Development of a fast and simple method for the isolation of superparamagnetic iron oxide nanoparticles protein corona from protein-rich matrices.

The adsorption of proteins onto the surface of nanoparticle (NP) leads to the formation of the so-called "protein corona" as consisting both loosely and tightly bound proteins. It is well established that the biological identity of NPs that may be acquired after exposure to a biological matrix is mostly provided by the components of the hard corona as the pristine surface is generally less accessible for binding. For that reason, the isolation and the characterisation of the NP-corona complexes and identification of the associated biomolecules can help in understanding its biological behaviour. Established methods for the isolation of the NP-HC complexes are time-demanding and can lead to different results based on the isolation method applied. Herein, we have developed a fast and simple method using ferromagnetic beads isolated from commercial MACS column and used for the isolation of superparamagnetic NP following exposure to different types of biological milieu. We first demonstrated the ability to easily isolate superparamagnetic iron oxide NPs (IONPs) from different concentrations of human blood plasma, and also tested the method on the corona isolation using more complex biological matrices, such as culture medium containing pulmonary mucus where the ordinary corona methods cannot be applied. Our developed method showed less than 20% difference in plasma corona composition when compared with centrifugation. It also showed effective isolation of NP-HC complexes from mucus-containing culture media upon comparing with centrifugation and MACS columns, which failed to wash out the unbound proteins. Our study was supported with a full characterisation profile including dynamic light scattering, nanoparticle tracking analysis, analytical disk centrifuge, and zeta potentials. The biomolecules/ proteins composing the HC were separated by vertical gel electrophoresis and subsequently analysed by liquid chromatography-tandem mass spectrometry. In addition to our achievements in comparing different isolation methods to separate IONPs with corona from human plasma, this is the first study that provides a complete characterisation profile of particle protein corona after exposure in vitro to pulmonary mucus-containing culture media.

The role of extracellular vesicles in non-small-cell lung cancer, the unknowns, and how new approach methodologies can support new knowledge generation in the field.

Extracellular vesicles (EVs) are nanosized particles released from most human cell types that contain a variety of cargos responsible for mediating cell-to-cell and organ-to-organ communications. Current knowledge demonstrates that EVs also play critical roles in many aspects of the progression of Non-Small-Cell Lung Cancer (NSCLC). Their roles range from increasing proliferative signalling to inhibiting apoptosis, promoting cancer metastasis, and modulating the tumour microenvironment to support cancer development. However, due to the limited availability of patient samples, intrinsic inter-species differences between human and animal EV biology, and the complex nature of EV interactions in vivo, where multiple cell types are present and several events occur simultaneously, the use of conventional preclinical and clinical models has significantly hindered reaching conclusive results. This review discusses the biological roles that EVs are currently known to play in NSCLC and identifies specific challenges in advancing today's knowledge. It also describes the NSCLC models that have been used to define currently-known EV functions, the limitations associated with their use in this field, and how New Approach Methodologies (NAMs), such as microfluidic platforms, organoids, and spheroids, can be used to overcome these limitations, effectively supporting future exciting discoveries in the NSCLC field and the potential clinical exploitation of EVs.

A Method for Culturing 3D Tumoroids of Lung Adenocarcinoma Cells at the Air-Liquid Interface.

Three-dimensional (3D) tumor spheroids and tumoroids are among the most exploited cell culture methods in the lung cancer field, finding applications in the investigation of tumor growth and proliferation, invasion, and drug screening. However, 3D tumor spheroids and tumoroids cannot fully mimic the architecture of the human lung adenocarcinoma tissue and, in particular, the direct contact of the lung adenocarcinoma cells with the air, as they lack polarity. Our method allows to overcome this limitation by enabling to grow tumoroids of lung adenocarcinoma cells and healthy lung fibroblasts at the Air-Liquid Interface (ALI). This ensures straightforward access to both the apical and basal surface of the cancer cell culture, with several advantages in drug screening applications.

The Current Status and Work of Three Rs Centres and Platforms in Europe.

The adoption of Directive 2010/63/EU on the protection of animals used for scientific purposes has given a major push to the formation of Three Rs initiatives in the form of centres and platforms. These centres and platforms are dedicated to the so-called Three Rs, which are the Replacement, Reduction and Refinement of animal use in experiments. ATLA's 50th Anniversary year has seen the publication of two articles on European Three Rs centres and platforms. The first of these was about the progressive rise in their numbers and about their founding history; this second part focuses on their current status and activities. This article takes a closer look at their financial and organisational structures, describes their Three Rs focus and core activities (dissemination, education, implementation, scientific quality/translatability, ethics), and presents their areas of responsibility and projects in detail. This overview of the work and diverse structures of the Three Rs centres and platforms is not only intended to bring them closer to the reader, but also to provide role models and show examples of how such Three Rs centres and platforms could be made sustainable. The Three Rs centres and platforms are very important focal points and play an immense role as facilitators of Directive 2010/63/EU 'on the ground' in their respective countries. They are also invaluable for the wide dissemination of information and for promoting the implementation of the Three Rs in general.

The Case for Modernizing Biomedical Research in Ireland through the Creation of an Irish 3Rs Centre.

Since its publication, the 3Rs principle has provided a cornerstone for more ethical and humane biomedical and regulatory research. In Europe, the 3Rs principle has been incorporated into the European Directive 63/2010/EU, with the ultimate aim of fully replacing the procedures on live animals for scientific and educational purposes as soon as it is scientifically possible to do so. Thus, a critical shift in the discussion on animal use in biomedical and regulatory research is undergoing in Europe, a discussion where satisfying the "replacement" principle is becoming more and more defined as a scientific rather than ethical need. 3Rs Centres have been established in recent years across Europe. To date, Ireland has no 3Rs Centre, and the uptake of the 3Rs principle, and in particular of the "replacement" aspect, has been slow. In this Commentary, we present the Irish context of the use of animal models in biomedical and regulatory research, and urge for what, in the authors' opinion, are the most critical actions that Ireland must undertake to align its biomedical (basic, applied and translational) research with the European 3Rs strategy.

The Rise of Three Rs Centres and Platforms in Europe.

Public awareness and discussion about animal experiments and replacement methods has greatly increased in recent years. The term 'the Three Rs', which stands for the Replacement, Reduction and Refinement of animal experiments, is inseparably linked in this context. A common goal within the Three Rs scientific community is to develop predictive non-animal models and to better integrate all available data from in vitro, in silico and omics technologies into regulatory decision-making processes regarding, for example, the toxicity of chemicals, drugs or food ingredients. In addition, it is a general concern to implement (human) non-animal methods in basic research. Toward these efforts, there has been an ever-increasing number of Three Rs centres and platforms established over recent years - not only to develop novel methods, but also to disseminate knowledge and help to implement the Three Rs principles in policies and education. The adoption of Directive 2010/63/EU on the protection of animals used for scientific purposes gave a strong impetus to the creation of Three Rs initiatives, in the form of centres and platforms. As the first of a series of papers, this article gives an overview of the European Three Rs centres and platforms, and their historical development. The subsequent articles, to be published over the course of ATLA's 50th Anniversary year, will summarise the current focus and tasks as well as the future and the plans of the Three Rs centres and platforms. The Three Rs centres and platforms are very important points of contact and play an immense role in their respective countries as 'on the ground' facilitators of Directive 2010/63/EU. They are also invaluable for the widespread dissemination of information and for promoting implementation of the Three Rs in general.

Three-dimensional (3D) liver cell models - a tool for bridging the gap between animal studies and clinical trials when screening liver accumulation and toxicity of nanobiomaterials.

Despite the exciting properties and wide-reaching applications of nanobiomaterials (NBMs) in human health and medicine, their translation from bench to bedside is slow, with a predominant issue being liver accumulation and toxicity following systemic administration. In vitro 2D cell-based assays and in vivo testing are the most popular and widely used methods for assessing liver toxicity at pre-clinical stages; however, these fall short in predicting toxicity for NBMs. Focusing on in vitro and in vivo assessment, the accurate prediction of human-specific hepatotoxicity is still a significant challenge to researchers. This review describes the relationship between NBMs and the liver, and the methods for assessing toxicity, focusing on the limitations they bring in the assessment of NBM hepatotoxicity as one of the reasons defining the poor translation for NBMs. We will then present some of the most recent advances towards the development of more biologically relevant in vitro liver methods based on tissue-mimetic 3D cell models and how these could facilitate the translation of NBMs going forward. Finally, we also discuss the low public acceptance and limited uptake of tissue-mimetic 3D models in pre-clinical assessment, despite the demonstrated technical and ethical advantages associated with them. 3D culture models for use as in vitro alternatives to traditional methods and conventional in vivo animal testing for testing liver accumulation and toxicity of nanobiomaterials.

Towards More Predictive, Physiological and Animal-free In Vitro Models: Advances in Cell and Tissue Culture 2020 Conference Proceedings.

Experimental systems that faithfully replicate human physiology at cellular, tissue and organ level are crucial to the development of efficacious and safe therapies with high success rates and low cost. The development of such systems is challenging and requires skills, expertise and inputs from a diverse range of experts, such as biologists, physicists, engineers, clinicians and regulatory bodies. Kirkstall Limited, a biotechnology company based in York, UK, organised the annual conference, Advances in Cell and Tissue Culture (ACTC), which brought together people having a variety of expertise and interests, to present and discuss the latest developments in the field of cell and tissue culture and in vitro modelling. The conference has also been influential in engaging animal welfare organisations in the promotion of research, collaborative projects and funding opportunities. This report describes the proceedings of the latest ACTC conference, which was held virtually on 30th September and 1st October 2020, and included sessions on in vitro models in the following areas: advanced skin and respiratory models, neurological disease, cancer research, advanced models including 3-D, fluid flow and co-cultures, diabetes and other age-related disorders, and animal-free research. The roundtable session on the second day was very interactive and drew huge interest, with intriguing discussion taking place among all participants on the theme of replacement of animal models of disease.

Preclinical Development of Orally Inhaled Drugs (OIDs)-Are Animal Models Predictive or Shall We Move Towards In Vitro Non-Animal Models?

Respiratory diseases constitute a huge burden in our society, and the global respiratory drug market currently grows at an annual rate between 4% and 6%. Inhalation is the preferred administration method for treating respiratory diseases, as it: (i) delivers the drug directly at the site of action, resulting in a rapid onset; (ii) is painless, thus improving patients' compliance; and (iii) avoids first-pass metabolism reducing systemic side effects. Inhalation occurs through the mouth, with the drug generally exerting its therapeutic action in the lungs. In the most recent years, orally inhaled drugs (OIDs) have found application also in the treatment of systemic diseases. OIDs development, however, currently suffers of an overall attrition rate of around 70%, meaning that seven out of 10 new drug candidates fail to reach the clinic. Our commentary focuses on the reasons behind the poor OIDs translation into clinical products for the treatment of respiratory and systemic diseases, with particular emphasis on the parameters affecting the predictive value of animal preclinical tests. We then review the current advances in overcoming the limitation of animal animal-based studies through the development and adoption of in vitro, cell-based new approach methodologies (NAMs).

Docetaxel gold complex nanoflowers: A chemo-biological evaluation for their use as nanotherapeutics.

Docetaxel (DTX) is an anticancer treatment widely used in the clinic for the treatment of various human malignancies, including Non-Small-Cell Lung Cancer (NSCLC). Its low water solubility and systemic toxicity, however, negatively impact the clinical application of such drug. In order to improve DTX solubility in biological fluids and decrease its adverse effects in patients, the scientific community is currently focusing on developing drug delivery systems where DTX is the payload. In this context, the present study aims at presenting a step forward in the development of platforms based on gold complexes for multifunctional approaches (theragnostic tools) and stimuli-responsive therapies. Tetrachloroauric acid (HAuCl4) were complexed with the antitumor drug and dicarboxylic acid-terminated polyethylene-glycol (PEG) to form the nanometric complex named DTX-Au-PEG. Following reduction with sodium borohydride (NaBH4), the DTX-Au-PEG complex formed hybrid-metal nanoparticles (DTX IN PEG-AuNPs), where DTX was protected in the gold core embedded within the polymer chains. To achieve therapeutic targeting, DTX-Au-PEG complex and DTX IN PEG-AuNPs were chemical combined with the human anti-EGFR polyclonal antibody, which recognizes the hERG1 channel aberrantly expressed on the membrane of human lung cancer cells. The active targeting was demonstrated by various analytical techniques (Raman and UV-vis spectroscopies); whereas, in vitro experiments on tissue-mimetic, three-dimensional (3D) tumoroids grown at the Air-Liquid Interface (ALI) demonstrated that DTX encapsulation within a gold core strongly influenced the drug efficacy, with a significant increase of the DTX therapeutic index when AuNPs were specifically targeted against EGFR. Collectively, our study demonstrated that a drug delivery system based on Au (III)-DTX complexes constitutes an encouraging chemical approach to build Au (III) complexes into real chemotherapeutic drugs for cancer treatment.

In vitro Alternatives to Acute Inhalation Toxicity Studies in Animal Models-A Perspective.

When assessing the risk and hazard of a non-pharmaceutical compound, the first step is determining acute toxicity, including toxicity following inhalation. Inhalation is a major exposure route for humans, and the respiratory epithelium is the first tissue that inhaled substances directly interact with. Acute inhalation toxicity testing for regulatory purposes is currently performed only in rats and/or mice according to OECD TG403, TG436, and TG433 test guidelines. Such tests are biased by the differences in the respiratory tract architecture and function across species, making it difficult to draw conclusions on the potential hazard of inhaled compounds in humans. Research efforts have been therefore focused on developing alternative, human-relevant models, with emphasis on the creation of advanced In vitro models. To date, there is no In vitro model that has been accepted by regulatory agencies as a stand-alone replacement for inhalation toxicity testing in animals. Here, we provide a brief introduction to current OECD test guidelines for acute inhalation toxicity, the interspecies differences affecting the predictive value of such tests, and the current regulatory efforts to advance alternative approaches to animal-based inhalation toxicity studies. We then list the steps that should allow overcoming the current challenges in validating In vitro alternatives for the successful replacement of animal-based inhalation toxicity studies. These steps are inclusive and descriptive, and should be detailed when adopting in house-produced 3D cell models for inhalation tests. Hence, we provide a checklist of key parameters that should be reported in any future scientific publications for reproducibility and transparency.

ALI multilayered co-cultures mimic biochemical mechanisms of the cancer cell-fibroblast cross-talk involved in NSCLC MultiDrug Resistance.

BACKGROUND: Lung cancer is the leading cause of cancer-related deaths worldwide. This study focuses on its most common form, Non-Small-Cell Lung Cancer (NSCLC). No cure exists for advanced NSCLC, and patient prognosis is extremely poor. Efforts are currently being made to develop effective inhaled NSCLC therapies. However, at present, reliable preclinical models to support the development of inhaled anti-cancer drugs do not exist. This is due to the oversimplified nature of currently available in vitro models, and the significant interspecies differences between animals and humans. METHODS: We have recently established 3D Multilayered Cell Cultures (MCCs) of human NSCLC (A549) cells grown at the Air-Liquid Interface (ALI) as the first in vitro tool for screening the efficacy of inhaled anti-cancer drugs. Here, we present an improved in vitro model formed by growing A549 cells and human fibroblasts (MRC-5 cell line) as an ALI multilayered co-culture. The model was characterized over 14-day growth and tested for its response to four benchmarking chemotherapeutics. RESULTS: ALI multilayered co-cultures showed an increased resistance to the four drugs tested as compared to ALI multilayered mono-cultures. The signalling pathways involved in the culture MultiDrug Resistance (MDR) were influenced by the cancer cell-fibroblast cross-talk, which was mediated through TGF-ß1 release and subsequent activation of the PI3K/AKT/mTOR pathway. As per in vivo conditions, when inhibiting mTOR phosphorylation, MDR was triggered by activation of the MEK/ERK pathway activation and up-regulation in cIAP-1/2 expression. CONCLUSIONS: Our study opens new research avenues for the development of alternatives to animal-based inhalation studies, impacting the development of anti-NSCLC drugs.

A protein corona study by scattering correlation spectroscopy: a comparative study between spherical and urchin-shaped gold nanoparticles.

The study of protein interactions with gold nanoparticles (GNP) is a key step prior to any biomedical application. These interactions depend on many GNP parameters such as size, surface charge, chemistry, and shape. In this work, we propose to use a sensitive technique named scattering correlation spectroscopy or SCS to study protein interactions with GNP. SCS allowed the investigation of the GNP hydrodynamic radius with a very high sensitivity before and after interaction with proteins. No labeling is needed. As a proof-of-concept, two of the most used morphologies of GNP-based nanovectors have been used within this work: spherical-shaped GNP (GNS) and branched-shaped GNP (GNU). The measurement of several parameters such as the number of proteins binding to one GNP, the binding affinity and the cooperativeness of binding for three different plasma proteins on the GNP surface was carried out. While GNS showed an increase in the hydrodynamic radius, indicating that each kind of protein binds on the GNS in a specific orientation, GNU showed different orientations of proteins due to their multi-oriented surfaces (tips) with a higher surface to volume area. Quantitative data based on the Hill model were extracted to obtain the affinity of the proteins to both GNS and GNU surfaces. Data variations can be understood in terms of the electrostatic properties of the proteins, which interact differently with the negatively charged GNP surfaces.

Multilayered Cultures of NSCLC cells grown at the Air-Liquid Interface allow the efficacy testing of inhaled anti-cancer drugs.

Evidence supports the advantages of inhalation over other drug-administration routes in the treatment of lung diseases, including cancer. Although data obtained from animal models and conventional in vitro cultures are informative, testing the efficacy of inhaled chemotherapeutic agents requires human-relevant preclinical tools. Such tools are currently unavailable. Here, we developed and characterized in vitro models for the efficacy testing of inhaled chemotherapeutic agents against non-small-cell lung cancer (NSCLC). These models recapitulated key elements of both the lung epithelium and the tumour tissue, namely the direct contact with the gas phase and the three-dimensional (3D) architecture. Our in vitro models were formed by growing, for the first time, human adenocarcinoma (A549) cells as multilayered mono-cultures at the Air-Liquid Interface (ALI). The in vitro models were tested for their response to four benchmarking chemotherapeutics, currently in use in clinics, demonstrating an increased resistance to these drugs as compared to sub-confluent monolayered 2D cell cultures. Chemoresistance was comparable to that detected in 3D hypoxic tumour spheroids. Being cultured in ALI conditions, the multilayered monocultures demonstrated to be compatible with testing drugs administered as a liquid aerosol by a clinical nebulizer, offering an advantage over 3D tumour spheroids. In conclusion, we demonstrated that our in vitro models provide new human-relevant tools allowing for the efficacy screening of inhaled anti-cancer drugs.

Towards the Identification of an In Vitro Tool for Assessing the Biological Behavior of Aerosol Supplied Nanomaterials.

Nanoparticles (NP)-based inhalation systems for drug delivery can be administered in liquid form, by nebulization or using pressurized metered dose inhalers, and in solid form by means of dry powder inhalers. However, NP delivery to the lungs has many challenges including the formulation instability due to particle-particle interactions and subsequent aggregation, causing poor deposition in the small distal airways and subsequent alveolar macrophages activity, which could lead to inflammation. This work aims at providing an in vitro experimental design for investigating the correlation between the physico-chemical properties of NP, and their biological behavior, when they are used as NP-based inhalation treatments, comparing two different exposure systems. By means of an aerosol drug delivery nebulizer, human lung cells cultured at air-liquid interface (ALI) were exposed to two titanium dioxide NP (NM-100 and NM-101), obtained from the JRC repository. In parallel, ALI cultures were exposed to NP suspension by direct inoculation, i.e., by adding the NP suspensions on the apical side of the cell cultures with a pipette. The formulation stability of NP, measured as hydrodynamic size distributions, the cell viability, cell monolayer integrity, cell morphology and pro-inflammatory cytokines secretion were investigated. Our results demonstrated that the formulation stability of NM-100 and NM-101 was strongly dependent on the aggregation phenomena that occur in the conditions adopted for the biological experiments. Interestingly, comparable biological data between the two exposure methods used were observed, suggesting that the conventional exposure coupled to ALI culturing conditions offers a relevant in vitro tool for assessing the correlation between the physico-chemical properties of NP and their biological behavior, when NP are used as drug delivery systems.

Culturing substrates influence the morphological, mechanical and biochemical features of lung adenocarcinoma cells cultured in 2D or 3D.

Alternative models such as three-dimensional (3D) cell cultures represent a distinct milestone towards capturing the realities of cancer biology in vitro and reduce animal experimentation in the preclinical stage of drug discovery. Significant work remains to be done to understand how substrates used in in vitro alternatives influence cancer cells phenotype and drug efficacy responses, so that to accurately link such models to specific in vivo disease scenarios. Our study describes how the morphological, mechanical and biochemical properties of adenocarcinoma (A549) cells change in response to a 3D environment and varying substrates. Confocal Laser Scanning (LSCM), He-Ion (HIM) and Atomic Force (AFM) microscopies, supported by ELISA and Western blotting, were used. These techniques enabled us to evaluate the shape, cytoskeletal organization, roughness, stiffness and biochemical signatures of cells grown within soft 3D matrices (PuraMatrix™ and Matrigel™), and to compare them to those of cells cultured on two-dimensional glass substrates. Cell cultures are also characterized for their biological response to docetaxel, a taxane-type drug used in Non-Small-Cell Lung Cancer (NSCLC) treatment. Our results offer an advanced biophysical insight into the properties and potential application of 3D cultures of A549 cells as in vitro alternatives in lung cancer research.

Cadmium nanoparticles citrullinate cytokeratins within lung epithelial cells: cadmium as a potential cause of citrullination in chronic obstructive pulmonary disease.

Objective: The objective of the study was to determine whether the cadmium-derived materials induce intracellular protein citrullination. Methods: Human A549 lung epithelial cells were exposed to cadmium in soluble and nanoparticulate forms represented by cadmium chloride (CdCl2) and cadmium oxide (CdO), respectively, and their combinations with ultrafine carbon black (ufCB) produced by high temperature combustion, imitating cigarette burning. Protein citrullination in cell lysates was analyzed by Western immunoblotting and verified by immunofluorescent confocal microscopy. Target citrullinated proteins were identified by proteomic analysis. Results: CdO, ufCB and its combination with CdCl2 and CdO after high temperature combustion induced protein citrullination in cultured human lung epithelial cells, as detected by immunoblotting with anti-citrullinated protein antibody. Cytokeratins of type II (1, 2, 5, 6A, 6B and 77) and type I (9, 10) were identified as major intracellular citrullination targets. Immunofluorescent staining confirmed the localization of citrullinated proteins both in the cytoplasm and cell nuclei. Conclusion: Cadmium oxide nanoparticle exposure facilitated post-translational citrullination of proteins.

Tunable Design of Gold(III)-Doxorubicin Complex-PEGylated Nanocarrier. The Golden Doxorubicin for Oncological Applications.

To date, the translation of Au (III) complexes into chemotherapeutic agents has been hindered by their low stability under physiological conditions, a crucial parameter in drug development. In this study, we report an innovative four-step synthesis of a stable Au (III)-doxorubicin (DOX) complex, acting as a key constitutive component of doxorubicin-loaded PEG-coated nanoparticles (DOX IN-PEG-AuNPs). For therapeutic purposes, such AuNPs were then functionalized with the anti-Kv11.1 polyclonal antibody (pAb), which specifically recognizes the hERG1 channel that is overexpressed on the membrane of human pancreatic cancer cells. The nature of the interactions between DOX and Au (III) ions was probed by various analytical techniques (Raman spectroscopy, UV-vis, and (1)H NMR), which enabled studying the Au (III)-DOX interactions during AuNPs formation. The theoretical characterization of the vibrational bands and the electronic transitions of the Au (III)-DOX complex calculated through computational studies showed significant qualitative agreement with the experimental observations on AuNPs samples. Stability in physiological conditions and efficient drug loading (up to to 85 w/w %) were achieved, while drug release was strongly dependent on the structure of DOX IN-PEG-AuNPs and on the pH. Furthermore, the interactions among DOX, PEG, and Au (III) ions in DOX IN-PEG-AuNPs differed significantly from those found in polymer-modified AuNPs loaded with DOX by covalent linkage, referred to as DOX ON-PEG-AuNPs. In vitro experiments indeed demonstrated that such differences strongly influenced the therapeutic potential of AuNPs in pancreatic cancer treatment, with a significant increase of the DOX therapeutic index when complexed to Au (III) ions. Collectively, our study demonstrated that Au (III)-DOX complexes as building blocks of PEGylated AuNPs constitutes a promising approach to transform promising Au (III) complexes into real chemotherapeutic drugs for the treatment of pancreatic cancer.

Towards a nanospecific approach for risk assessment.

In the current paper, a new strategy for risk assessment of nanomaterials is described, which builds upon previous project outcomes and is developed within the FP7 NANoREG project. NANoREG has the aim to develop, for the long term, new testing strategies adapted to a high number of nanomaterials where many factors can affect their environmental and health impact. In the proposed risk assessment strategy, approaches for (Quantitative) Structure Activity Relationships ((Q)SARs), grouping and read-across are integrated and expanded to guide the user how to prioritise those nanomaterial applications that may lead to high risks for human health. Furthermore, those aspects of exposure, kinetics and hazard assessment that are most likely to be influenced by the nanospecific properties of the material under assessment are identified. These aspects are summarised in six elements, which play a key role in the strategy: exposure potential, dissolution, nanomaterial transformation, accumulation, genotoxicity and immunotoxicity. With the current approach it is possible to identify those situations where the use of nanospecific grouping, read-across and (Q)SAR tools is likely to become feasible in the future, and to point towards the generation of the type of data that is needed for scientific justification, which may lead to regulatory acceptance of nanospecific applications of these tools.