker. This study provided an example of indirect effects of NMs in the aquatic environment as well as possible routes of exposure to perovskite NMs. Finally, bioavailability and gene expression have proven sensitive and environmentally relevant analytical tools for investigation of NMs toxicity in the aqueous phase. Sorption of co-contaminants onto NMs was successfully identified and lead-halide perovskite NMs toxicity was attributed to lead ion dissolution.
The hazard studies conducted with the Microtox assay in a miniaturized 96-well format, using synthetic media containing 2% NaCl, revealed that NMs constituted of the same elements may have different toxicity. This difference in their toxicity was mainly correlated to their stability, and, for some of them a higher ion release in the media. An additional valuable information was that the shape of the NMs affect their toxicity. Furthermore the great number of NMs that were tested during this project by using this Microtox assay suggest that this test, which is already used for chemical toxicity testing, seems to be a good tool for fast screening of NMs toxicity in saline conditions (water with 2% of NaCl).
Chronic studies with Daphnia magna were compared to the results of short term acute studies. Results indicate that during acute tests the smaller NMs showed highest toxicity whereas chronic tests showed higher toxicity on exposures to larger NMs. This indicates the importance of test in defining endpoints.
The different NMs were analysed using nanomaterial tracking analysis (NTA) in different environmental media (MilliQ water; synthetic Daphnia medium or NaCl 2%) immediately after resuspension or after 7 days of incubation at room temperature. Most of the NMs studied had the tendency to aggregate. It was observed that NMs in deionised water (very low IS) were much less aggregated compared to NMs in synthetic Daphnia medium or NaCl 2%. The present results show that various cations and anions present in synthetic medium had strong impact on the NM stability and to aggregation/agglomeration.
The processes of aggregation and agglomeration significantly influence the fate and behaviour of NMs in the environment, with an impact on particle properties (e.g. size, chemical composition, surface charge) as well as environmental conditions (e.g. mixing rates, pH, and natural organic matter). The present results show that the IS affected the particle sizes of the NMs and led to higher aggregation/agglomeration of the NMs.
Work Package 8: Key elements of dissemination, potential changes to risk assessment approach for future nanomaterials
Task 8.1 was addressed by facilitating the content and format evolution of the CODATA Uniform De3scription System for Materials on the Nanoscale www.codata.org/nanomaterials (UDS) into a form that can be used for developing categorization schemes for nanomaterials in a variety of contexts. Specifically, the UDS provides a systematic and cohesive methodology for describing and identifying engineered and natural nanomaterials. The UDS allows data repositories, databases, and research publications to integrate disparate measurement results, such that categorization schemes and read-across techniques can be done on a scientifically sound foundation. What UDS does, in short, is provide an effective technology ensuring that referenced ENMs are correctly described and identified.
Furthermore, RIVM obtained significant results within Tasks 8.2 and 8.3 during the reporting period in relation to the development of the constituents of the tools, either of those that have already been established or of those that are still being set in place for the safe innovation using novel generations of ENMs. These tools are to be integrated in a systemic approach towards reducing uncertainties in risk assessment, and include:
•Predictive modelling of the fate of ENMs and their toxicity based on NP properties (e.g. size, shape, composition), as developed within FNN for both in vitro and in vivo endpoints;
•Operationalization of grouping and read-across approaches to facilitate safety assessment while avoiding extensive experimental testing;
•Integration of in vitro and in vivo approaches for safety assessment
The tool integration in the general assessment of ENMs’ safety involves four tasks in a multidisciplinary systemic approach, as follows:
1)Systemic plan for ENMs production, analysis and classification;
2)Study of ENM evolution in the environments to which they are exposed;
3)Study of ENMs biological effects and complex biological responses in both healthy and compromised states;
4)Understanding the fate, transformation and effects of ENMs in relevant environmental scenarios.
Subsequently, a model infrastructure that assists industry (especially SMEs) in systematically addressing safety in innovation was set in place. It proved to be a suitable model for implementing scientific findings in innovation that takes into account safety considerations from the early stages of product development.
TNO provided input for WP8, especially for Tasks 8.2 8.4 and 8.5 regarding cross-cutting themes between WP3 and WP8, considering an overarching program that specifically takes into account nanosafety investment actively promoting innovation. Two distinct types of activities were used in WP3 to provide the information relevant for WP8: i) release, exposure and risk assessment of ENMs and ii) road-mapping of the value chain. The crucial link between different WPs in FNN is represented by the interplay between ENM release, exposure and hazard, and this point was emphasized, as it determines occupational and environmental risk. TNO highlighted the importance the safe-by-design (SbD) approaches and risk management measures for the control of particulate matter emission and proposed a link with Nanoreg2 for the Safe Innovation Approach. Furthermore, the Stage Gate Idea-to-Launch model was suggested as an appropriate basis for adopting SbD activities at various stages of the innovation process.
A close connection with industry representatives was established within WP8 by TUL, for example with remediation companies (Aquatest AS, Aecom CZ and Microchem), membrane producers (KST Membrade) and façade companies (Progotherm). These collaborations are ongoing and TUL’s activity within the Advanced Materials Industrial Association (AMIA) platform facilitated the exchange of information and ideas on approaches for safe nanotechnology between industry and researchers.
Within D8.3 TUL described the influence that environmental media and endotoxins present in ENM samples have on risk assessment. Further, TUL took part in D8.5 by describing a green synthesis of Au NPs using natural polymers and by subsequently studying the characteristics and biological effects of these NPs.
The effects of graphene oxide prepared at UCD in collaboration with FIAT, were investigated by TUL, as the material is highly relevant for value chain 4, Transportation/Industry. Using the soil bacterium Pseudomonas putida and nitrifying bacteria for wastewater treatment plant activated sludge as test systems, results that can help the translation to market of a novel nano-lubricant were obtained and included in Deliverable 8.6. They provide specific information for the risk assessment and regulation of a novel ENM. Furthermore, studies on two perovskite materials obtained from EPFL – FALI and MAFA(67PC)LI – relevant for value chain 1, Energy harvesting, were performed and the results, which facilitate the potential translation to market of new perovskite ENMs, were also included in D8.6.
Although SMEs are extensively involved in research of nanotech-based products, steps in commercializing have high uncertainty, risk of delay or even low returns during the initial phase, therefore large investments by investors and multinational companies are required. An accurate risk assessment of nanotech-based products may reduce uncertainties and risk margins, which in turn may save time and resources. By addressing safety before marketing along the innovation chain, risk assessment may attract investor's interest and funds, thus ensuring good returns to SMEs and industries. The partners aimed to address these issues in Task 8.4. Silver nanoparticles (Ag NPs) are some of the most widely used ENMs in commercial products. A key step in the risk assessment of Ag NPs is their physicochemical characterization. Among others, parameters like size, size distribution, agglomeration or aggregation status of colloidal silver may provide useful indications on either worker or consumer exposure. FILARETE investigated a reliable protocol to assess the colloidal stability of Ag NPs of different sizes and coatings in two biological media, to correlate the Ag NPs physicochemical properties with their in vitro/in vivo behaviour and support the comprehension of Ag NPs toxicological data. Noteworthy, the Ag NPs characterization reported in this protocol require a standard analytical instrument (UV-vis spectrophotometer) that is widely used in industry and is expected to provide useful insights to evaluate current and potential impact of Ag NPs in the industrial landscape, as well as to identify the need for specific actions in the (risk) management of these nanomaterials.
Within Tasks 8.2 and 8.4 CEA explored and evaluated alternatives to current exposure metrics under the frame of the OECD general tiered approach for exposure assessment. The possible parameters to be used in the exposure assessment scheme were defined, along with a list of available methods, tools and devices for their measurement. Furthermore, within Task 8.5 innovative methods and devices that could overcome some of the current difficulties of field measurement were developed. Specifically, CEA established and evaluated the concept of a novel, easy-to-calibrate and transportable device that could provide a size distribution in real time, as well as the chemical composition for each size bin off-line. Several prototypes were designed, built and evaluated using both simulation tools (Finite Elements Analysis - COMSOL Multiphysics) and experimental approaches with controlled aerosols in a laboratory setting. The devices were iteratively improved and are currently reaching TRL 4. Two patent applications filled in July 2015 were extended in 2017. The innovation is to sort and collect charged particles in specific places of a circular substrate based on their electrical mobility. The concept developed through this task allows correlating size and chemical composition of airborne particles and it holds the promise of facilitating the discrimination of secondary sources of airborne particles in occupational settings.
Several field measurements campaigns along with Tier 1, Tier 2 and Tier 3 evaluations were conducted during the project to generate new information on the operative conditions and levels of exposure for several case studies on next-generation nanomaterials:
•Production of nano-enabled electrodes for Li-ion batteries
•End-of-life of nano-enabled electrodes for Li-ion batteries
•Production of thermoelectric generators based on nanostructured silicides
•Additive manufacturing
These campaigns allowed identifying experimental challenges related to emissions measurement in occupational settings and contributed to confirm that research on the development of personal devices was required. Such tools could be particularly useful for Tier 2 evaluation in SMEs and small-scale production lines.
The best practices identified throughout the technical activities conducted during the project were regrouped in a manual along with generic guidelines for ensuring exposure-driven safety of the next generation nanomaterials being developed for industrial applications. This manual will allow workplace hygienist, workers and executives to make appropriate decisions to tackle efficiently risks associated with ENMs.
Work Package 9: Liaison with NMP projects, national and other international projects
The activities within RIVM focussed in particular on outreach of the results obtained within WPs 6, 7, and 8. RIVM contributed to three smaller workshops: one on classification of the next generation of nanomaterials (Tasks 9.1 and 9.2) one workshop organized within the 2017 SETAC meeting in Brussels, and a small workshop organized within the EuroNanoforum meeting that was organized in Malta in 2017. The workshop organized within the broader SETAC annual meeting focussed on dissemination of the key results obtained within WP7, whereas the workshop organized during the EuroNanoforum focussed on options for categorisation of future generations of nanomaterials. The latter workshop also served as the preparatory workshop for the bigger workshop on categorisation, organised in November 2017 in Brussels. Apart from these activities, project results were disseminated by means of nine publications in peer-reviewed scientific journals, one book chapter, 11 oral presentations during scientific conferences, two presentations during regulatory meetings, and three poster presentations during scientific conferences.
The major scientific result for CODATA partner was progress on developing a consensus on categorization of next generation nanomaterials. Through two workshops that brought together international experts on this subject, FNN fostered a more detailed level of discussion of the need for, contexts of, and approaches to categorization.
Potential Impact:
Work Package 3: Roadmapping of value chain materials platforms, and consequently their exposure scenarios
WP3 outcomes will help to identify hotspot areas of concern, facilitate measurement strategies and address improvement of working conditions. As mentioned by IUTA, TNO and various other partners, the project had a substantial impact within the scientific community as is shown by various contributions to scientific conferences. All partners were able to maintain and further develop its scientific base with regard to release and exposure assessment. Even though no directly exploitable results are obtained, the knowledge gained, especially for the mass flow analysis, leads to further reputation possibly resulting in new scientific or industrial projects. In view of nanosafety the work conducted leads to a better understanding of release hot spots, not only for novel next generation nanomaterials, and possible countermeasures, thus contributing to the safety-by-design principle. Different concepts for release assessment were integrated into a coherent approach and not only occupational hygienists working in the field of nanomaterials can make use of the concepts conceived. The overview of methods and devices with regard to exposure assessment of nanomaterials is a valuable tool to decide on the best choice for an appropriate and cost-efficient measurement approach to assess release of and exposure to (next-generation) nanomaterials.
The work conducted during the project was to be beneficial in FutureNanoNeeds but more importantly beyond, as for instance in other EU projects, to the scientific community, regulators and stakeholders. Information on the methods, devices, tools, and exposure assessment strategies, that were developed, used and evaluated throughout the project, will allow regulators and industry to make appropriate choices to implement efficiently harmonized approaches for specific exposure situations to next generation nanomaterials. Manual of best practice will be directly applicable in laboratories and in industry to promote a safer, sustainable and responsible development of nanotechnology.
For instance, the technical activities conducted, and the experience gathered on the thermoelectric generators (TEG) value chain will be directly applicable in the EU pilot project INTEGRAL which started in January 2017. It’s ambition to scale-up safely the European production of TEG modules through partnerships between public research institutes, industrial research teams and SMEs can be aided by our work.
The results and deliverables produced on WP3 are expected to be useful for NANOGAP in terms of comparing data for similar products and for the better design of new nanomaterials that will develop in the middle and long term. This will increase products quality and could be useful in terms of commercialization. On the other hand, the implication on the VCAC allowed us to obtain useful commercial information from the different value chains and the relative position of VC7 (AgNW, which we are producers) on the market. Consequently, all the information related to best practices (D3.11) and hot spots for the VC7 (D3.4) will improve our practices handling and producing nanomaterials, what will reduce environmental and workers exposure. Future spin-offs are anticipated for work that was performed within WP3, in particular in areas where there is an interest in approaches that address data poor scenarios. For example, possibilities to test the BBN shredding model and to subject the model to a more extensive expert elicitation and refinement. Further development of the thermo-electric VC in EU Integral pilot project has already been initiated.
Short-term impacts would be the use of best practices in laboratory, industry, and spin-offs for further development (e.g. refinement of MFA and BBN models). Long-term impacts could be the further development of measurement devices and their introduction in practice, and eventual implementation of matured methods and models. And further in the future, the harmonization to close the gap between release/exposure and hazard potential of NMs (based on outcomes of other FNN WPs) would be advantageous. Further research is therefore required to map and predict changes in particle morphology for different types of NMs and different release and exposure scenarios (in particular for end-of-life, mechanical recycling)
Overall, FNN provided a platform for WP3 partners to explore new innovative ideas on next generation NMs, e.g. release of NMs. It provided an opportunity to collaborate & exchange expertise between partners, but also with other EU projects (NanoReg, BUONAPART-E, etc).
Work Package 4: Synthesis and exhaustive characterization of materials of the future
The work done enables a high potential for scientific and technical innovation. This applies to the development of new production methods of new nanomaterials, such as complex hybrid NCs, NCs with doping, and shaped nanostructures. The developed nanomaterials will enable innovative and environmentally sound products.
The scientific and economic results generate a base of knowledge, which means that the WP4 partners strengthens their national and international role in the field of nanomaterial production, characterization, metrology, and assessment. As well as in the field of the transfer of functionalized materials in industrially processable products.
Within the framework of this project, innovative and future applications were investigated. These can go into series production in the subsequent period and have a high market potential. The pursuit of environmentally friendly and consumer-safe products is the goal here. The development of competence in this project expands the capabilities for risk assessment of nanomaterials, thus protecting consumers and securing jobs for many partners. The development of new technologies and methods has great potential for the industry.
Work Package 5: Biological and environmental interactions of future nanomaterials
The main impact emerging from this series of studies is the standardization of an integrated platform of preclinical imaging combining both in vivo (two-photon confocal microscopy, optical imaging) and ex vivo (confocal and super-resolution microscopy) approaches. This platform can be easily extended to other materials, if associated with fluorescent dyes, and other models of study. This could be extremely important to repeat other studies, even more related to the evaluation of the fate of NPs modifying parameters such as the geometry and/or topology of the components.
The overall effect of these studies in this work package are represented in novel nanomaterial’s interactions with biomolecules which has paramount importance. As we have shown in this work package, size and shape of nanomaterials can be critical parameters for such interactions, thus driving variety of biological identities for these nanomaterials. Our prospective is that the protocols and the structure-property relationship built in this work package will pave the way for better designed materials with established characterization methodologies.
Work Package 6: Biological identity, processes and human health hazards of future nanomaterials
Nanotechnologies are viewed as being the driving force behind a new industrial revolution which is expected to have profound socio-economic effects. Engineered NMs are the main players in this growth, and therefore they offer tremendous opportunities for industrial development in many different areas. However, any new technology could mean a risk to human health, so knowledge of its potential impact is a priority. Understanding the safety of NMs is therefore an issue for future nano-based products and a means to achieve greater public acceptance of nanotechnology and awareness of the overall benefits that it can bring. In accordance with the present project, this WP has been mainly focused on the development of an in vitro/in vivo platform for ranking the toxicological hazard associated with nanotechnology products. In designing and implementing our approach different classes of NMs were compared, in particular we paid a great attention on the potential influence of basic physico-chemical parameters (e.g. size, shape, surface, curvature) characterizing NPs. The end-points of the various screening tests applied were not only limited to macroscopic toxic effects such as cell death or alteration of morpho-functional features, but also to more subtle perturbation of the cell homeostasis and activation toward a “reactive” phenotype. This is a great point of innovation and could be extremely important for future studies aimed at predicting the first “prognostic” markers upon a low but chronic NM exposure (e.g. food, workplaces, pollution). Since this platform has been developed to the identification of highly reproducible and reliable hazard factors, useful for the future categorization of a wide range of industrial, biomedical or environmental NPs, the presence of industrial partners and regulatory bodies in the Consortium was instrumental to critically evaluate the data produced and drive to a more responsible and coherent control exploiting this integrated approach. We therefore strongly believe that this aspect will be particularly important to determine the relative hazard of newly-synthesized NMs before their large-scale production and market distribution. Another important impact raised fromm the platform generated within this WP, is the achievement of a deeper insight about nanomaterials-biomolecule interactions, with once again a great attention on the relevance of geometry and surface (coating). This aspect may be of extraordinary relevance for the prediction of the impact of NMs for both medical and toxicological purposes. The association between the exposure to different types of Nanomaterial with the increased incidence of alteration on the different biological targets (fluids, cells, animals) provided through this WP may give an interesting and original contribution towards the discovery of new diagnostic hallmarks and or risk/factors associated with NPs and also support the development of even more faster and cheaper and reliable methods of screening of new nanodrugs.
To briefly recapitulate the main impact emerging from this WP we can say that:
1) In general, the different tested NMs did not show acute toxicity. In a few cases, some acute toxicity was observed, but this can be easily explained on the base of the release of ions or oxidative burst (e.g. perovskites or CdSe). The main conclusion is that there are no big reasons to consider NMs able to induce acute toxicity when they are stable and not releasing ions. This is crucial to reduce the threshold of perplexity of citizens toward these materials and to create a greater feeling on this field.
2) Regarding chronic toxicity, some nanomaterials have shown the potential to induce inflammasome activation. Health effects of chronic exposure to nanomaterials that can be related to their inflammasome activating properties are as yet largely unknown, although using nanomaterial libraries a correlation between induction of inflammasome activation in vitro and lung fibrosis in vivo was in fact shown. Inflammasome activation as such has been related to various chronic diseases, albeit with different degrees of certainty. Our proposal would be to include inflammasome activation as one of the assays in the Immunotoxicity element of the flow chart as proposed in the nanospecific approach for risk assessment that has been developed within the EU NANoREG project. Moreover, we proposed to include this assay in the safety evaluation of nanomedicinal products. This would be crucial to give a deeper and broader view of the effects of NMs and to furthermore restrict the possibility to produce and diffuse at the large scale dangerous materials containing NPs.
3) The observed shape- and doping related effects can be used within a Safe-by-Design/Safe Innovation approach as exemplified recently by us for graphene. Interestingly, their geometries can greatly influence their sub-lethal effect on critical biological processes, which may contribute to disease development. However, in the real world the effects played by nanoparticles almost always occur in the presence of bio-active compounds, such as biological immunogens, chemical allergens or surfactants, which may undergo a complex interplay with the nanoparticles in their environment, and thus give rise to unexpected toxicity mechanisms. For this reason our integrated platform could be a great resource even for regulatory items in terms of nanosafety.
4) Finally, the approach developed in this platform is not exclusively limited to be exploited for industrial and biomedical aims but could be a strong added-value in many academic or industrial projects, including some of the next H2020 NMBP.
Work Package 7: Exposure and environmental interactions of future nanomaterials
The large numbers and diversity of next generation NMs preclude having comprehensive studies carried out on the environmental data and effects of all emerging NMs. Therefore, scientifically sound methods and approaches, as the ones developed under Tasks 7.2 and 7.4 will enable researchers, industry and government agencies to assess the potential for hazard (and therefore, risk) more efficiently and effectively. Furthermore, studies undertaken under Tasks 7.1 and 7.3 allow an improved understanding of modifications novel NM may undergo and therefore will facilitate interpretation of exposure conditions.
The key results from WP7 were primarily disseminated by means of scientific publications, presentations at scientific meetings, and meetings with regulatory authorities (OECD, ECHA), industry and SMEs. It was thus warranted that the key results are directly implemented in for instance the design of novel classes of NMs. In this case this implementation of key project finings dealt with the safety assessment of biobased NMs consisting of nanocellulose (impact of size and shape on ecotoxicity of nanocellulose), and the safety assessment of novel types of metallic-based NMs used in photovoltaic cells, including nanowires consisting of In/Ga/P. In both cases, SMEs were assisted in the development of the novel materials indicated, this inducing a direct socio-economic impact. Thereupon, the project results are already included in nano-specific guidance related to future regulation of NMs. This includes the development of draft OECD guidance documents on fate and effect assessment of NMs, and nano-specific guidance on regulatory assessment of NMs within REACH.
In summary, the project had a relevant impact within the scientific community as is shown by various contributions to scientific conferences. Furthermore, the collaboration with international partners was strengthened.
Work Package 8: Key elements of dissemination, potential changes to risk assessment approach for future nanomaterials
The large numbers and diversity of next generation nanomaterials preclude having comprehensive measurements done on all emerging nanomaterials. Consequently, scientifically sound categorization methods, as the ones developed under Task 8.1 will enable researchers, industry and government agencies to assess the potential for risk more efficiently and effectively.
The key results from WP8 were primarily disseminated by means of scientific publications, presentations at scientific meetings, and meetings with regulatory authorities (OECD, ECHA), industry and SMEs. Furthermore, two patent applications were filed, and other peer-reviewed papers are in the process of being published. By interacting with and making our work and results known to different stakeholders, it was warranted that the key technical results are directly implemented in, for instance, the design of novel classes of ENMs and in safe product innovation. RIVM applied this approach in a case study that focussed on safety assessment of newly developed biobased (cellulose) nanomaterials by SMEs that worked on the development of new applications for nanocellulose.
Furthermore, the technical activities conducted by CEA and the experience gathered on the thermoelectric generators (TEG) value chain will be directly applicable in the EU pilot project INTEGRAL that started in January 2017. This project aims at safely scaling-up the European production of TEG modules through partnerships between public research institutes, industrial research teams and SMEs.
The work conducted during the project was to be beneficial in FutureNanoNeeds but, more importantly beyond, for instance in other EU projects, to the scientific community at large, to regulators and industrial stakeholders. Information on the methods, devices, tools, and exposure assessment strategies, that were developed, used and evaluated throughout the project, will allow regulators and industry to make appropriate choices to implement efficiently harmonized approaches for specific exposure situations to next generation nanomaterials. The manual of best practice will be directly applicable in laboratories and in industry to promote a safer, sustainable and responsible development of nanotechnology. The main beneficiaries span industry (cosmetics, food, biocidal products, medical devices, pharmaceuticals), waste management bodies, authorities who regulate occupational exposure to ENMs, the workers affected by said regulations, as well as the environment at large.
Work Package 9: Liaison with NMP projects, national and other international projects
The main dissemination activities within RIVM focussed on outreach of the results obtained within WPs 6, 7, and 8. RIVM contributed to three smaller workshops: one on classification of the next generation of nanomaterials (Tasks 9.1 and 9.2) one workshop organized within the 2017 SETAC meeting in Brussels, and a small workshop organized within the EuroNanoforum meeting that was organized in Malta in 2017. The workshop organized within the broader SETAC annual meeting focussed on dissemination of the key results obtained within WP7, whereas the workshop organized during the EuroNanoforum focussed on options for categorisation of future generations of nanomaterials. The latter workshop also served as the preparatory workshop for the bigger workshop on categorisation, organised in November 2017 in Brussels. Apart from these activities, project results were disseminated by means of nine publications in peer-reviewed scientific journals, one book chapter, 11 oral presentations during scientific conferences, two presentations during regulatory meetings, and three poster presentations during scientific conferences, and a stand at Nanotexnology 2017.
The large numbers and diversity of next generation nanomaterials preclude having comprehensive measurements done on all emerging nanomaterials. Consequently, scientifically sound categorization methods (CODATA and NIUD UCD) will enable researchers, industry and government agencies to assess the potential for risk more efficiently and effectively.
List of Websites:
https://www.futurenanoneeds.eu/
Contact: admin@cbni.ucd.ie
Tel: +35317162416
FutureNanoNeeds was designed to develop a novel framework to enable the naming, classification, hazard, and environmental impact assessment of the next generation nanomaterials (NM’s) prior to their widespread industrial use. The goal was to develop a suite of integrated tools and concepts which will form the basis of a “value chain” regulatory process which allows each new nanomaterial to be assessed for different applications on the basis of available data and the specific exposure and life cycle concerns for that application. The project has succeeded in doing so.
Roadmaps for future nanomaterial value chains were developed by promoting multi-stakeholder dialogues between researchers from industry, SME’s and other advisors. Eight value chains (VCs) were identified and prioritised by a Value Chain Advisory Committee (VCAC) and subjected to a detailed analysis to determine value chains with a high social and economic impact in the medium to long term, and with a focus on nanomaterials which have a significant research interest and/or synthesis and manufacturing capacities. Policy recommendations for future European cofounded innovation and research actions, or coordination and support actions, were released and validated through an online survey open to external stakeholders. A framework was developed to forecast the potential release of the selected VCs during different life cycle stages. For this purpose, an iterative tiered approach was developed by using mass flow analysis and Bayesian Belief Network methodologies to predict NM release, in each instance using case studies of different VCs. These methods considered the associated data gaps and uncertainty to account for data rich/poor scenarios. An important outcome was that these methods identified the End-of-Life (EoL) mechanical recycling processes as important hotspots in the future. Several measurement studies were conducted to identify and characterize next-generation nanomaterials in the workplace setting. To keep pace with the development of novel structures and functionalities, two new innovative, easy-to-calibrate and transportable devices were developed. In addition, a literature review was conducted on new concepts of exposure metrics and their applicability through the OECD (2015) measurement strategy. Based on real-world case studies, a practical manual on best practice for safe nanotechnology action plan was completed.
Responding to the VCAC, the consortium developed and produced a variety of homologous series, each one varying in a controlled manner to explore the impact of structural details on biology. It has involved the synthesis of nanocrystals with radically different shapes, geometries, compositions, and surface treatments. Special attention has been paid to the purity, monodispersity, and shape control (production of different geometries of the same chemical substance) to avoid confounding the biological outcomes with different active species in the same sample. Each particle type was made by at least two different laboratories. Nanocrystals sizes ranged from few atoms to hundreds of nanometres, and they presented with different dimensionalities (0D, 1D, 2D, 2D and intermediate dimensions as stars (2.5 D). Attention was paid to the control of synthetic environments and processes to obtain ‘reproducible’ particles, monodisperse in size and shape, free from chemical and biological (endotoxin) contamination. Remarkably, internal and small informal round robins and quality assurance checks were made to provide a complete audit of the processes underpinning quality and material supply. The success in these processes was considered a milestone of the program. Samples produced were analysed with state-of-the art characterization techniques. Beyond synthesis, partners carefully studied and analysed the evolution of nanocrystals during time and storage conditions, and in different working media. Nanoconstructs were distributed across the FutureNanoNeeds consortium for subsequent biological eco-tox evaluation. Significant effort has been spent in the proper labelling of nanoparticles as appropriate for biological studies.
The project carried out a series of experiments on different types of polymer nanoparticles (NPs) and studied their bioavailability and targeting. PCL3 NPs are deemed an interesting tool for the development of nanodrugs or to predict the fate of NPs in organs and cells. Studies were carried out on the dispersion states of NMs in biofluids and found that in general the materials were stable. The project also studied the effect of the protein corona on the stability of NMs. In situ and in-line monitoring of the fate of the particles has been studied and the results show NP stability in biofluids containing proteins only at certain concentrations. The physical size and shape of NMs was found to influence the orientation of protein adsorption. Finally, the degradation of NMs in a variety of environmental fluids has been studied.
Intentional (drugs, foods, cosmetics) and unintentional (pollution, workplaces) exposure to NMs has been enormously increased over the last decades. The perception of risk is controversial and the correlation between the main physico-chemical parameters characterizing NMs and their effect on cells and organs is still far from being elucidated. To answer to this pressing need, an innovative nanosafety platform was built within the project. Three main biological matrices with an increasing level of complexity (biological fluids, cells, healthy rodents) were considered to achieve reliable and transferable results for human health. Briefly, we confirmed that independent to the material, other parameters such as size, shape, and surface, can strongly influence the biological response. Overall, the results from this project provide useful suggestions to drive the production of safer industrial NMs and also to predict the potential impact of nano-pollutants. The approach could help develop new way of considering and critically evaluating the risks emerging from nanotechnology, in order to create a more responsible and easily transferable strategy of validation.
The project, in its investigation of environmental impact, found that the nanoparticle dispersion state in the aqueous phase was related to nanoparticle toxicity. Lead ion release was found to be the cause of toxicity of perovskite nanomaterials in adult zebrafish through a dietary exposure. Organ uptake and internal distribution should be monitored more closely to provide more in depth information of the toxicity of particles. In soil, the low mobility of perovskite nanomaterials may lead to an accumulation in the upper soil layers. The consortium found that toxicity study exposure must be expressed on the basis of time weighted average particle concentrations. This must explicitly take particle dissolution as well as sedimentation into account. Particle shape and size were found to be the key properties affecting the toxicity of nanoparticles. The coating of nanoparticles may determine toxicity, while the toxicity of metal nanoparticles may be via a ‘trojan horse effect’ whereby dissolution is achieved following nanoparticle-organism interaction or nanoparticle internalisation.
Durable links to other key EU projects, stakeholders, and regulatory bodies, were created during the course of FutureNanoNeeds, ensuring the communication and sharing of new understanding and findings beyond the project end. The project had significant success in the organization of several workshops to disseminate key results and open discussions on topics such as the standardization, classification, and categorization for present and future nanomaterials. In particular, the Conference on the Categorization of Next Generation Nanomaterials in Brussels, was successfully organized in 2017, preceded by several satellite meetings and the involvement of worldwide experts.
Project Context and Objectives:
Work Package 2 Scientific Coordination
The main objectives of Work Package 2 were the coordination and leadership of the overall program and the in-depth management and administration of workflows, and the administration of materials flow and QA. Throughout the project, regular communication between the work packages was facilitated through teleconferences, face to face meetings, emails, phone calls, and the FNN website (https://www.futurenanoneeds.eu). This work package was responsible for the initial round-robin process, compiling and updating SOP’s, and compiling the annual, periodic, and final reports, gathering and submitting deliverables, organising reviews etc.
In terms of materials flow, teleconferences and discussions were held between relevant partners to make sure the flow of materials in the project was effective, including the implementation of a spreadsheet to help predict Nanoparticle requirements (type and quantity) in the medium term. While there was concern regarding the quantities of nanoparticles available for some experiments, through effective communication and cooperation between partners, this was resolved in the final half of the project allowing the research to progress to a successful conclusion.
Work Package 3: Roadmapping of value chain materials platforms, and consequently their exposure scenarios
Work in WP3 contributes to the development of a novel framework to enable naming, classification, hazard and environmental impact assessment of the next generation nanomaterials prior to their widespread industrial use. To achieve this, new and existing concepts have to be integrated to produce a robust, versatile and an adaptable framework and methods to forecast release of next generation NMs and to ensure its safe use. Different life cycle stages and the characterization of NM release in each instance are important issues to consider for this purpose. The scope within WP3 includes occupational and environmental exposures as a starting point, although the focus for measurement studies and the best practices action plan relates to occupational exposure.
In terms of objectives, the central objective that relates to WP3 and all other WPs within the FutureNanoNeeds projects was to develop roadmaps for future nanomaterial value chains by promoting multi-stakeholder dialogues between
researchers from industry, SME’s, and other advisors. A Value Chain Advisory Committee (VCAC) was appointed to maintain, develop and update those decisions made within the VCAC, and to translate them into action, and therefore encourages an intensive engagement involving many specialists from different stakeholders, advisors, extending from industry to innovators and venture capitalists and others. The main objective was to prioritise value chains for FutureNanoNeeds within the Value Chain Advisory Committee (VCAC), and to analyse them in detail for range of nanomaterials likely to be used, thereby proposing homologous series of representative nanomaterials that exemplify the value chain chosen.
Another crucial objective relevant for the entire work package was to develop a framework that allows for the forecasting of potential release during production and other stages of these chosen next-generation nanomaterials/product value chain. For this purpose Bayesian networks are proposed to model complex systems with many related variables, while considering associated uncertainties and to model complex systems with many related variables. It was of importance to keep pace with development of novel structures and functionalities by exploring concepts of new methods
and techniques to monitor release or exposure. Also, the identification of potential for (occupational) exposure to the next generation of nanomaterials and development of (innovative) methods/tools to identify and control possible exposure in the workplace and in ambient air along the value chain should be considered. And lastly, the development of a manual on best practice for safe nanotechnology action plan should ensure that control measures are implemented and tested to ensure optimal performance.
Work Package 4: Synthesis and exhaustive characterization of materials of the future
Responding to the Value Chain Advisory Committee (VCAC) the main activity of WP4 has been the development and production of a variety of nanoparticle homologous series corresponding to value chains (see table). Taken as a basic starting point the available nanomaterials structures routinely made in the laboratories of the Partners in WP4 and considering the inputs from VCAC, series of NCs were developed. In detail, high quality homologous series of NCs have been prepared, each series varying selected parameters in a controlled manner to explore the impact of structural detail on biological and other outcomes. It has involved the synthesis of different series with radically different shapes, geometries, compositions, and surface treatments. A full list is available in the attached report.
Within WP4, Task 4.1 focused on the synthesis of novel geometries and shapes of nanomaterials, particularly those structures plus variants around them that allowed the study of biological and environmental interactions with a wide variety of different geometries. A variety of techniques were applied allowing the kinetic control of specific crystal planes to make different geometries of the same chemical substance. Task 4.2 was intended to explore variations going far beyond those envisaged in Task 4.1. In this regard, hybrid nanoparticles composed of a combination of different materials in core-shell, hollow or heteromer-like configurations were produced. These new hybrids represented a powerful strategy for modifying nanoparticle properties, combined the properties of the individual components, or even exhibit novel and unique properties that originate from collective interactions between constituents. Task 4.3 evolved the nanomaterials formed in Task 4.1 by (post-) modifying their surface, particularly with different charge and hydrophilicity/hydrophobicity. Samples were characterized both dry (where appropriate) and dispersed in various simple media within Task 4.4. The most basic characterization of particles involved (average) size/dimensions, ‘charge’ (averaged mobility) and surface area. In some cases new instrumental developments (or evolution of existing ones) were required to meaningfully characterize these novel shapes and geometries.
During the lifetime of the project special attention has been paid to the control of synthetic environments and processes to obtain ‘reproducible’ particles, monodisperse in size and shape control, free from chemical and biological (endotoxin) contamination to avoid confounding the biological outcomes with different shapes in the same sample. Each particle type was made by at least two different laboratories, in order to provide a comparative study of the synthetic processes and expecting to provide a significant achievement in the refinement of synthetic methodology of shape control synthesis. Remarkably, internal and small informal round robins and quality assurance checks were made throughout the lifetime of WP4 activity aiming to provide a complete audit of the processes underpinning quality and materials supply. The success in these process was considered a milestone of the program. Samples produced were analysed with state-of-the art characterization using established methods, and introduction of novel approaches appropriate for new material.
Beyond synthesis, WP4 partners carefully studied and analysed the evolution of the physicochemical properties along the time and storage conditions and/or in different media.
NPs were distributed across the FutureNanoNeeds consortium for subsequent biological (WP5, WP6) and eco-tox (WP7) evaluation. Full list of samples prepared and delivered can be found in D4.5 D4.6 D4.7 D4.8 and D4.9. Significant effort has been spent in the proper labelling of nanoparticles as appropriate for biological studies.
Work Package 5: Biological and environmental interactions of future nanomaterials
In this workpackage we successfully created high quality, characterized (and realistic) dispersions of the nanoparticles decided by the VCAC in different biological media, as appropriate for WP6, We also investigated and created examples of high quality, characterized (and realistic) dispersions of nanomaterials in different environmental media. To make sure these scenarios were realistic we applied a balanced portfolio of strategies (high content screening, uncovering biological pathways and mechanisms, hypothesis driven detailed study, including in vivo). We also advanced the understanding of the state of organization of non-standard-geometry (shape, topology) nanoparticles in biological media both in relation to dispersion, and in relation to the biomolecules adsorbed to their surfaces (‘ biomolecular corona’). This was done by altering the kinetics of internalization and sub-cellular localization. We also modified the activation of the main systems of metabolism and degradation. This work package had multiple objectives (early alert to hazard, understanding of connection between structure and action, future design principles) which we feel we have addressed fully.
Work Package 6: Biological identity, processes and human health hazards of future nanomaterials
The main goal of this WP was to understand how physico-chemical parameters of novel NMs (e.g. shape, size, curvature) could influence their interaction with biological targets at different levels of complexity (biological fluids, cells, animals). Specific hallmarks, such as the protein corona formation, the cell penetration, the kinetics and accumulation in cells and organs, and, finally, the potential triggering of detrimental effects, have been carefully evaluated along the 6 tasks of the WP. We were mainly interested to understand if and how parameters such as shape, size, curvature, surface might influence the interaction with biological fluids. This is relevant for both nanotoxicology and nanomedicine. The interaction with proteins, sugars, lipids and other components may indeed strongly influence the behaviours of NMs once penetrated in the host tissues. To this end a deep analysis with biochemical, bio-molecular and morphological assays was carried out before moving to single cell investigations.
The in vitro studies were the second, and most extensive part of this WP. They concern at least four different tasks, devoted to the determination of the influence of specific physico-chemical features of NMs on different cell types. The comprehension of the potential alterations of morpho-functional parameters (not exclusively associated with death or high toxicity) after exogenous compounds exposure is always important. However, it becomes even more important in nanotoxicological studies, due to the peculiar ability of nanoparticles (NPs) to be efficiently internalized inside the cells through an endocytotic mechanism commonly defined as “Trojan horse effect”. The first part was therefore aimed at measuring the NP uptake and localization inside the cells depending on the dose, the material, the shape, the size, the curvature. Afterwards, a deep investigation of the “fate” of internalized NPs was carried out. To this end we planned a series of experiments matching different approaches, such as EM, fluorescence imaging, ICPMS and cytoViva, to investigate the presence and dynamics of processes of intracellular dissolution, degradation kinetics and exocytosis of different NMs in different cell types. The rate of penetration into the cell and the consequent accumulation, cannot be automatically translated to a toxic effect. In fact, there may be materials that, although penetrate and accumulate inside the cell, are easily isolated from cytoplasmic structures or are chemically inert. The studies of uptake and accumulation, are therefore necessary but not sufficient to define the potential hazard. The following part of the study was therefore devoted to the effect of NPs after internalization. However, we did not exclusively investigated the survival, but also looked for upstream and subtler alterations, such as immunomodulation, inflammation, oxidative stress and other sub-lethal processes. Finally, for a restricted type of materials (gold and TiO2) we evaluated the effect of size and shape on organ biodistribution, accumulation, clearance and potential detrimental effects (inflammation, immune response, histopathology) after single administration in healthy immunocompetent mice. This last part is crucial to understand if the results coming from in vitro studies can be somehow transferred to more complex systems and to complete the development of this integrated platform devoted to nanotoxicology.
Work Package 7: Exposure and environmental interactions of future nanomaterials
Project FutureNanoNeeds (FNN) studied the fate and effects of novel nanomaterials (NMs) along selected value chains in close collaboration with key stakeholders. Within FNN Workpackage 7 (WP7) addressed “Exposure and environmental interactions of future nanomaterials” and its main aim was to study environmental fate and effects of the so-called new generation NMs. This aim was addressed via these key objectives:
1. To translate the general exposure scenarios (WP3) considered most important along selected value chains and life cycle of future NMs into an understanding of the in situ exposure conditions;
2. To investigate the nature of surface adsorbed molecules derived from product formulation modifications to the NMs (e.g. surface modification derived from the embedding matrix of the product) for exemplary value chains as decided by the VCAC (WP4);
3. To characterize the nature and structure of adsorbed molecules, and other surface modifications, derived from the natural aquatic environment (freshwaters), from solids as well as from wastes or effluents (characterisation of for instance the specific interactions with Dissolved Organic Matter, but also the specific interactions with selected contaminants present in the natural medium (like metals, PAHs, etc.);
4. To develop suitable exposure models for environmentally relevant species, and sentinel species, and to investigate the interactions of novel NMs with them.
The above objectives were fulfilled in this WP by research structured around the following tasks:
• Task 7.1 Environmental binding and ecoidentity of future NMs;
• Task 7.2 Development and application of environmentally relevant organism exposure protocols;
• Task 7.3 Material transformation (including degradation) in the environment;
• Task 7.4 Interaction with environmentally relevant species, including ‘sentinel’ species.
Research carried out within WP7 was undertaken by 7 partners, namely, NUID-UCD (P1); IUTA (P5); HWU (P6); LIST (P7); UoB (P8); MNI (P11); RIVM (P12) TUL (P15), with different and complementary expertise and techniques. The practical approach adopted followed the conceptual model presented in Figure 1 of the attached report.
Results from the work undertaken in WP7 was comprehensively reported in the following deliverables:
D7.1 Identification of the composition of the ‘eco-corona’ formed in specific environmental media on next-generation NMs;
D7.2 Report on the propensity of next-generation NMs to provide a ‘Trojan horse’ effect of environmental pollutants;
D7.3 Report on proposed standardized exposure conditions and protocols for environmental species, building upon the general exposure scenarios identified in WP3;
D7.4 Identification of possible ‘hot-spots’, where material transformations are most likely to occur, along several value chain platforms;
D7.5 Material transformations along several value chains and their consequences identified;
D7.6 Report on methods and protocols for measuring NMs interactions with environmental compartments;
D7.7 Preliminary results on NMs interactions with environmental compartments for selected value chain platforms;
D7.8 Report highlighting any novel toxicity or toxicity mechanisms derived from standardized exposure conditions.
Work Package 8: Key elements of dissemination, potential changes to risk assessment approach for future nanomaterials
The FutureNanoNeeds project comes into existence at a time when research, industry and society (through, among others, regulatory bodies and consumers) are beginning to come in contact with what we here have termed next generation nanomaterials. More complex, versatile, engineered nanomaterials (ENMs) are characterized by parameters beyond their size and core composition, such as shape, surface chemistry, biological and environmental identity. These materials hold great promise for innovation, but they also present unique challenges for their safe use and commercialization of the products that incorporate them. The FutureNanoNeeds project aims to address these emerging issues by, among others, creating the framework for a fruitful conversation among all of the stakeholders involved in the research, commercialization, use and regulation of next generation nanomaterials. WP8 focuses on the dissemination aspects related to the many facets of this conversation.
Tasks 8.1 through 8.5 in WP8 were carried out by a unique mixture of partners. They brought into discussion nanosafety and the development of novel nanomaterials in a very broad and inclusive context, aiming for i) the development of classifications systems for the next generations of engineered nanomaterials (Task 8.1) ii) assessing the impacts of uncertainties surrounding nanosafety and facilitating innovation and commercialisation on the basis of safety assessment, with the collaborative efforts of researchers, regulators and industry (Tasks 8.2 8.3 and 8.4) and iii) the development of cost-cutting tools for SMEs in the context of safety in innovation (Task 8.5).
Work Package 9: Liaison with NMP projects, national and other international projects
Nanotechnology and engineered nanomaterials hold the potential to make qualitative improvements in rapidly developing markets such as energy harvesting and storage, construction, advanced materials for electronics and biomedical devices. FutureNanoNeeds project has produced a totally new workflow for the classification of nanomaterials and complex nanomaterials of the future by reading across physical-chemical properties, biological hazard and environmental impact assessment. This complex framework for the next generation nanomaterials will allow to better regulate and therefore facilitate the introduction of engineered nanomaterials for a widespread industrial use.
Two main goals, strictly connected each other have emerged along the project:
• To ensure a smooth benchtop-to-market transition for nanomaterials in future industries by predicting the nature of future nanomaterials and by providing reliable tools to assess potential health and environmental hazards ahead of time.
• To promote a standardized system of classification and regulation across EU and worldwide for nanomaterials.
The data generated along the process will form the basis of a “value chain” regulatory process. The nanomaterials will be assessed for different applications (and for each step of the production value chain) on the basis of FNN findings and the specific exposure scenarios and life cycle concerns for that application. In this context it is of paramount importance to manage the data and the findings emerged from the project, making them accessible and available for the scientific community, the stakeholders and regulators, beyond FNN. In this sense, the role of WP9 has been to collect, summarize and disseminate the main results obtained for the other WPs as well to create the basis for a broader discussion involving key players in the EU and international regulation in the world stage. The main urgency now is to ensure a future to all the project findings and discussion on promoting strategies to reach consensus. This can be done beyond FNN, with the creation of databases, bridging together FNN and other relevant EU projects and the EU regulation organs.
Project Results:
Work Package 3: Roadmapping of value chain materials platforms, and consequently their exposure scenarios
NfA (RINA-C, supported by MBN Nanomaterialia and Prodintec) ensured the integration of the outcomes of a VCAC web meeting on FNN policy recommendations, the link between FNN deliverables and selected value chains, a revision of selected patent and market analyses and finally the integration of the results of an online survey open to external stakeholders aimed at validating key findings of the deliverable. A list of 8 Value Chains centred on key nanomaterials has been identified and analysed though a vigorous discussion by all consortium partners (especially the material researchers in WP4) and by selected external experts. General descriptions of nanomaterials in terms of key properties and of the target applications and final products were provided. Advantages with respect to state-of-the art materials or competing nanomaterials were summarized. Current Technology Readiness Level (TRL) of the value chain was also estimated. Market analysis was described in detail for each value chain, estimating market size, trends, geographical distribution, and segmentation by technology/material and by application and top companies. Patent and literature analyses were provided, aimed to enhance the development phase of the value chains and the innovation potential. A summary of the “hot spots” was also provided for each value chain: these are likely points of exposure to nanomaterials during the life cycle. Finally, policy recommendations for future European cofounded innovation and research actions or coordination and support actions were released and validated through an online survey, open to external stakeholders. The survey showed that for the majority of value chains, the most critical VC step was considered the recycling and disposal step, followed by nanomaterial synthesis.
To provide input for the roadmapping activities, TUL was involved with UCD/ /CRF in a conjoint project: the biological and environmental impact of reduced graphene oxide (rGO) nanomaterials (gemmate s.r.l.) intended to be used as motorcar lubricant. CRF was active in the definition and updating of value chains for relevant nanomaterials and was responsible for keeping up to date the nano-lubricant value chain. That activity was based on the two types of reduced graphene oxide procured by CRF and supplied by Gemmate Technologies. The two materials were thermally and chemically reduced graphene oxides (T-rGO and C-rGO). These results demonstrate the potential of the applications of r-GO as friction modifier in industrial lubricants and enforce the industrial interest in the nano-lubricant value chain. For this reason UCD, TUL and CRF studied the biological and environmental impact r-GO nanomaterials intended to be used as an automotive lubricant. IUTA had been involved into the VCAC actions, for example initiating two major VCs including Li-Ion Battery Technology and the thermoelectric value chain. IUTA played a major role to develop a powerpoint template that provides a brief overview of the value chains and the corresponding results. NANOGAP has provided useful information for the inclusion of one of our products (AgNW) as value chain material, including market and production process information.
A two-tiered iterative approach is proposed to prioritize the focal point and to forecast the potential release and emissions at different levels of detail. The framework assesses material flows along different life cycle stages. The developed Tier 1 approach (an adapted EUSES-based methodology) was expanded to include a more in-depth assessment (Tier 2). The Tier 2 includes two levels to assess release of nanomaterials. The level 1 assessment applies a Material Flow Analysis (MFA) intended for a selected ‘hotspot’ Life Cycle stage. The methodology for the analysis is based on previous work conducted by IUTA within the FP7 Project BUONAPRT-E and was extended to close the mass balance during the possibly different end-of-life steps. Three case studies were worked out to illustrate its application, i.e. (i) Silicon nanoparticles for battery applications (VC2), (ii) Silicon-germanium powder to be used in thermoelectric generators (VC3) and (iii) MoS2 fullerenes used as lubricant additive for automotive applications (VC 4). IUTA received input from various partners incl. CEA and TNO for these cases. The developed simple spreadsheet software allows an easy identification of possible release hotspots and the final compartments in which the nanomaterials end up after the life cycle is closed. It was revealed that currently a lot of work has been conducted and data complied with regard to possible release and exposure during the synthesis, compounding and assembly of nano-enhanced products (occupational settings) but that tremendous knowledge gaps exists for release during the use and especially also for the recycling phase.
The overall mass flow analysis showed that approximately 20% of the nanoparticles will end up in environmental compartments with the majority going into air and soil, which is mainly due to the use phase within the motor oil. About 15% end up in landfills and another approximately 65% are being fed into recycling processes. Overall, the detailed mass flow analyses revealed, that during the production/assembly phase a major release of the nanomaterials is generally unlikely. Uncontrolled release occurs during the use phase and for the end-of-life phase there are still many unknowns with regard to recycling pathways. Also, possible transformations of the materials are not covered by this kind of analysis.
The level 2 involves the application of a Bayesian Belief Network (BBN) to forecast release of NMs at process level. The process “shredding” was selected because mechanical recycling during End-of-Life was found to be an important ‘hotspot’ (from Tier 1) for next generation NMs. The following value chains were selected for this development: (i) perovskites in solar panels, (ii) graphene/Si in lithium-ion batteries and (iii) quantum dots in display technologies. TNO lead the development of this model and invited a BBN expert from TNO to facilitate the development process. IUTA, CEA and external experts actively participated in the development of the Bayesian Belief Network during two intensive (two day) F2F workshops and five (half-day) follow-up teleconferences over a period of 9 months. The model is presented in the form of a graphical representation of the BBN and is underpinned with probability tables based on the expert elicitation procedure obtained during the workshops and follow-up meetings. Different influencing factors on release with regard to the specific activity were identified, showing the general applicability and versatility of the approach also with regard to future nanomaterials not yet covered. The model was tested with shredding data from CEA (i.e. shredding electrodes of batteries).
To define ‘hotspots’ or focal points, the developed Tier 1 approach (an adapted EUSES-based methodology) was applied. The main objective of these assessments are to identify hotspots, that flag potential specific areas of concern in the nanomaterial life cycle and may relate to substantial release, emission or exposure, or where unknown changes and alterations of the material are expected. The following NM Value Chains were identified by the Value Chain Advisory Committee (VCAC) as reported in Deliverable 3.3 and subsequently subjected to a Tier 1 assessment and the identification of focal points (as presented in Deliverable 3.7): (i) VC Energy harvesting: focal points Perovskites, (ii) VC Energy harvesting: focal points Boron doped silicon, (iii) VC Energy storage: focal points Silicon Nanowires, (iv) VC Lubricants: focal points fullerene and graphene flakes, (v) VC Thermoelectrics: focal points of nanocomposites in thermoelectric generators and (vi) VC Display screens: focal points Quantum Dots (CdSe). For each VC, a flow chart was developed with mass flows, incl. information sources and assumptions are indicated. Based on the outcome this flow and the criteria, the focal points of concern were identified and summarized. The tier 1 assessment indicates semi-quantitatively the emission to the environmental compartments, which could be useful for benchmarking worst-case concentration assumptions for toxicological studies. TNO conducted the material flow analysis for the VCs, VC owners, i.e. EPFL, PUM, IUTA, CFR and task partners, i.e. IUTA and CEA, reviewed the assessments. Various partners were involved, incl. TUL who provided information on the biological and environmental impact of reduced graphene oxide (rGO) nanomaterials intended to be used as motorcar lubricant (FNN value chain 4 Transportation/ Industry). The resulting toxicological assessment is intended to help the translation to the market for the novel nano-lubricant, providing specific information for the risk assessment and regulation of a novel nanomaterial.
For methods and devices to detect, characterise and quantify exposure to next-generation nanomaterials, a review was conducted of new methods. IUTA provided valuable input for the overall Deliverable. Of all the detection techniques available, a common drawback identified is that most of the approaches are particle-dependent and therefore a versatile tool would require a multi-sensor approach to be effective and efficient. TUL and TNO worked on a literature review on new exposure metrics and involved in state of the art on “new concepts of exposure metrics” for next generation nanomaterials. It is currently not possible to obtain the size distribution and the chemical composition of the released particles into the air using affordable portable devices. Personal monitors provide the lung-deposited surface area (LDSA) of the nanoparticles in few seconds while personal samplers provide the quantitative chemical composition of the collected particles over a certain period of time using among other X-ray fluorescence spectroscopy. Measuring the chemical composition of the collected particles allows dealing with the nanoparticle background which is known to be variable in time and space. However, it is very difficult and time consuming to correlate size and chemical composition.concepts of exposure metrics are explored in this task through a literature review.
CEA and IUTA was involved in the selection of methods and devices to detect, quantify and characterize exposure to next-generation nanomaterials. IUTA also actively participated in the discussions on best practices to ensure the safety of these nanomaterials. To disseminate the results with regard to release and exposure several oral presentations at international conferences on a framework of release (based on work conducted within the FP7 Project MARINA and jointly extended by TNO, CEA and IUTA) had been given. CEA continued the development of its electrostatic precipitator which is an innovative device, easy-to-calibrate and transportable. It provides a size in real time and the chemical composition for several size ranges off-line in order to give a response for ENMs that could be easily differentiated from the atmospheric background or when there is several sources of nanoparticle that can lead to exposure. IUTA participated in the discussion on test methods and devices able to detect next generation nanomaterials. The work included the further development of the OECD tiered approach for exposure assessment in occupational settings in view of possibly unknown particle properties. TUL worked on a literature review on new exposure metrics methods devices to detect, characterise and quantify exposure to next-generation nanomaterials. Alternatives to current exposure metrics were explored and evaluated under the frame of the OECD general tiered approach for exposure assessment. The possible parameters to be used in the exposure assessment scheme were defined along with a list of available methods, tools and devices for their measurement. Moreover, innovative methods and devices that could overcome some of the current difficulties observed during field measurement were developed during the project. In particular, CEA developed and evaluated the concept of a novel device, easy-to-calibrate and transportable which could provide a size distribution in real time and the chemical composition for each size bin off-line. Several prototypes were designed, built and evaluated using both simulation tools (Finite Elements Analysis - COMSOL Multiphysics) and experiments performed in laboratory with controlled aerosols. The devices were improved iteratively and are currently reaching TRL 4. Two patent applications were filled in July 2015 and were extended in 2017. The innovation is to sort and collect charged particles in specific places of a circular substrate based on their electrical mobility. The concept developed through this task allowing to correlate size and chemical composition of airborne particles is promising since it would allow the discrimination of secondary sources of airborne particles in occupational settings.
CEA performed several field measurements campaigns along with Tier1, Tier 2 and Tier 3 evaluations were conducted during the project to generate new information on the operative conditions and levels of exposure for several case studies on next-generation nanomaterials:
•Production of nanoenabled electrodes for Li-ion batteries
•End-of-life of nanoenabled electrodes for Li-ion batteries
•Production of thermoelectric generators based on nanostructured silicides
•Additive manufacturing
Those visits and campaigns also allowed the identification of experimental challenges related to the measurement of emissions in occupational settings and contributed to confirm that research activity on the development of personal devices was required. This is particularly the case since those tools could be useful for Tier 2 evaluation in SMEs and small scale production lines.
CEA developed a manual on best practice for safe nanotechnology action plan. The best practices identified throughout the technical activities conducted during the project were regrouped in a manual along with generic guidelines for ensuring exposure-driven safety of the next generation nanomaterials being developed for industrial applications. It is also based on extensive literature review and considers the life cycle stage and hierarchy of control (source to worker). Best practices were identified using inputs from other WP3 outcomes (VC roadmapping, field study data, etc). It presents an approach on best practices for ensuring safety of the next generations of nanomaterials being developed for industrial applications. Those selected value chains are representative of the 3rd and 4th generation nanomaterials. The action plan will be beneficial to the scientific community, regulators and stakeholders outside of the project since there is a strong need to reach consensus on methods and devices to assess exposure in practice along the whole value chain (workplace, consumer and environment). This manual will also allow workplace hygienist, workers and executives to make appropriate decisions to tackle efficiently risks associated to manufactured nanomaterials.
The work was directed towards a safer and more responsible development of nanotechnologies. It allows workplace hygienist, workers and executives to make appropriate choices to efficiently address risks associated to manufactured nanomaterials. A user-friendly action plan table was developed that summarizes the best practices for each risk management controls and life cycle stages. CEA, IUTA, NANOGAP and TNO participated in discussions and review of the work.
Work Package 4: Synthesis and exhaustive characterization of materials of the future
The main activity of WP4 has been the synthesis of novel materials relevant in future value chains as agreed with the VCAC. The work has been structured in 4 different tasks. The main science and technological results are detailed below. At the end of the document a summary of nanomaterials structures made in the laboratories of the Partners in WP4 classified by specificity (size, dimension, composition, shape, and application) and partner production is provided.
Task 4.1 Synthesis of novel materials as analogues of those relevant (or likely to be) in future value chains
Within this task WP4 has synthesized nanoparticle classes as agreed with the VCAC. We focused on the synthesis of novel geometries and shapes of nanomaterials, particularly those structures plus variants around them that allowed the study of biological and environmental interactions with a wide variety of differential geometries. For the first time synthetic laboratories were asked to produce a relatively uniform distribution in shape to avoid confounding the biological outcomes with different shapes in the same sample. Within this task we achieved significant advance in the refinement of synthetic methodology of shape control synthesis. A variety of techniques were applied allowing the kinetic control of specific crystal planes to make different geometries of the same chemical substance.
Task 4.2 (VHIR, TCD, NUID-UCD) Combinatorial nanomaterial libraries and collections
This task was intended to explore variations going far beyond those envisaged in Task 4.1. Assembling several materials into a single nanostructure was the strategy to design and produce systems possessing diverse physical and chemical properties. In particular, WP4 partners succeeded in producing hybrid nanoparticles composed of a combination of different materials in core-shell or heteromer-like configurations. These new hybrids represented a powerful strategy for modifying nanoparticle properties, combined the properties of the individual components, or even exhibit novel and unique properties that originate from collective interactions between constituents.
Among the different hybrid structures, WP4 partners succeeded in producing magneto-plasmonic and metal-semiconductor nanoparticles with more complex function and shapes. Magneto-plasmonic particles have been proposed as bi-functional systems for a variety of applications in theranostics, in which the metal shell acts as a protecting agent for the magnetic core and as a highly functionalizable surface. Similarly, metal-semiconductor nanoparticles are ideal candidates for solar energy harvesting applications, in particular photocatalysis, in which light energy is stored into chemical bonds. Thus, the semiconductor part of the particle can be tailored for optimal light absorption while the combination with the metal component facilitates charge separation and the reactive metal surface with a large surface area can serve as the proper catalytic substrate for the intended reactions. This combination leads to novel electronic properties which may ultimately be expressed in biological or other interactions.
CBNI-UCD in WP4 mainly focus on the synthesis and advanced characterisation of gold nanoparticles with complex nanostructures for biological applications (see gold library in Figure 2). Despite the almost limitless possibility in the synthesis of “gold shapes”, most of the methodology reported in literature are not well reproducible or not well described often leading to low quality products. Aiming to understand the effect of different shapes in biology CBNI-UCD invested a great effort in order to carefully select and make reproducible SOPs and high-quality NPs. For this reason, round robins on different synthetic methods and advanced physico-chemical characterisation have been performed on several batches. To perform a deeper characterisation on shaped GNPs CBNI-UCD developed an advanced TEM analysis able to establish a kind of “shape distribution” (more discussed in D8.2) and (in collaboration with FILARETE, Nanoscale, 2017, 9, 2778–2784) SPES analysis was also adopted. However, these techniques concern mainly the study of the core structures in water suspension of the NPs but this is not enough toward biological applications. Several batches of shaped NPs with different synthetic surface chemistry were prepared to be tested for their stability in relevant biological media (as discussed in WP5). The new surface chemistry (biomolecular corona) of the NPs exhibiting good stability in biological fluid were investigated by mass spectrometry (collaboration with Prof. Gabriella Tedeschi UNIMI) and also by QCM mapping technique. Significant differences in the biomolecular corona were found in both composition and orientation.
Figure 2: Gold shape library synthesized by CBNI-UCD
We believe the biomolecular corona can represent the biological identity of the NPs in vivo influencing their cell trafficking, biodistribution and immunological effects (Nanoscale Horiz., 2, 187-198 (2017). For this reason, it is fundamental to exclude biocorona contaminations. It is now well known that endotoxins (as LPS) can adsorb on the surface of the NPs and can be recognised by specific cell receptor (for example in the liver) possibly modifying the original biodistribution or activating certain specific immuno responses. In CBNI-UCD a special effort has been dedicated to build up an endotoxin-free synthesis laboratory (see Figure 3). In this laboratory (with restricted access) strict SOPs are followed to guarantee an almost 100 % rate of LPS-free sample production. All the sample produced are tested for the LPS content by LAL and western-blot (LPS testing protocols are described in WP2).
Figure 3: Endotoxin-free synthesis laboratory in CBNI-UCD
The LPS-free samples were characterised as previously described and sent to several partners for biological studies, including in vivo studies (ACS Nano, 2017, 11 (6), pp 5519–5529) in collaboration with Istituto Mario Negri (Milano) leading to significant biological outcomes (see WP6).
Task 4.3 (PUM, EPFL, TCD, NUID-UCD, Filarete, IUTA, SolarPrint) Surface treatment and modification of novel materials
This task evolved the nanomaterials formed in Task 4.1 by (post-) modifying their surface. Series of nanoparticles with different charge (electrophoretic mobility) and hydrophilicity/hydrophobicity (including superhydrophobic and superhydrophilic modifications) were created using standard surface chemistries.
Task 4.4 (NUID-UCD, IUTA, EPFL, TCD, ICN, USC, PUM, Filarete, LIST/CRP-GL, SolarPrint) Dispersion and characterization of novel materials.
Samples were characterized both dry (where appropriate) and dispersed in various simple media. The most basic characterization of particles involved (average) size/dimensions, ‘charge’ (averaged mobility) and surface area. In some cases new instrumental developments (or evolution of existing ones) were required to meaningfully characterize these novel shapes and geometries. Full characterization of the materials was provided with respect to elemental/chemical composition, morphology and structure. Obtained nanomaterials were characterized for their elemental/chemical composition (UV-Vis-, emission- infrared-, Raman-spectroscopy, EDX, EELS, ICPMS) and as well as for their colloidal properties such as hydrodynamic and geometric size and size distribution (dynamic light scattering (DLS), nanoparticle tracking analysis (NTA)), surface area (gas adsorption - BET method), shape (TEM, STEM, HAAF-STEM, HRSEM, and WDX), crystallinity (x-ray diffraction), porosity (BET/SEM), surface charge (Zeta-potential), isoelectric point (Zeta potential) as well as their dispersability and stability. All characterization protocols will be carefully prepared following OECD, NIST and ISO standards in order to ensure the comparability of experimental data and therefore fulfill information standards. The quality/stability of the NCs was monitored throughout the project to identify potential variations in the material properties. Optical properties of NC-based photocatalysts, in particular the emission, was a central focus of attention. We systematically measured the quantum yield of NPs and hybrid systems using organic dyes such as Rhodamine B and Rhodamine G6 as a standard probe as well as NC life-time. Besides, modeling tools (NanoHub platform) were used to correlate and model the properties of the NPs. Due to the large set of different types of nanoparticles prepared in this project, appropriate high-throughput screening methods for surface characterization was of interest. The use of high throughput methods for studying surface interactions significantly aided in the identification of the basic interaction mechanisms between nanostructures and biological systems.
Summary of nanomaterials structures classified by composition and shape.
-Composition: A wide range of different nanomaterial types were produced: inorganic, metals, inorganic-metal composites, polymeric, metal-polymer, inorganic polymer particles.
-Shapes: Nanoparticles with different sizes and shapes, and different geometry families were prepared from metals and semiconductors. Some of these series of particles corresponded to series of conventional regular geometries (spherical) where only one feature was varied within the series (e.g. a size dimension). Others corresponded to radically different geometries. For example, particles with precisely tuned cavities/pores were prepared. This was the case of hollow shapes (hollow cubes, hollow spheres, hollow wires -tubes-, hollow triangles and hollow rods) and semiconductor-like subnanometric clusters of metals with different sizes, displaying very different physicochemical properties than the corresponding metal nanoparticles. Some materials (Au/Cu/Se/Te/S) have permitted the fabrication of spiky nanoparticles which were of interest because of their different optical and electronic transport properties, suggesting possible future use in optoelectronics, solar cells and light emitting devices. Spiky metal nanoparticles have improved plasmonic properties, which is very promising for surface enhanced Raman scattering (SERS) and thus for applications as molecular sensors.
-Doping of particles: Doped semiconductor systems have been explored due to their broader access to visible light and enhancing photocatalytic activity. Crucially doping changed not just the optical or electronic properties of nanoparticles (e.g. fluorescent semiconductor quantum dots), but also tuned dissolution of the nanoparticles and degradation of the particles, allowing such aspects as the rate of degradation to be controlled as part of a series.
Summary of nanomaterials structures classified by Partner.
-NUID-UCD Contribution. NUID-UCD developed a shape reference library of anisotropic gold nanoparticles (GNPs) with monodisperse distributions of particle sizes and shapes. The library included the following geometries: spherical, rod-shaped, nanocubes, nanostars (and other branched GNPs), nanotrisoctahedra and gold nanoprisms. The particles were produced in large scale and were applied in subsequent cross-collaborative studies of biocompatibility involving other partners of the FNN project. To render the particles biocompatible, and minimise surfactant-induced cytotoxicity surface of the particles several of these were custom functionalised. Successful surface modification was succeeded in the case of spherical, rod shaped and star shaped gold nanoparticles, where the surface chemistry was normalised using a biocompatible carboxy-PEG-thiol ligands. These ligands provided exceptional stability and biocomplatibility of gold particles of the reported size range in aqueous and biological media. A great effort was also done in order to produce endotoxin-free NPs by developing a platform/laboratory specific for the production of “clean nanomaterials”.
-ICN2/VHIR Contribution. ICN2/VHIR developed a comprehensive reference library of nanomaterials with monodisperse distributions of particle sizes and shapes. The library included the following nanomaterials: i) Shape reference library of TiO2 NPs with narrow size distributions of particle sizes and shapes: spherical, rods, bipyramids and platelets; ii) Homologous series of NPs with different compositions and surface chemistries for co-exposure experiments: TiO2, ZnO, CeO2, FexOy, CdSe, Ag and Au; iii) Shape reference library of hollow Au NPs with narrow size distributions of particles sizes and shapes: spherical, rods, cubes, triangles and wires; iv) Inorganic fullerene-like (IF) MoS2 nanoparticles and V) metallic Ni nanoparticles. In all cases, the surface chemistry of NPS was precisely adjusted in order to ensure their colloidal stability and dispersability.
-PUM Contribution. PUM has synthetized different homologous series of NPs libraries including water soluble gold shapes, Fe2O3, CdS and CdS doped with Mn, ZnS and ZnS dopped with Mn NPs. Some of these NPs were firstly synthesized in organic solvents. Afterwards, all the NPs were transferred to water using an amphiphilic polymer. This approach is based on the use a polymer which contains a hydrophilic backbone and a hydrophobic domain formed by aliphatic chains. This hydrophobic domain interacts with the aliphatic chains that are stabilizing the NPs after their synthesis in organic solvent. Finally, raising the pH, the rings of the hydrophilic domain of the polymer are opened. This process creates a surface rich on carboxylic groups on the NP that provides colloidal stability.
Figure 4. TEM micrographs reporting some example form the GNPs library prepared by UCD and further functionalized by PUM from up to down and left to right: FNN_PUM_Au_#3; FNN_PUM_Au_#4; FNN_PUM_Au_#5; FNN_PUM_Au_#6; FNN_PUM_Au_#7; FNN_PUM_Au_#8; FNN_PUM_Au_#9; FNN_PUM_Au_#10 and FNN_PUM_Au_#11. NPs were distributed across the FutureNanoNeeds consortium for subsequent biological (WP5, WP6) or eco-tox (WP7) evaluation.
-IUTA Contribution. IUTA has worked on the synthesis of materials on the pilot-plant scale along several value chains. Within WP 4 IUTA has used its pilot plant including three different reactor concepts (hot-wall reactor, flame reactor, microwave assisted plasma reactor) to generate materials.
-LIST/CRP-GL Contribution. CRP-GL has worked on the synthesis of Fe3O4, ZnO and PEG-coated Ag Nanoparticles. Fe3O4 nanoparticles were synthesized by a co-precipitation method and stabilized by an oleic acid coating and further dispersed in hexane. ZnO nanorods were synthesized by hydrothermal synthesis. Polymers were used during the synthesis to control the aspect ratio. Citrate-stabilized Ag nanoparticles (20, 40, 80 nm) were functionalized by using the well-known thiol-silver chemistry PEG-SH (MW 3000).
-TCD Contribution: TCD has worked on the synthesis and characterization of CdS, CdS doped with Mn2+ and TiO2 NPs. CdS nanotetrapods of approximately 15 nm size were produced through the use of an aqueous reflux in the presence of the enantiomerically pure stabilising ligand D or L penicillamine. CdS nanotetrapods doped with Mn2+ ions were synthesized in a similar fashion to the synthesis of pristine CdS nanotetrapods above with a number of changes. TiO2 nanoparticles were produced through a non-aqueous reflux.
-FILARETE Contribution: TCD has worked on SPES (Single Particle Extinction & Scattering) technology which is capable of characterizing nano and micro particles in complex fluids with unprecedented high-quality results.
Figure 5- TEM/STEM images TiO2 NCs synthesized by ICN2/VHIR from up to down and left to right: FNN_ICN2_TiO2_#2; FNN_ICN2_TiO2_#3; FNN_ICN2_TiO2_#4; FNN_ICN2_TiO2_#7; FNN_ICN2_TiO2_#8¸ FNN_ICN2_TiO2_#9. NPs were distributed across the FutureNanoNeeds consortium for subsequent biological (WP5, WP6) or eco-tox (WP7) evaluation.
-NANOGAP Contribution: NANOGAP has synthesized and sent to partners both, fluorescent Au clusters with enhanced Stokes shift, and surfactant-free ultrasmall Ag clusters, according with Task 4.1. Results from partners have shown that clusters synthesized in NANOGAP were endotoxin-free, as required for toxicology testing. IN detail, they succeeded in the development of synthetic and purification techniques for the production of Ag AQCs with a specific number of atoms with a concentration of different species low enough to avoid interference in the observed properties. The species produced by NANOGAP in an almost pure form are Ag3 and Ag5, with very different biological effects one from the other. Besides, a much simpler synthetic approach has been developed for the production of complexes of Au AQCs which, due to their radically new luminescent properties, have enormous potential in any application concerning fluorescence. This fact makes very interesting the study of their biological interactions and the potential toxicity of the product.
Figure 6 - TEM images homologous of series of NPs with different compositions, and surface chemistries. The library includes the following compositions: TiO2, ZnO, CeO2, FexOy, CdSe, Ag and Au. NPs were distributed across the FutureNanoNeeds consortium for subsequent biological (WP5, WP6) or eco-tox (WP7) evaluation
-USC Contribution: USC has worked on the preparation of silver nanoclusters with sizes slightly larger (6-10 atoms) than those prepared by Nanogap (2-5 atoms) and with the highest monodispersity in size. They used different methods for achieving this objective: i) Direct reduction of Ag(I) salt by UV irradiation (254nm wavelength);ii) Chemical reduction of Ag(I) salt with strong reducing agents (e.g. sodium borohydride); iii) Electrochemical synthesis using two Ag foils; iv) Two-step synthesis: electrochemical synthesis of small Ag clusters followed by chemical reduction with strong reducing agents; and v) Two-step synthesis: electrochemical synthesis of small Ag clusters followed by UV irradiation (254nm wavelength). From these previous methods, the one-step methods showed long-term stability problems (aggregation and settling) and some polydispersity in sizes. The results were much better with the two-step methods, being the last method the best one.
Figure 7. TEM images of Au-Ag hollow nanoparticles after reaction of Ag templates of different shapes with different volumes of HAuCl4: (A) Au-Ag hollow spheres (FNN_ICN2_AuHollow_#1, 53nm in diameter), (B) Au-Ag hollow cubes (FNN_ICN2_AuHollow_#2, boxes) (36 nm side) (C) Au-Ag hollow rods (FNN_ICN2_AuHollow_#3, 60x27x27 nm), (D) Au-Ag hollow prims (FNN_ICN2_AuHollow_#4, 45x45x10 nm), and (E) Au-Ag hollow wires (FNN_ICN2_AuHollow_#5, tubes) (> 1000 x 50 x 50 nm).
Work Package 5: Biological and environmental interactions of future nanomaterials
Within WP5 TNO performed an analysis of the protein corona formation around nanoparticles and investigated the implications for safe design. In order to control the activation of specific biological pathways, it is therefore essential to understand the formation of the biocorona in terms of composition and amount of adsorbed proteins and its relation to the physicochemical properties of nanomaterials and proteins. In this way, it would be possible to develop structure-activity relationships, allowing the engineering of nanomaterials in order to develop nanostructures with controlled fate and bioactivity/toxicity. It was found that the profile of corona composition around different nanoparticles is basically the same, and that this depends on the plasma concentration and molecular weight of the proteins. For the most abundant proteins, we also find that electrostatic interactions play a role in determining the amount of adsorbed proteins. For surface modification of nanomaterials, in order to selectively bind or repel specific proteins, may not be functional to control specific biological effects or avoid biological pathways.
Partner CBNI-UCD: The stability of several nanomaterials (including, but to not limited to different gold nanoparticles with different size and shapes (as spheres, rod-like, urchin-like and star-like GNPs) and different carbon based nanoparticles (as graphene and nanodiamonds) dispersed in relevant biological media have been investigated. To this purpose several biofluids and biological media have been selected, ranging from full human plasma or serum (to mimic in vivo scenarios) to complete growth medium (RPMI and MEM supplemented with different concentrations of faetal bovine and human serum, in order to mimic in vitro conditions). The nanoparticles have been therefore suspended in these milieu and incubated at 37 °C for different time points (up to 24 hours). Optimised suspension protocols have been developed. Generally, the NP dispersions in biological media of the analysed nanoparticles turned out to be stable in “realistic biological conditions”.
A further work was performed in collaboration with Filarete. DCS and SPES were both employed to successfully analyse in situ and in line the stability of different GNPs shapes in biofluids. This is an important achievement allowingthe study of NPs suspension in real conditions without washing/isolation of the NPs. Several NPs at certain concentration are well stabilised by the large excess of proteins present in biological media but lack of stability below a certain protein concentration (i.e. after washing/centrifugation).
CBNI also investigated the biomolecular corona composition of different shaped nanomaterials (synthetized in the group / WP4) exposed to relevant biological fluids. Some of these shaped nanoparticles were also sent to FILARETE for investigation of the corona by SPES and shotgun mass spectrometry (in collaboration with UNIMI).
CBNI-UCD also investigated how the shape can influence the orientation of the protein during the adsorption on the NPs surface. The study was performed by QCM (quartz microbalance) using nanospheres and nanostars of same size with transferrin corona. The analysis showed a significantly higher number of Tf with the “right orientation” for nanostars compared to nanospheres. These results confirmed an effect of the shape not simply in the biocorona composition but also in the epitope presentation and therefore potentially different biological recognition. Also the prepared surface chemistry was shown to parametrize the NP-protein complexation. Indeed, for gold nanostars functionalised with BSPP the recognition was much higher than the one observed for the very same nanostars functionalised with PEG. This is probably due to the reduced interaction of the proteins with the PEGylated NPs surface.
Moreover, the influence of the shape (and related biomolecular corona) have been tested in vitro. Briefly, CBNI developed a cell platform to assess the role of specific receptors (mostly express in the liver) in the uptake of NPs. Human Embryonic Kidney 293 T cells (HEK-293T cells) have been used as a model for the transfection of the specific receptors of interest, due to its high efficiency of transfection and protein production.
Furthermore, in collaboration with TUL, LIST and FILARETE, CBNI-UCD investigated the stability/degradation of different NPs suspensions in different environmental fluids as peatbog water, sea water and reservoir water. Preliminary data suggest that again combining SPES, DCS (as complementary techniques) nad UV/Vis it is possible to follow the overtime evolution of the NPs degradation.
TUL leaded D5.2 Report on novel methods to assess degradation, which included examples on assessment of nanomaterial degradation in most important biological and environmental media, particularly in FBS, hepes buffer, PBS buffer, artificial lysosomal fluid, simulated body fluid, bacterial growth media, algal growth media, in soil, contaminated groundwater, and freshwaters. This deliverable served as a basis for WP6 and WP7 partners performing toxicity and ecotoxicity studies in terms for providing recent advances in methodology for characterisation of nanomaterials. Moreover, TUL developed a new standard (nAu) for differential centrifugal sedimentation analysis, because commercial PVC standard was found to be unsuitable for all types of nanomaterials. A scientific article is now under preparation (Mikšíček. et al. Quality control of differential centrifugal sedimentation analysis and its use for determining the stability of synthesized with a gold and iron oxide nanoparticles).
Further we performed research on transformation of new FNN nanomaterials in biological media, particularly in algal and bacterial growth media and in activated sludge from wastewater treatment plant. Results are present in D5.3 Data inventory of solubility and degradation kinetics of novel nanomaterials in biological milieu. Generally, this study helped us to understand behaviour of the materials is different exposure media and thus better understanding of ecotoxicological data (within WP7). Several scientific articles are now under preparation in collaboration with RIVM, UoB, and UCD.
Dispersion, aggregation and stability of FNN nanomaterials (VHIR_AgNP_15 nm_060516, mesoporous silica NPs coated by APTES with magnetic cores, and Si@Gra01) in reservoir water, media of low and high pH and media of different ionic strength were tested at TUL. Results were described in D5.6: Characterisation of dispersions in several environmental media and in conference extended abstract (Wacławek et al. 2016). Finally, TUL leaded a study on FNN ZnO materials obtained from LIST (nanoscale ZnO-HP1, ZnO-HP1-Mirj S40, LNP ZnO-HP1, ZnO nanowires) in natural waters (reservoir representing normal pH and ionic strength conditions, peatbog representing acidic environment, and seawater representing higher ionic strength environment) in collaboration with MNI (EOS Instruments) and NUID-UCD. Results are being analysed and an article intended for journal with high IF will be prepared.
Contribution from MNI:A limited series of experiments has been performed to evaluate the influence of the material on cell internalization mechanism. To this aim NPs with similar size, shape, z-potential and surface functionalization (PEG) but differentiating among them for the chemical nature of the Polymer (PMMA, PCL3 and PLA8 respectively) were incubated in breast cancer cells MDA-MB231.1833. Rhodamine-B was added to the monomer and the process of polymerization was undertaken in order to have a strong signal and to avoid the biological elution of the dye. Representative images showing the pattern of RhB distribution of NPs are shown in figure 8.
Figure 8: Representative images showing the accumulation of red signal (associated with NPs) in MDA-MB231.1833 cells (identified by the presence of nuclei in blue). All field of views were acquired by exciting the sample with a laser at 405 nm and immediately after with a laser at 546 of λ. The merge between the two signals is reported in these images. Scale bar: 75 µm. Scatterograms showing the merge of events selected by TissueQuest in each experimental group. For each group nine different fields of view, obtained by two different experiments, were processed. In the X-axis the fluorescence intensity for each single spot is reported. In the Y-axis the intensity of fluorescence for each single spot is reported.
The quantification of fluorescence is reported in figure 9. Our data showed that the NP accumulation significantly increase from at each time-point for each type of materials but the uptake was faster and stronger in MDA-MB231.1833 cells incubated with PCL3.
Figure 9.A: Histograms showing the fluorescent area for each experimental group (n = 20 fields of views). The same fields of view processed for the analysis of RhB were analyzed to determine if the time of incubation and/or the type of nanomaterial may influence the number of cells.
In figure 10 A depicts the cell internalization of the three materials is shown.
Figure 10A: Representative pictures showing the staining associated with the nuclei (left column), the staining for RhB (central column) and the merge between the two signals (left column) in MDA-MB231.1833 cells incubated for 24 hours without NP (upper line), PMMA-NP (second line)PLA8-NP (third line) and PCL3-NP (lower line).
B: High magnified pictures showing the different localization of NPs (red) 24 after incubation in MDA-MB231.1833 cells. PMMA-NP incubation (LEFT panel) led to a massive internalization of NP (see green arrow) and a weak staining surrounding the peripheral rig of the cell (yellow arrow). PLA8-NP (central panel) mainly remained confined on the external surface of NP (yellow arrow) with a weak penetration inside the cell (green arrow). A homogenous distribution of red spots was instead observed in cells exposed to PCL3-NP (right panel).
The vehicle-treated cells (upper panels) were added to demonstrate that, by this procedure, the red signal associated with was almost completely broken down (the black arrow show a signal apparently unrelated to the nuclei). Both PMMA-NP and PCL3-NP incubation was characterized by separated dots and mainly concentrated close to the perinuclear region. On the other hand, the staining for RhB after 24 hours of PLA8 was almost exclusively confined to the external part of the cells.
Experiments with blockers of endocytosis figured out that, whilst both PMMA and PCL3 staining was greatly reduced after treatment, no significant alterations were found in cells treated with PLA8. This result furthermore confirmed the lack of endocytosis by PLA8-NP.
Overall, the present results furthermore confirm the dramatic changes in bio-nano interaction after modification of a single parameter of NPs (in the case the softness of the material). This is an extremely important point to be considered to both evaluate the efficacy of a treatment or, opposite, to predict its potential toxic effect.
Work Package 6: Biological identity, processes and human health hazards of future nanomaterials
As described above, in this WP the biological impact of several NMs having different physico-chemical properties has been carefully evaluated by a multi-step platform starting from the basic interaction with molecules or fluids and, passing through analyses in cells, reaching the in vivo characterization in rodents.
A first period was devoted to set-up and standardize the experimental procedures required to this platform development. This approach is in line with the whole philosophy of the project, in which there has been extensive effort to make reliable and reproducible the data generated throughout the lifetime of the study. This has included a centrally managed workflow of materials produced and distributed to partners for testing. The existence of a wide variety of data from previous FP programs already suggested guidelines and rules to be followed along the whole duration of the project among all partners involved in this WP. To this end standard operating procedures (SOP) have been tested in a number of Round Robin and interlaboratory comparisons and compared to those tested with the reference materials. In detail, viability assays, mainly through MTS assay experiments, were carried out using the same stock of cells representative of the alveolar epithelium (A549 cells). The results, that were then uploaded to Teamlab to be easily shared with other partners, unveiled a high level of reproducibility among the different labs involved in this WP. Further to this, the results are in good agreement with previous interlaboratory comparisons performed using the same SOP and materials within the QualityNano Infrastructure. This indicates a high level of confidence in the reproducibility of results within WP6 and the potential for read-across of a number of biologically relevant endpoints across numerous cell lines. Another important point to be assessed before the different effect of NPs in biological fluids, cells and animals is the level of contamination. To this end TUL an accurate analysis of endotoxins in new NM samples obtained from FNN project partners was carried out. This was important for WP6 partners performing toxicity studies with mammal cell lines, because some toxic or inflammatory effects, which might be attributed to NPs, can be in fact caused by the presence of endotoxins. Therefore, we newly established chromogenic Limulus amoebocyte lysate (LAL) assay and fluorogenic EndoLisa assay for detection of endotoxins. Both methods were developed for medical purpose and had to be adjusted for NMs. For example, presence of NPs can cause false positive and false negative results. Hence, a control displaying possible inhibition or enhancement of the signal was included. During screening of FNN NMs for endotoxins, we found out that considerable number of samples was contaminated by endotoxins and we promptly communicated these results to the other WP partners involved on the toxicological studies. Guidelines for FNN WP4 partners on how to minimize the introduction of endotoxins during NM synthesis were then prepared and circulated. A study to compare four methods for detection of endotoxins was therefore performed. Experiments with E. coli lipopolysascharide (LPS) spiked samples of ultraclean Au-NPs and SiO2-NPs LPS were done using LAL assay and EndoLISA to find out the limits of the methods and possibilities for further optimization. Another important aspect that had to be considered before biological tests was the behaviour of NMs over time. Particle storage and aging, dissolution in short and long term and in relevant media may lead to a change in the physico-chemical properties of materials and, consequently, modify their biological identity. In this WP we focused on the attention on the selection of the best procedures to follow the fate of the different NMs after their synthesis. This was pivotal to control the quality of the products and to remove impurities in a broad range of material types. This allowed us to undertake the next phases of the project with an inner control based on the purity and the maintenance of the basal characteristics of the different NP before the next part of the WP, devoted to the in vitro studies.
The first feature we evaluated in cells was the ability to uptake NPs and the consequent intracellular accumulation. The mechanism of uptake and localization of novel materials, graphene and graphene oxide (of interest for VC4), long-tipped gold nanostars (due to unorthodox shape parameters), gold nanorods (possible shape of interest for some applications) and spheres (reference material) was investigated in detail in this WP. The study of NP-cell membrane interactions is the more realistic way but is a really complex system. To get results we performed these experiments with high speed spinning Disk confocal LASER microscopy. Utilising spinning disc confocal LASER microscopy it is possible to create ‘movies’ of NPs as they interact with the cell membrane. Our results highlight that interaction between a NP and the cell include several processes such as, the diffusion near the cell membrane, the diffusion to the cell membrane and the endocytosis. It is difficult to completely separate those phenomena as some of them might be time based, e.g. particles which stick to the membrane could be internalized at different time periods. We found that the time a particle spends on the membrane prior to internalization can range across a large period of time, internalization events could be seen to range from time scales of seconds to upwards of 30 minutes and that that the rarity of combination between NP and receptor is not directly dependent on its physico-chemical details, such as shape and material, but rather depend on particle/cell concentration and conditions of the study. It is known the uptake can be influenced by the percentage of serum conditions. The achieved results indicate that there are a higher number of events under the lower serum condition for the same time period per unit length of membrane. Hence it is more likely to engage in an interaction event with the membrane, meaning a higher probability to potentially undergo endocytosis compared to the same type of NPs incubated in a higher concentration of serum.
To further investigate the involvement of the receptor receptor-dependent uptake of NPs, we focused our attention on the action played by scavenger receptors. Macrophage scavenger receptors are a family of trimeric membrane glycoproteins responsible for the endocytosis of a large range of macromolecules, likely proteins linked to NPs. To this end, the human embryonic kidney cell line (HEK293) was induced to express specific scavenger receptors on their surface to evaluate the importance of each individual member of these scavenger receptors. In this study graphene, silica and gold NPs were extensively used to test the efficiency of uptake. In a receptor, a transmembrane protein devoted to recognition of bacteria fragments or other non-self-agents circulating in the bloodstream, a progressive reduction of the overall uptake was observed when the serum concentration increased. However, the 33% of NPs were still uptake by the scavenger receptor 1 even at high level of serum in the medium. These results suggest that despite the low interaction due to the presence of human serum proteins the receptor is still able to recognize NPs in the medium and internalized them. Using another scavenger receptor, we found that both star and spherical gold NPs behave in the same way and that the complexity of the cell surface increase when the cells are exposed to gold NPs. This suggests that NPs can adhere to the cell membrane inducing relevant alterations in the homeostasis of the cell surface.
Once determined the most critical parameters influencing the cell surface-NP interaction and the consequent uptake, the attention was pointed at to their localization inside the cell. We used TEM to evaluate the subcellular localization in macrophages of gold NPs incubated after 24 hours. As expected, our study revealed that NPs mainly localized into the cytoplasm, in organelles probably related with the endocytic pathways and vesicles. After NP uptake by endocytosis the primary location is in acidic organelles with different levels of maturation; this can go from primary endosomes, late endosomes, lysosomes and lamellar bodies. To combine observational to quantitative results, ICP-MS experiments were carried out. Results showed a different level of uptake by the two cell types which varied between different materials, sizes and shapes. Very interestingly, two different sizes of the gold nanospheres were mixed (at 5% v/v of the respective stock suspensions) the uptake by A549 cells appeared to be determined by the uptake rate of the largest NPs, whereas in Caco-2 cells uptake remained similarly low for both sizes.
Once internalized, NPs can be either dissolved or put out from the cells (exocytosis) or remain inside the cells leading to a progressive accumulation. It is clear that both NP characteristics and cell type may greatly influence their fate after penetration. In this multivalent process, lysosomes play a pivotal role in processing because of their ability in digesting and controlling the release of materials out of the cells. Using A549 cells, exposed to different types of NPs for two hours and then maintained for a longer window of time (weeks), we found that the isolated lysosomal fraction showed a trend of decrease of NPs over the time. Obviously, this decrease was tightly associated with the nature of the NM and their main physic-chemical features. In order to understand if this reduction was due either a real dissolution of the NM or related to a clearance by exocytosis, the NPs were separated from isolated lysosomes and TEM analyses were carried out. Quite interestingly we found that gold and silica NPs did not show any sign of dissolution whereas magnetite core NPs had a significant change on particle structure. This is a very important finding to predict what could happen once ingested or inhaled by humans and penetrated in the cells of digestive or respiratory tract. The interaction between NPs and lysosomes was also evaluated by super resolution microscopy using fluorescent polymeric NPs in human breast cancer cells. This study showed that 2 hours after incubation NPs are already in contact with mature lysosomes, that hey actively enter inside the lysosome passing through its membrane to be degraded. Very interestingly several agglomerates of lysosomes containing NPs, resembling a multivescicualr bodies, were found at the periphery of the cells starting from 24 hours of NPs. One week after NPs injection the signal associated with them almost completely disappeared and lysosome organization was similar to the resting state. This strongly suggested that a complete clearance of polymeric NPs from the cells was occurred. To mimic the in vivo condition, spheroids form A459 were added to this platform to check the processes of NP degradation in a 3D model. Our results revealed a mild but progressive decrease of silica NPs over time. In contrast, the rate of decrease for non-degradable polystyrene showed a significant reduction within the first week but a maintenance of these levels at the latter times, indicating that there was no further dilution of the particles. This is a crucial point in case of repeated exposure, accumulated NPs could indeed lead to relevant functional alteration, first a lower efficiency of lysosomes but not only. To better evaluate this relationship between uptake-accumulation and downstream effects, a careful investigation on the systematic toxicity of NMs was carried in the next step of this integrated platform for the nanosafety assessment. The combination of MTS assay and High Content Imaging high-throughput method was applied to test the viability and other parameters of toxicity after exposure to 30 different NMs at the three different concentrations in three different cell lines. This analysis generated a very large number of data. However, it is possible summarize that many parameters, such as the geometry of NPs, their concentration, the nature of the material and the cell type may lead to different results. In addition, it is also important to underline that relevant hallmarks of toxicity (e.g. ROS production, genotoxicity, mitochondrial impairment) found with specific NMs were not always strictly associated with the lethal effect of them. This is a very important point because it underlines that the mere evaluation of cell survival is not such a reliable approach to deeply evaluate their potential effect to the human health.
Another important question is whether NP accumulation can be safe for the target but, at the same time can induce on it some physiological alterations that can be detrimental for neighbouring cells. This always happens in pathological condition such as automminune disorders or reactive gliosis in neuroinflammatory diseases. To this aim NP-dependent immunomodulation was investigated by inflammasome activation. Inflammasomes comprise a group of intracellular multiprotein complexes readily triggered by exogenous stimuli. They mainly lead to cytokine expression and release and activation of a pro-inflammatory phenotype from a resting to an activated macrophage. Among the several results achieved we interestingly found that CeO2 nanospheres had a dose-dependent activation of Il-1β without changes in cell viability (in blood monocytes THP-1), whereas CeO2 stamps sharply decreased the viability but did not increase the cytokine release. Other NMs, such SiO2 or gold, showed a shape dependent infammasome activation. All this data is extremely important both in nanotxicology but, above all, in nanomedicine. To know that an inflammatory activation of circulating cells can be somehow associated with a specific physic-chemical feature of injected nanocarrier would be extremely important to develop NPs for pharmacological purposes.
Although this in vitro screening is very important to maximally reduce preclinical studies, they cannot completely exclude the use of rodents to predict crucial bio-nano parameters such as organ distribution, the clearance and the occurrence of detrimental multy-systemic effects. To this end, on a very restricted number of mice, a comparative evaluation of the effect of intravenously injected NPs was carried out. In a first study the role of the size and the shape of endotoxin-free gold NPs was evaluated. We selected this kind of pure NPs to be close to the clinical practice. The animals were sacrificed 1 hour, 1 day and 5 days after the treatment because these three time points are representative of three different situations, filter organ penetration, clearance and long-term accumulation respectively. Both shape and size strongly influenced the filter organ accumulation and the excretion, no relevant improvement of biological barrier passage was instead observed for any kind of geometry tested. Very interestingly, the injection of endotoxin-free gold NP did not lead to either any evident histopathological alternation or signs of inflammatory activation. A similar approach was used to define the shape-dependent behaviour of TiO2 NPs. In this case we found a lower ability of accumulation, and the shape did not lead to significant difference, but a marked but transient response of blood, liver and lungs (the three organs with the higher presence of NPs) accompanied to a pro-inflammatory activation. In particular rods, gave a better effect in spite of a lower persistence and bio-accumulation thus indicating these two latter factors are not always tightly correlated.
Although the present platform has been mainly exploited to evaluate the behaviour of NPs with a well-defined shape, it can be easily transferable to the evaluation of the effect of NMs with unusual geometry. In the past three decades, nanostructures with a variety of morphologies, components, chemistries, and functional molecules have been extensively developed, and the tuning of these physicochemical properties can be directly used to influence nano-bio interactions. Given the significant nano-immune interactions for all types of nanostructures and all delivery routes, we have decided to focus our attention on understanding the impact of NP with irregular shapes on the innate immunity. First, we examined the role of surface geometry on the gold (spheres or stars) NP uptake in human macrophages. We noticed that both spherical and star-shaped gold nanoparticle could be internalized by macrophages, however, it was observed for different concentrations that the uptake of gold nanospheres (GNP) is more efficient than that of gold nanostars (GNS). Compared to GNP, the uptake rate of GNS was much lower at the initial exposure and remained steady until 16 h, where it showed a trend of reaching a plateau. In order to identify the signalling pathways and mechanisms that contribute to the immune responses to different shapes of gold nanoparticles, a dual-colour transcriptome microarray was performed in THP-1 cells 24 hours after incubation. Gold NPs exposure triggered to a significant alteration in gene expression. Overall, we observed that gene expression regulation was more significantly affected by GNP, compared to GNS in the macrophage model.
The immunomodulant effect of the shape was also seen by a chronic repeated subcutaneous administration of endotoxin free GNP and GNS in healthy rats. An increase in the circulating IgG levels was indeed observed in certain animals treated with GNS suggesting again that there might be a differential processing in vivo compared to GNP. Moving from the stars and spheres, we decided to further synthesize more complex branched nanostructures and screen the adjuvant effects of the geometry in mice, previously immunized by Ovalbumin and then treated with spheres, urchin, long-trip urchin and flowers respectively. Mice were sacrificed at 42th day, axillary lymph nodes, Bone marrow from hind limbs and spleen were harvested and homogenized in a single cell suspension, PBMCs were extracted from blood samples. Mice treated with spheres showed a very weak production of antibodies almost comparable to the ovalbumin alone whereas urchin-like NPs did not show any production of antibodies. Long-urchin like NPs seemed to be more efficient in stimulating antigen-specific antibody response than free ovalbumin and an increase of IgG production after second booster comparable to free ovalbumin. Although other experiments should be carried out to better define the immunogenic power of these different shapes, including the ability to migrate into the sentinel lymph-node (axillary) an evidence of a shape-dependent influence is emerging from this study. These preliminary results suggest that GN shape convey the adjuvanticity, which is consistent with previous observations with nanostars and nanospheres. It is important to understand the mechanism underlying the shape-dependent adjuvanticity. The ongoing work by NUID-UCD is focusing on further phenotyping and analysis of different antigen-presenting cells to understand the quality of these adjuvanticity.
Work Package 7: Exposure and environmental interactions of future nanomaterials
Task 7.1 Environmental binding and ecoidentity of future NMs
Work carried out within WP7 pioneered the investigation of the role of organism-secreted biomolecules in modulating the acquired biomolecule corona and thus the implications and impacts of NMs on living systems. Figure 11 shows schematically the approach used to collect the secreted biomolecules (conditioning of the medium), in which NMs are incubated prior to their presentation to organisms for assessment of uptake, retention and impacts.
Figure 11. Schematic illustration of the process of conditioning medium, incubation of NMs in the conditioned medium, and subsequent presentation of the eco-corona coated NMs to organisms, in this case the aquatic crustacean, Daphnia magna.
Thus, the secreted corona arises from the fact that organisms secrete biomolecules such as proteins, carbohydrates and lipids, which adsorb selectively to the NMs to create an eco-corona or layer of biomolecules around the NM which changes the identity of the NM (i.e. confers and environmental identity) and alters their interactions, stability, uptake by, and toxicity to organisms.
Notably, in cellular systems the reduction of surface energy through binding of biomolecules and corona formation always leads to reduced NM uptake and cytotoxicity, while in the case of environmental organism, with their myriad of uptake modalities, this is not always so straightforward. The impacts from secreted coronas observed within FNN include:
1. Biomolecules can have destabilising or stabilising effects on NMs, which can cause them to agglomerate and become a more or less attractively sized food source for D. magna which in turn can drastically affect their toxicity. It was observed that the secreted proteins caused agglomeration of polystyrene NMs, which lead to increased retention of the NMs in the D. magna gut, and thus our working hypothesis is that bigger particles (closer to the algae size) are more recognisable and potentially more attractive as a food source, though NM dissolution also needs to be taken into account for many NM types. The addition of natural biomolecules during NM toxicity studies should be considered in NM exposure protocols. Furthermore, whole organisms such as D. magna are themselves dynamic systems and therefore potential changes to the NMs may occur once consumed in, for example, the gut, which can be considered as a sub-environment where NMs may be altered.
2. It is a well-established fact that D. magna decrease their grazing if they are already fed. It has been found that NMs may become lodged within the gut, in proximity to the bush border, which appears to be causing the D. magna to feel full, resulting in reduced uptake over the subsequent period. A wide range of NMs, or different sizes and compositions have been found to be retained by D. magna, over at least 6 hours post-exposure to NMs, in the absence of feeding. It is well-known that D. magna require a food source to push out previously ingested material so that the amount remaining in the gut may be cleared in real environmental scenarios where food is present. At the same time, NMs can bind to a food source incidentally increasing their uptake so that a food source acts as a modulator of both uptake and excretion. Thus, more realistic exposure scenarios need to be developed, that account for the physiological features of the test organisms and the unique considerations of NMs. Assays to assess the fate of the NMs following uptake also need to be developed.
3. The presence of the corona and secreted proteins in the medium can have a profound effect on the dissolution of NMs, and specifically on the mobilisation of specific metals contained within some NMs. Extensive work with lead iodide perovskite particles, comparing their toxicity in standard media versus conditioned media again found enhanced toxicity in the case of the conditioned media. However, the mode of toxicity in this case was related primarily to the ability of the secreted proteins to sequester lead and thus drive the release of lead from the perovskite NMs and thus enhance its bioavailability to the organisms. Design of the NM surface to minimise contact with the specific lead-binding proteins may be one potential route forward to reduce the toxicity of these materials during use and end-of-life. Work is ongoing to confirm whether the mobilisation of lead occurs outside the organisms or is enhanced at the low pH of the D. magna gut.
Limited work on the development of a workflow and methodology for assessment of the other constituents of the eco-corona, beyond the proteins, was also undertaken. Assays for total carbohydrate concentration and for identification of the functional groups in the carbohydrates were developed, given the strong prevalence of polysaccharides in the environment. Additionally, a workflow to identify the small-molecule corona, also called the metabolite corona, have been developed based on mass spectrometry again utilising the conditioned medium as the starting point, with limited success. However, this is a very promising area of future research, and several different avenues are being pursued in parallel to optimise the small-molecule corona workflow.
In summary, research undertaken to address task 7.1 focussed on characterisation of the secreted corona (proteins, polysaccharides, small molecules, including method development for small molecule (metabolite corona) and evaluation of the impact of the route of exposure to NMs (with/without food before, during and after exposure) on the resulting effects of the NMs. Specific features of NMs linked with toxicity included the release of toxic ions (e.g. lead from perovskites) or effects from cationic charges and rod shaped particles that lead to excessive reactive oxygen species production, which required the organisms to divert energy usually required for moulting to fighting the oxidative stress, resulting in delayed development through delayed shedding of the carapace. In the absence of the conditioning step, D. magna were able to mitigate the oxidative stress induced by cationic particles within 24 hours. By contrast, the same NMs exposed at the same concentrations but following incubation in the D. magna conditioned medium, were found to be unable to recover with >90% dead within the 8 hours of exposure (at the EC40 concentration for the bare NMs). The role of the corona in enhancing oxidative stress, or preventing the functioning of anti-oxidant molecules is an important aspect worthy of additional research.
Task 7.3 Material transformation (including degradation) in the environment
In WP7 partners conducted leaching experiments in soils with different perovskites as well as nanoscale MoS2 and non nanoscale MoS2, as for these materials a release into soils are suggested. The primary particle size and the hydrodynamic diameter, zeta potential as well as the solubility of the materials was tested before the leaching experiments. For the solubility experiments the NM mixture was shaken for 24 h (overhead shaker). After this the suspension was centrifuged (5000 g) for 30 min and filtered by using a 0.02 µm syringe filter. The filtrate was analysed with ICP MS (Mo for MoS2 and Pb for the perovskites).
For the soil experiments, glass columns with an inner diameter of 4 cm were filled with soil (dystric cambisol) to a height of 10 cm. Afterwards the soil was pre-wetted from the bottom to the top with 0.01 M CaCl2 solution and drained again before the NM suspension was applied on the top of the soil column as suspension. After this "artificial rain" 0.01 M CaCl2 solution was applied on the top for 48 h. The eluate was sampled after 3 h, 6 h, 24 h and 48 h and analysed to the Mo and Pb concentration. After the experiments different segments of the soil column were analysed (NM conc.) with ICP MS.
No breakthrough was observed for the three tested perovskites (FALi, MAFA, MALB), these materials show a low mobility. FALi shows a slightly higher mobility, followed by MAFA and MALB. The MoS2 materials show a breakthrough with a significant higher mobility of the nanoscale material. No correlation was observed with the agglomerate size or solubility. For the agglomerate size the material with the highest size the highest mobility was observed and no ranking between the perovskite materials. As both MoS2 materials show a slightly higher agglomerate size, compared to the perovskites we can conclude that the agglomerate size did not significantly affect the mobility. For the zeta potential a trend was indicated showing the highest mobility for the materials with the highest zeta potential and vice versa.
WP7 was able to develop mobility experiments of the next generation of NMs in soil columns. The work conducted leads to a better understanding of the mobility of novel NMs in soil columns, helping to understand the expected possible behaviour in the environment as well as to identify exposure hot spots in soils relevant for hazard assessment for soil organisms, plants and assess groundwater threats.
Task 7.2 Development and application of environmentally relevant organism exposure protocols and Task 7.4 Interaction with environmentally relevant species, including ‘sentinel’ species.
When assessing the impacts of new generations of NMs, it is essential to combine quantitative exposure assessment with effect assessment, independent of the type of toxicity assessment. Amongst others, time weighted average (TWA) particle concentrations are well suited to express the effective exposure. Although TWA concentrations are commonly based in mass-units, it is also possible to express TWA in alternative dose expressions like number of particles and total surface area to which the biota are exposed. When assessing TWA concentrations, it was found to be essential to explicitly take particle dissolution as well as sedimentation into account as both processes can (dependent on the composition of the medium in terms of especially ionic strength and intrinsic particle properties like chemical composition of particle core and particle coating) be extremely fast with half-live as low as just a few minutes.
Homologies series of NMs allowed to assess that particle shape and particle size are the key properties affecting acute toxicity of NMs. In most cases, the impact of shape and size is related to the kinetics of ion release of the particles. Studies using homologues series of Pb-based perovskites, Ag, Cu and ZnO NMs indicated that in most cases, toxicity is dictated by the release of toxic metal ions whereas in general the contribution to acute of the NMs is limited. No acute effects were found for stable NMs like TiO2.
An ex vivo exposure of embryos of the waterflea Daphnia magna to polystyrene NMs (PSNMs) demonstrated a similar accumulation of PSNMs in or on lipophilic cells, illustrating the likelihood of brood pouch-mediated PSNMs uptake by embryos. By demonstrating embryo PSNM uptake via the brood pouch, novel insights in bioaccumulation of NMs and likely other lipophilic contaminants, were obtained. Since this uptake route can occur within a diverse array of aquatic organisms, it is clear that brood pouch-mediated accumulation needs to be considered in studies of the hazards and risks of NMs contamination. Up till now, this exposure route is ignored in such kind of studies despite the fact that the embryos thus affected may well be inherently sensitive to NMs.
Particle uptake of PSNMs in zebrafish embryos was found to be restricted to oral exposure, whereas the dermal route resulted in adsorption to the epidermis and gills only. Ingestion followed by biodistribution was observed to be size-dependent with uptake possible only for relative small particles of 25 and 50 nm. The particles spread through the body and eventually accumulated in specific organs and tissues such as the eyes. Particles larger than 50 nm were predominantly adsorbed onto the intestinal tract and outer epidermis of zebrafish embryos. Embryos exposed to particles via both epidermis and intestine showed highest uptake and eventually accumulated particles in the eye, whereas uptake of particles via the chorion and epidermis resulted in marginal uptake. Organ uptake and internal distribution should be monitored more closely to provide more in depth information of the toxicity of NMs.
One important focus of WP7 was to investigate relevant exposure conditions. One way of assessing this was to evaluate hazard via aqueous and dietary exposures on endpoints at various levels of biological organization. Another important area of investigation was to assess interactions between NMs and possible co-contaminants.
The main findings were: 1) sorption of environmental contaminants on NMs can change the bioavailability of the contaminant in the aqueous phase. Specifically, sorption of copper and benzo(a)pyrene (under fluorescent light) on NMs reduced the adsorbent bioavailability. In contrast, benzo(a)pyrene and anthracene, when adsorbed on TiO2 or Si NMs, were photo-catalysed under UVA and in the case of benzo(a)pyrene, highly toxic photo-by-products showed increased bioavailability in the presence of the NMs; 2) higher toxicity was found when NMs were in dispersion, indicating toxicity is related to the dispersion state of NMs; 3) lead-halide perovskite acute toxicity was attributed to lead ion dissolution based on induction of metallothionein 2 gene expression through aqueous and dietary exposure, and 4) the perovskite-spiked diets did not disrupt zebrafish gut microbiome after a 14-d exposure while disruption of gut microbiota by equivalent Pb(NO3)2 diets was observed.
As a general conclusion of the co-contaminant study (‘trojan-horse’), sorption of copper was confirmed on negatively charged NMs with sorption being closely related to surface area. Sorption of photo-labile organic compounds occurred on semi-conductor NMs which under UVA promoted photo-catalysis of the organic compounds. Toxicity of adsorbed contaminants in aquatic organisms strongly related to sorption efficiency and while copper sorption on NMs showed a protective mechanism reducing copper bioavailability in larval zebrafish and C. vulgaris, the NMs under UVA can catalyse organic compounds to toxic photo-by-products and obtained in this study indicate that benzo(a)pyrene photo-by-product bioavailability was increased in the presence of NMs. This study also indicated that controlled conditions of NM exposure in the aqueous phase lead to consistent results and higher toxicity of NM in zebrafish larvae indicating that NM toxicity depends on NM aqueous dispersion. Ag nano-prisms and CeO2 NMs showed highest toxicity in larval zebrafish and C. vulgaris compared to other FNN NMs used. Regarding possible modified assay conditions for improved exposure, the designed exposure chamber can provide the start for a standardised methodology that would limit variation in results among laboratories and will enable to critically compare data and conduct correct risk assessment. It would be interesting to further investigate the physicochemistry of NM during the controlled conditions of the fish larvae toxicity test using the exposure chamber, hence link toxicity with NM physicochemistry.
The perovskite studies indicate that lead dissolution is responsible for lead-halide perovskite NM toxicity observed in all studies, using different test models. The perovskite NMs dissolved lead in the aqueous phase and via dietary exposure in zebrafish inducing a metal bioavailability specific biomarker. This study provided an example of indirect effects of NMs in the aquatic environment as well as possible routes of exposure to perovskite NMs. Finally, bioavailability and gene expression have proven sensitive and environmentally relevant analytical tools for investigation of NMs toxicity in the aqueous phase. Sorption of co-contaminants onto NMs was successfully identified and lead-halide perovskite NMs toxicity was attributed to lead ion dissolution.
The hazard studies conducted with the Microtox assay in a miniaturized 96-well format, using synthetic media containing 2% NaCl, revealed that NMs constituted of the same elements may have different toxicity. This difference in their toxicity was mainly correlated to their stability, and, for some of them a higher ion release in the media. An additional valuable information was that the shape of the NMs affect their toxicity. Furthermore the great number of NMs that were tested during this project by using this Microtox assay suggest that this test, which is already used for chemical toxicity testing, seems to be a good tool for fast screening of NMs toxicity in saline conditions (water with 2% of NaCl).
Chronic studies with Daphnia magna were compared to the results of short term acute studies. Results indicate that during acute tests the smaller NMs showed highest toxicity whereas chronic tests showed higher toxicity on exposures to larger NMs. This indicates the importance of test in defining endpoints.
The different NMs were analysed using nanomaterial tracking analysis (NTA) in different environmental media (MilliQ water; synthetic Daphnia medium or NaCl 2%) immediately after resuspension or after 7 days of incubation at room temperature. Most of the NMs studied had the tendency to aggregate. It was observed that NMs in deionised water (very low IS) were much less aggregated compared to NMs in synthetic Daphnia medium or NaCl 2%. The present results show that various cations and anions present in synthetic medium had strong impact on the NM stability and to aggregation/agglomeration.
The processes of aggregation and agglomeration significantly influence the fate and behaviour of NMs in the environment, with an impact on particle properties (e.g. size, chemical composition, surface charge) as well as environmental conditions (e.g. mixing rates, pH, and natural organic matter). The present results show that the IS affected the particle sizes of the NMs and led to higher aggregation/agglomeration of the NMs.
Work Package 8: Key elements of dissemination, potential changes to risk assessment approach for future nanomaterials
Task 8.1 was addressed by facilitating the content and format evolution of the CODATA Uniform De3scription System for Materials on the Nanoscale www.codata.org/nanomaterials (UDS) into a form that can be used for developing categorization schemes for nanomaterials in a variety of contexts. Specifically, the UDS provides a systematic and cohesive methodology for describing and identifying engineered and natural nanomaterials. The UDS allows data repositories, databases, and research publications to integrate disparate measurement results, such that categorization schemes and read-across techniques can be done on a scientifically sound foundation. What UDS does, in short, is provide an effective technology ensuring that referenced ENMs are correctly described and identified.
Furthermore, RIVM obtained significant results within Tasks 8.2 and 8.3 during the reporting period in relation to the development of the constituents of the tools, either of those that have already been established or of those that are still being set in place for the safe innovation using novel generations of ENMs. These tools are to be integrated in a systemic approach towards reducing uncertainties in risk assessment, and include:
•Predictive modelling of the fate of ENMs and their toxicity based on NP properties (e.g. size, shape, composition), as developed within FNN for both in vitro and in vivo endpoints;
•Operationalization of grouping and read-across approaches to facilitate safety assessment while avoiding extensive experimental testing;
•Integration of in vitro and in vivo approaches for safety assessment
The tool integration in the general assessment of ENMs’ safety involves four tasks in a multidisciplinary systemic approach, as follows:
1)Systemic plan for ENMs production, analysis and classification;
2)Study of ENM evolution in the environments to which they are exposed;
3)Study of ENMs biological effects and complex biological responses in both healthy and compromised states;
4)Understanding the fate, transformation and effects of ENMs in relevant environmental scenarios.
Subsequently, a model infrastructure that assists industry (especially SMEs) in systematically addressing safety in innovation was set in place. It proved to be a suitable model for implementing scientific findings in innovation that takes into account safety considerations from the early stages of product development.
TNO provided input for WP8, especially for Tasks 8.2 8.4 and 8.5 regarding cross-cutting themes between WP3 and WP8, considering an overarching program that specifically takes into account nanosafety investment actively promoting innovation. Two distinct types of activities were used in WP3 to provide the information relevant for WP8: i) release, exposure and risk assessment of ENMs and ii) road-mapping of the value chain. The crucial link between different WPs in FNN is represented by the interplay between ENM release, exposure and hazard, and this point was emphasized, as it determines occupational and environmental risk. TNO highlighted the importance the safe-by-design (SbD) approaches and risk management measures for the control of particulate matter emission and proposed a link with Nanoreg2 for the Safe Innovation Approach. Furthermore, the Stage Gate Idea-to-Launch model was suggested as an appropriate basis for adopting SbD activities at various stages of the innovation process.
A close connection with industry representatives was established within WP8 by TUL, for example with remediation companies (Aquatest AS, Aecom CZ and Microchem), membrane producers (KST Membrade) and façade companies (Progotherm). These collaborations are ongoing and TUL’s activity within the Advanced Materials Industrial Association (AMIA) platform facilitated the exchange of information and ideas on approaches for safe nanotechnology between industry and researchers.
Within D8.3 TUL described the influence that environmental media and endotoxins present in ENM samples have on risk assessment. Further, TUL took part in D8.5 by describing a green synthesis of Au NPs using natural polymers and by subsequently studying the characteristics and biological effects of these NPs.
The effects of graphene oxide prepared at UCD in collaboration with FIAT, were investigated by TUL, as the material is highly relevant for value chain 4, Transportation/Industry. Using the soil bacterium Pseudomonas putida and nitrifying bacteria for wastewater treatment plant activated sludge as test systems, results that can help the translation to market of a novel nano-lubricant were obtained and included in Deliverable 8.6. They provide specific information for the risk assessment and regulation of a novel ENM. Furthermore, studies on two perovskite materials obtained from EPFL – FALI and MAFA(67PC)LI – relevant for value chain 1, Energy harvesting, were performed and the results, which facilitate the potential translation to market of new perovskite ENMs, were also included in D8.6.
Although SMEs are extensively involved in research of nanotech-based products, steps in commercializing have high uncertainty, risk of delay or even low returns during the initial phase, therefore large investments by investors and multinational companies are required. An accurate risk assessment of nanotech-based products may reduce uncertainties and risk margins, which in turn may save time and resources. By addressing safety before marketing along the innovation chain, risk assessment may attract investor's interest and funds, thus ensuring good returns to SMEs and industries. The partners aimed to address these issues in Task 8.4. Silver nanoparticles (Ag NPs) are some of the most widely used ENMs in commercial products. A key step in the risk assessment of Ag NPs is their physicochemical characterization. Among others, parameters like size, size distribution, agglomeration or aggregation status of colloidal silver may provide useful indications on either worker or consumer exposure. FILARETE investigated a reliable protocol to assess the colloidal stability of Ag NPs of different sizes and coatings in two biological media, to correlate the Ag NPs physicochemical properties with their in vitro/in vivo behaviour and support the comprehension of Ag NPs toxicological data. Noteworthy, the Ag NPs characterization reported in this protocol require a standard analytical instrument (UV-vis spectrophotometer) that is widely used in industry and is expected to provide useful insights to evaluate current and potential impact of Ag NPs in the industrial landscape, as well as to identify the need for specific actions in the (risk) management of these nanomaterials.
Within Tasks 8.2 and 8.4 CEA explored and evaluated alternatives to current exposure metrics under the frame of the OECD general tiered approach for exposure assessment. The possible parameters to be used in the exposure assessment scheme were defined, along with a list of available methods, tools and devices for their measurement. Furthermore, within Task 8.5 innovative methods and devices that could overcome some of the current difficulties of field measurement were developed. Specifically, CEA established and evaluated the concept of a novel, easy-to-calibrate and transportable device that could provide a size distribution in real time, as well as the chemical composition for each size bin off-line. Several prototypes were designed, built and evaluated using both simulation tools (Finite Elements Analysis - COMSOL Multiphysics) and experimental approaches with controlled aerosols in a laboratory setting. The devices were iteratively improved and are currently reaching TRL 4. Two patent applications filled in July 2015 were extended in 2017. The innovation is to sort and collect charged particles in specific places of a circular substrate based on their electrical mobility. The concept developed through this task allows correlating size and chemical composition of airborne particles and it holds the promise of facilitating the discrimination of secondary sources of airborne particles in occupational settings.
Several field measurements campaigns along with Tier 1, Tier 2 and Tier 3 evaluations were conducted during the project to generate new information on the operative conditions and levels of exposure for several case studies on next-generation nanomaterials:
•Production of nano-enabled electrodes for Li-ion batteries
•End-of-life of nano-enabled electrodes for Li-ion batteries
•Production of thermoelectric generators based on nanostructured silicides
•Additive manufacturing
These campaigns allowed identifying experimental challenges related to emissions measurement in occupational settings and contributed to confirm that research on the development of personal devices was required. Such tools could be particularly useful for Tier 2 evaluation in SMEs and small-scale production lines.
The best practices identified throughout the technical activities conducted during the project were regrouped in a manual along with generic guidelines for ensuring exposure-driven safety of the next generation nanomaterials being developed for industrial applications. This manual will allow workplace hygienist, workers and executives to make appropriate decisions to tackle efficiently risks associated with ENMs.
Work Package 9: Liaison with NMP projects, national and other international projects
The activities within RIVM focussed in particular on outreach of the results obtained within WPs 6, 7, and 8. RIVM contributed to three smaller workshops: one on classification of the next generation of nanomaterials (Tasks 9.1 and 9.2) one workshop organized within the 2017 SETAC meeting in Brussels, and a small workshop organized within the EuroNanoforum meeting that was organized in Malta in 2017. The workshop organized within the broader SETAC annual meeting focussed on dissemination of the key results obtained within WP7, whereas the workshop organized during the EuroNanoforum focussed on options for categorisation of future generations of nanomaterials. The latter workshop also served as the preparatory workshop for the bigger workshop on categorisation, organised in November 2017 in Brussels. Apart from these activities, project results were disseminated by means of nine publications in peer-reviewed scientific journals, one book chapter, 11 oral presentations during scientific conferences, two presentations during regulatory meetings, and three poster presentations during scientific conferences.
The major scientific result for CODATA partner was progress on developing a consensus on categorization of next generation nanomaterials. Through two workshops that brought together international experts on this subject, FNN fostered a more detailed level of discussion of the need for, contexts of, and approaches to categorization.
Potential Impact:
Work Package 3: Roadmapping of value chain materials platforms, and consequently their exposure scenarios
WP3 outcomes will help to identify hotspot areas of concern, facilitate measurement strategies and address improvement of working conditions. As mentioned by IUTA, TNO and various other partners, the project had a substantial impact within the scientific community as is shown by various contributions to scientific conferences. All partners were able to maintain and further develop its scientific base with regard to release and exposure assessment. Even though no directly exploitable results are obtained, the knowledge gained, especially for the mass flow analysis, leads to further reputation possibly resulting in new scientific or industrial projects. In view of nanosafety the work conducted leads to a better understanding of release hot spots, not only for novel next generation nanomaterials, and possible countermeasures, thus contributing to the safety-by-design principle. Different concepts for release assessment were integrated into a coherent approach and not only occupational hygienists working in the field of nanomaterials can make use of the concepts conceived. The overview of methods and devices with regard to exposure assessment of nanomaterials is a valuable tool to decide on the best choice for an appropriate and cost-efficient measurement approach to assess release of and exposure to (next-generation) nanomaterials.
The work conducted during the project was to be beneficial in FutureNanoNeeds but more importantly beyond, as for instance in other EU projects, to the scientific community, regulators and stakeholders. Information on the methods, devices, tools, and exposure assessment strategies, that were developed, used and evaluated throughout the project, will allow regulators and industry to make appropriate choices to implement efficiently harmonized approaches for specific exposure situations to next generation nanomaterials. Manual of best practice will be directly applicable in laboratories and in industry to promote a safer, sustainable and responsible development of nanotechnology.
For instance, the technical activities conducted, and the experience gathered on the thermoelectric generators (TEG) value chain will be directly applicable in the EU pilot project INTEGRAL which started in January 2017. It’s ambition to scale-up safely the European production of TEG modules through partnerships between public research institutes, industrial research teams and SMEs can be aided by our work.
The results and deliverables produced on WP3 are expected to be useful for NANOGAP in terms of comparing data for similar products and for the better design of new nanomaterials that will develop in the middle and long term. This will increase products quality and could be useful in terms of commercialization. On the other hand, the implication on the VCAC allowed us to obtain useful commercial information from the different value chains and the relative position of VC7 (AgNW, which we are producers) on the market. Consequently, all the information related to best practices (D3.11) and hot spots for the VC7 (D3.4) will improve our practices handling and producing nanomaterials, what will reduce environmental and workers exposure. Future spin-offs are anticipated for work that was performed within WP3, in particular in areas where there is an interest in approaches that address data poor scenarios. For example, possibilities to test the BBN shredding model and to subject the model to a more extensive expert elicitation and refinement. Further development of the thermo-electric VC in EU Integral pilot project has already been initiated.
Short-term impacts would be the use of best practices in laboratory, industry, and spin-offs for further development (e.g. refinement of MFA and BBN models). Long-term impacts could be the further development of measurement devices and their introduction in practice, and eventual implementation of matured methods and models. And further in the future, the harmonization to close the gap between release/exposure and hazard potential of NMs (based on outcomes of other FNN WPs) would be advantageous. Further research is therefore required to map and predict changes in particle morphology for different types of NMs and different release and exposure scenarios (in particular for end-of-life, mechanical recycling)
Overall, FNN provided a platform for WP3 partners to explore new innovative ideas on next generation NMs, e.g. release of NMs. It provided an opportunity to collaborate & exchange expertise between partners, but also with other EU projects (NanoReg, BUONAPART-E, etc).
Work Package 4: Synthesis and exhaustive characterization of materials of the future
The work done enables a high potential for scientific and technical innovation. This applies to the development of new production methods of new nanomaterials, such as complex hybrid NCs, NCs with doping, and shaped nanostructures. The developed nanomaterials will enable innovative and environmentally sound products.
The scientific and economic results generate a base of knowledge, which means that the WP4 partners strengthens their national and international role in the field of nanomaterial production, characterization, metrology, and assessment. As well as in the field of the transfer of functionalized materials in industrially processable products.
Within the framework of this project, innovative and future applications were investigated. These can go into series production in the subsequent period and have a high market potential. The pursuit of environmentally friendly and consumer-safe products is the goal here. The development of competence in this project expands the capabilities for risk assessment of nanomaterials, thus protecting consumers and securing jobs for many partners. The development of new technologies and methods has great potential for the industry.
Work Package 5: Biological and environmental interactions of future nanomaterials
The main impact emerging from this series of studies is the standardization of an integrated platform of preclinical imaging combining both in vivo (two-photon confocal microscopy, optical imaging) and ex vivo (confocal and super-resolution microscopy) approaches. This platform can be easily extended to other materials, if associated with fluorescent dyes, and other models of study. This could be extremely important to repeat other studies, even more related to the evaluation of the fate of NPs modifying parameters such as the geometry and/or topology of the components.
The overall effect of these studies in this work package are represented in novel nanomaterial’s interactions with biomolecules which has paramount importance. As we have shown in this work package, size and shape of nanomaterials can be critical parameters for such interactions, thus driving variety of biological identities for these nanomaterials. Our prospective is that the protocols and the structure-property relationship built in this work package will pave the way for better designed materials with established characterization methodologies.
Work Package 6: Biological identity, processes and human health hazards of future nanomaterials
Nanotechnologies are viewed as being the driving force behind a new industrial revolution which is expected to have profound socio-economic effects. Engineered NMs are the main players in this growth, and therefore they offer tremendous opportunities for industrial development in many different areas. However, any new technology could mean a risk to human health, so knowledge of its potential impact is a priority. Understanding the safety of NMs is therefore an issue for future nano-based products and a means to achieve greater public acceptance of nanotechnology and awareness of the overall benefits that it can bring. In accordance with the present project, this WP has been mainly focused on the development of an in vitro/in vivo platform for ranking the toxicological hazard associated with nanotechnology products. In designing and implementing our approach different classes of NMs were compared, in particular we paid a great attention on the potential influence of basic physico-chemical parameters (e.g. size, shape, surface, curvature) characterizing NPs. The end-points of the various screening tests applied were not only limited to macroscopic toxic effects such as cell death or alteration of morpho-functional features, but also to more subtle perturbation of the cell homeostasis and activation toward a “reactive” phenotype. This is a great point of innovation and could be extremely important for future studies aimed at predicting the first “prognostic” markers upon a low but chronic NM exposure (e.g. food, workplaces, pollution). Since this platform has been developed to the identification of highly reproducible and reliable hazard factors, useful for the future categorization of a wide range of industrial, biomedical or environmental NPs, the presence of industrial partners and regulatory bodies in the Consortium was instrumental to critically evaluate the data produced and drive to a more responsible and coherent control exploiting this integrated approach. We therefore strongly believe that this aspect will be particularly important to determine the relative hazard of newly-synthesized NMs before their large-scale production and market distribution. Another important impact raised fromm the platform generated within this WP, is the achievement of a deeper insight about nanomaterials-biomolecule interactions, with once again a great attention on the relevance of geometry and surface (coating). This aspect may be of extraordinary relevance for the prediction of the impact of NMs for both medical and toxicological purposes. The association between the exposure to different types of Nanomaterial with the increased incidence of alteration on the different biological targets (fluids, cells, animals) provided through this WP may give an interesting and original contribution towards the discovery of new diagnostic hallmarks and or risk/factors associated with NPs and also support the development of even more faster and cheaper and reliable methods of screening of new nanodrugs.
To briefly recapitulate the main impact emerging from this WP we can say that:
1) In general, the different tested NMs did not show acute toxicity. In a few cases, some acute toxicity was observed, but this can be easily explained on the base of the release of ions or oxidative burst (e.g. perovskites or CdSe). The main conclusion is that there are no big reasons to consider NMs able to induce acute toxicity when they are stable and not releasing ions. This is crucial to reduce the threshold of perplexity of citizens toward these materials and to create a greater feeling on this field.
2) Regarding chronic toxicity, some nanomaterials have shown the potential to induce inflammasome activation. Health effects of chronic exposure to nanomaterials that can be related to their inflammasome activating properties are as yet largely unknown, although using nanomaterial libraries a correlation between induction of inflammasome activation in vitro and lung fibrosis in vivo was in fact shown. Inflammasome activation as such has been related to various chronic diseases, albeit with different degrees of certainty. Our proposal would be to include inflammasome activation as one of the assays in the Immunotoxicity element of the flow chart as proposed in the nanospecific approach for risk assessment that has been developed within the EU NANoREG project. Moreover, we proposed to include this assay in the safety evaluation of nanomedicinal products. This would be crucial to give a deeper and broader view of the effects of NMs and to furthermore restrict the possibility to produce and diffuse at the large scale dangerous materials containing NPs.
3) The observed shape- and doping related effects can be used within a Safe-by-Design/Safe Innovation approach as exemplified recently by us for graphene. Interestingly, their geometries can greatly influence their sub-lethal effect on critical biological processes, which may contribute to disease development. However, in the real world the effects played by nanoparticles almost always occur in the presence of bio-active compounds, such as biological immunogens, chemical allergens or surfactants, which may undergo a complex interplay with the nanoparticles in their environment, and thus give rise to unexpected toxicity mechanisms. For this reason our integrated platform could be a great resource even for regulatory items in terms of nanosafety.
4) Finally, the approach developed in this platform is not exclusively limited to be exploited for industrial and biomedical aims but could be a strong added-value in many academic or industrial projects, including some of the next H2020 NMBP.
Work Package 7: Exposure and environmental interactions of future nanomaterials
The large numbers and diversity of next generation NMs preclude having comprehensive studies carried out on the environmental data and effects of all emerging NMs. Therefore, scientifically sound methods and approaches, as the ones developed under Tasks 7.2 and 7.4 will enable researchers, industry and government agencies to assess the potential for hazard (and therefore, risk) more efficiently and effectively. Furthermore, studies undertaken under Tasks 7.1 and 7.3 allow an improved understanding of modifications novel NM may undergo and therefore will facilitate interpretation of exposure conditions.
The key results from WP7 were primarily disseminated by means of scientific publications, presentations at scientific meetings, and meetings with regulatory authorities (OECD, ECHA), industry and SMEs. It was thus warranted that the key results are directly implemented in for instance the design of novel classes of NMs. In this case this implementation of key project finings dealt with the safety assessment of biobased NMs consisting of nanocellulose (impact of size and shape on ecotoxicity of nanocellulose), and the safety assessment of novel types of metallic-based NMs used in photovoltaic cells, including nanowires consisting of In/Ga/P. In both cases, SMEs were assisted in the development of the novel materials indicated, this inducing a direct socio-economic impact. Thereupon, the project results are already included in nano-specific guidance related to future regulation of NMs. This includes the development of draft OECD guidance documents on fate and effect assessment of NMs, and nano-specific guidance on regulatory assessment of NMs within REACH.
In summary, the project had a relevant impact within the scientific community as is shown by various contributions to scientific conferences. Furthermore, the collaboration with international partners was strengthened.
Work Package 8: Key elements of dissemination, potential changes to risk assessment approach for future nanomaterials
The large numbers and diversity of next generation nanomaterials preclude having comprehensive measurements done on all emerging nanomaterials. Consequently, scientifically sound categorization methods, as the ones developed under Task 8.1 will enable researchers, industry and government agencies to assess the potential for risk more efficiently and effectively.
The key results from WP8 were primarily disseminated by means of scientific publications, presentations at scientific meetings, and meetings with regulatory authorities (OECD, ECHA), industry and SMEs. Furthermore, two patent applications were filed, and other peer-reviewed papers are in the process of being published. By interacting with and making our work and results known to different stakeholders, it was warranted that the key technical results are directly implemented in, for instance, the design of novel classes of ENMs and in safe product innovation. RIVM applied this approach in a case study that focussed on safety assessment of newly developed biobased (cellulose) nanomaterials by SMEs that worked on the development of new applications for nanocellulose.
Furthermore, the technical activities conducted by CEA and the experience gathered on the thermoelectric generators (TEG) value chain will be directly applicable in the EU pilot project INTEGRAL that started in January 2017. This project aims at safely scaling-up the European production of TEG modules through partnerships between public research institutes, industrial research teams and SMEs.
The work conducted during the project was to be beneficial in FutureNanoNeeds but, more importantly beyond, for instance in other EU projects, to the scientific community at large, to regulators and industrial stakeholders. Information on the methods, devices, tools, and exposure assessment strategies, that were developed, used and evaluated throughout the project, will allow regulators and industry to make appropriate choices to implement efficiently harmonized approaches for specific exposure situations to next generation nanomaterials. The manual of best practice will be directly applicable in laboratories and in industry to promote a safer, sustainable and responsible development of nanotechnology. The main beneficiaries span industry (cosmetics, food, biocidal products, medical devices, pharmaceuticals), waste management bodies, authorities who regulate occupational exposure to ENMs, the workers affected by said regulations, as well as the environment at large.
Work Package 9: Liaison with NMP projects, national and other international projects
The main dissemination activities within RIVM focussed on outreach of the results obtained within WPs 6, 7, and 8. RIVM contributed to three smaller workshops: one on classification of the next generation of nanomaterials (Tasks 9.1 and 9.2) one workshop organized within the 2017 SETAC meeting in Brussels, and a small workshop organized within the EuroNanoforum meeting that was organized in Malta in 2017. The workshop organized within the broader SETAC annual meeting focussed on dissemination of the key results obtained within WP7, whereas the workshop organized during the EuroNanoforum focussed on options for categorisation of future generations of nanomaterials. The latter workshop also served as the preparatory workshop for the bigger workshop on categorisation, organised in November 2017 in Brussels. Apart from these activities, project results were disseminated by means of nine publications in peer-reviewed scientific journals, one book chapter, 11 oral presentations during scientific conferences, two presentations during regulatory meetings, and three poster presentations during scientific conferences, and a stand at Nanotexnology 2017.
The large numbers and diversity of next generation nanomaterials preclude having comprehensive measurements done on all emerging nanomaterials. Consequently, scientifically sound categorization methods (CODATA and NIUD UCD) will enable researchers, industry and government agencies to assess the potential for risk more efficiently and effectively.
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