Toxic effects of nanomaterials for health applications: How automation can support a systematic review of the literature?

Systematic reviews of the scientific literature can be an important source of information supporting the daily work of the regulators in their decision making, particularly in areas of innovative technologies where the regulatory experience is still limited. Significant research activities in the field of nanotechnology resulted in a huge number of publications in the last decades. However, even if the published data can provide relevant information, scientific articles are often of diverse quality, and it is nearly impossible to manually process and evaluate such amount of data in a systematic manner. In this feasibility study, we investigated to what extent open-access automation tools can support a systematic review of toxic effects of nanomaterials for health applications reported in the scientific literature. In this study, we used a battery of available tools to perform the initial steps of a systematic review such as targeted searches, data curation and abstract screening. This work was complemented with an in-house developed tool that allowed us to extract specific sections of the articles such as the materials and methods part or the results section where we could perform subsequent text analysis. We ranked the articles according to quality criteria based on the reported nanomaterial characterisation and extracted most frequently described toxic effects induced by different types of nanomaterials. Even if further demonstration of the reliability and applicability of automation tools is necessary, this study demonstrated the potential to leverage information from the scientific literature by using automation systems in a tiered strategy.

Bridging communities in the field of nanomedicine.

An early dialogue between nanomedicine developers and regulatory authorities are of utmost importance to anticipate quality and safety requirements for these innovative health products. In order to stimulate interactions between the various communities involved in a translation of nanomedicines to clinical applications, the European Commission's Joint Research Centre hosted a workshop titled "Bridging communities in the field of Nanomedicine" in Ispra/Italy on the 27th -28th September 2017. Experts from regulatory bodies, research institutions and industry came together to discuss the next generation of nanomedicines and their needs to obtain regulatory approval. The workshop participants came up with recommendations highlighting methodological gaps that should be addressed in ongoing projects addressing the regulatory science of nanomedicines. In addition, individual opinions of experts relevant to progress of the regulatory science in the field of nanomedicine were summarised in the format of a survey.

The agglomeration state of nanoparticles can influence the mechanism of their cellular internalisation.

BACKGROUND: Significant progress of nanotechnology, including in particular biomedical and pharmaceutical applications, has resulted in a high number of studies describing the biological effects of nanomaterials. Moreover, a determination of so-called "critical quality attributes", that is specific physicochemical properties of nanomaterials triggering the observed biological response, has been recognised as crucial for the evaluation and design of novel safe and efficacious therapeutics. In the context of in vitro studies, a thorough physicochemical characterisation of nanoparticles (NPs), also in the biological medium, is necessary to allow a correlation with a cellular response. Following this concept, we examined whether the main and frequently reported characteristics of NPs such as size and the agglomeration state can influence the level and the mechanism of NP cellular internalization. RESULTS: We employed fluorescently-labelled 30 and 80 nm silicon dioxide NPs, both in agglomerated and non-agglomerated form. Using flow cytometry, transmission electron microscopy, the inhibitors of endocytosis and gene silencing we determined the most probable routes of cellular uptake for each form of tested silica NPs. We observed differences in cellular uptake depending on the size and the agglomeration state of NPs. Caveolae-mediated endocytosis was implicated particularly in the internalisation of well dispersed silica NPs but with an increase of the agglomeration state of NPs a combination of endocytic pathways with a predominant role of macropinocytosis was noted. CONCLUSIONS: We demonstrated that the agglomeration state of NPs is an important factor influencing the level of cell uptake and the mechanism of endocytosis of silica NPs.

Identification of new Presenilin-1 phosphosites: implication for γ-secretase activity and Aß production.

An important pathological hallmark of Alzheimer's disease (AD) is the deposition of amyloid-beta (Aß) peptides in the brain parenchyma, leading to neuronal death and impaired learning and memory. The protease γ-secretase is responsible for the intramembrane proteolysis of the amyloid-ß precursor protein (APP), which leads to the production of the toxic Aß peptides. Thus, an attractive therapeutic strategy to treat AD is the modulation of the γ-secretase activity, to reduce Aß42 production. Because phosphorylation of proteins is a post-translational modification known to modulate the activity of many different enzymes, we used electrospray (LC-MS/MS) mass spectrometry to identify new phosphosites on highly purified human γ-secretase. We identified 11 new single or double phosphosites in two well-defined domains of Presenilin-1 (PS1), the catalytic subunit of the γ-secretase complex. Next, mutagenesis and biochemical approaches were used to investigate the role of each phosphosite in the maturation and activity of γ-secretase. Together, our results suggest that the newly identified phosphorylation sites in PS1 do not modulate γ-secretase activity and the production of the Alzheimer's Aß peptides. Individual PS1 phosphosites shall probably not be considered therapeutic targets for reducing cerebral Aß plaque formation in AD. In this study, we identified 11 new phosphosites in Presenilin-1 (PS1), the catalytic subunit of the Alzheimer's γ-secretase complex. By combining a mutagenesis approach with cell-based and cell-free γ-secretase assays, we demonstrate that the new phosphosites do not modulate the maturation and activity of γ-secretase. Individual PS1 phosphosites shall thus not be considered therapeutic targets for reducing cerebral Aß plaque formation in Alzheimer's Disease. Aß, amyloid beta.

Stress reaction of kidney epithelial cells to inorganic solid-core nanoparticles.

A route of accumulation and elimination of therapeutic engineered nanoparticles (NPs) may be the kidney. Therefore, the interactions of different solid-core inorganic NPs (titanium-, silica-, and iron oxide-based NPs) were studied in vitro with the MDCK and LLC-PK epithelial cells as representative cells of the renal epithelia. Following cell exposure to the NPs, observations include cytotoxicity for oleic acid-coated iron oxide NPs, the production of reactive oxygen species for titanium dioxide NPs, and cell depletion of thiols for uncoated iron oxide NPs, whereas for silica NPs an apparent rapid and short-lived increase of thiol levels in both cell lines was observed. Following cell exposure to metallic NPs, the expression of the tranferrin receptor/CD71 was decreased in both cells by iron oxide NPs, but only in MDCK cells by titanium dioxide NPs. The tight association, then subsequent release of NPs by MDCK and LLC-PK kidney epithelial cells, showed that following exposure to the NPs, only MDCK cells could release iron oxide NPs, whereas both cells released titanium dioxide NPs. No transfer of any solid-core NPs across the cell layers was observed.

Induction of oxidative stress, lysosome activation and autophagy by nanoparticles in human brain-derived endothelial cells.

Different types of NPs (nanoparticles) are currently under development for diagnostic and therapeutic applications in the biomedical field, yet our knowledge about their possible effects and fate in living cells is still limited. In the present study, we examined the cellular response of human brain-derived endothelial cells to NPs of different size and structure: uncoated and oleic acid-coated iron oxide NPs (8-9 nm core), fluorescent 25 and 50 nm silica NPs, TiO2 NPs (21 nm mean core diameter) and PLGA [poly(lactic-co-glycolic acid)]-PEO [poly(ethylene oxide)] polymeric NPs (150 nm). We evaluated their uptake by the cells, and their localization, generation of oxidative stress and DNA-damaging effects in exposed cells. We show that NPs are internalized by human brain-derived endothelial cells; however, the extent of their intracellular uptake is dependent on the characteristics of the NPs. After their uptake by human brain-derived endothelial cells NPs are transported into the lysosomes of these cells, where they enhance the activation of lysosomal proteases. In brain-derived endothelial cells, NPs induce the production of an oxidative stress after exposure to iron oxide and TiO2 NPs, which is correlated with an increase in DNA strand breaks and defensive mechanisms that ultimately induce an autophagy process in the cells.

Tiered testing of micro- and nanoplastics using intestinal in vitro models to support hazard assessments.

The uncertainty of potential risks associated with micro- and nanoplastics (MNPs) are of growing public concern. However, the diversity of MNPs in the environment makes a systematic analysis of potential health effects challenging. New tools and approaches are necessary to investigate biological effects of MNPs. With this quick scoping review, we aim to analyse the suitability of in vitro models for assessing the interaction of MNPs with intestinal cells. Our analysis revealed that currently the majority of in vitro tests are based on the three cell lines Caco-2, HT-29, and HCT-116. They have particularly been used to assess endpoints related to basal cytotoxicity, the internalisation of MNPs and effects on the intestinal barrier. When co-cultured with various cell lines, they also allow to investigate additional effects such as inflammation, metabolic actions and the relevance of the intestinal mucus. However, methodological gaps remain regarding the assessment of a potential accumulation of MNPs, leaching of additives/impurities and in resulting long-term effects as well as cell-type specific toxicities. In addition, only few in vitro studies investigated effects of MNPs on the microbiome. Stem cell-based assays using, for example, the emerging organoid technology are promising for analysing MNP effects on tissue-like structures, while avoiding the particular characteristics of the currently used cancer derived cell lines. The various cell lines and culture techniques can be combined in testing strategies, to better elucidate potential biological interaction of MNPs with biological systems. We suggest to implement a tiered testing strategy, in which monocultures can serve as a tool for high-throughput testing of MNPs. In the next steps co-cultures can be used to assess the potential of a systemic uptake of MNPs and organ-on-a-chip models will provide more reliable insights into relevant doses triggering biological effects. Finally, organoids can help to discover new and more complex reactions initiated by MNPs.

Future perspectives for advancing regulatory science of nanotechnology-enabled health products.

The identification of regulatory challenges for nanotechnology-enabled health products, followed by discussions with the involved stakeholders, is the first step towards a strategic planning of how such challenges can be successfully addressed in the future. In order to better understand whether the identified regulatory needs are sector-specific for health products or might also hinder the progress in other domains, the REFINE consortium reached out to communities representing other sectors that also exploit the potential of nanotechnology, i.e. industrial chemicals, food and cosmetics. Through a series of trans-sectorial workshops, REFINE partners identified common as well as sector-specific challenges and discussed possible ways forward. Potential solutions lie in a more strengthen collaboration between regulatory and research communities resulting in a targeted production and exploitation of academic data for the regulatory decision-making. Furthermore, a coordinated use of knowledge sharing platforms and databases, trans-sectorial standardisation activities and harmonisation of regulatory activities between geographical regions are possible ways forward, in line with the upcoming European political initiatives such as the Chemical Strategy for Sustainability (CSS). Finally, we also discuss the perspectives for further development and sustainability of methods and tools developed in the REFINE project.

Emerging technologies and their impact on regulatory science.

There is an evolution and increasing need for the utilization of emerging cellular, molecular and in silico technologies and novel approaches for safety assessment of food, drugs, and personal care products. Convergence of these emerging technologies is also enabling rapid advances and approaches that may impact regulatory decisions and approvals. Although the development of emerging technologies may allow rapid advances in regulatory decision making, there is concern that these new technologies have not been thoroughly evaluated to determine if they are ready for regulatory application, singularly or in combinations. The magnitude of these combined technical advances may outpace the ability to assess fit for purpose and to allow routine application of these new methods for regulatory purposes. There is a need to develop strategies to evaluate the new technologies to determine which ones are ready for regulatory use. The opportunity to apply these potentially faster, more accurate, and cost-effective approaches remains an important goal to facilitate their incorporation into regulatory use. However, without a clear strategy to evaluate emerging technologies rapidly and appropriately, the value of these efforts may go unrecognized or may take longer. It is important for the regulatory science field to keep up with the research in these technically advanced areas and to understand the science behind these new approaches. The regulatory field must understand the critical quality attributes of these novel approaches and learn from each other's experience so that workforces can be trained to prepare for emerging global regulatory challenges. Moreover, it is essential that the regulatory community must work with the technology developers to harness collective capabilities towards developing a strategy for evaluation of these new and novel assessment tools.

Mapping of the available standards against the regulatory needs for nanomedicines.

Appropriate documentary standards and reference materials are crucial building blocks for the development of innovative products. In order to support the emerging sector of nanomedicine, relevant standards must be identified and/or developed before the products will enter into the regulatory approval process. The anticipation of standardization needs requires a good understanding on the regulatory information requirements that can be triggered by the particularities of nanomedicines. However, robust datasets allowing firm conclusions on regulatory demands are not yet available due to a lack of regulatory experience with innovative products. Such a catch-22 situation can only be advanced in an iterative process by monitoring continuously the scientific evidence and by promoting intensive knowledge exchange between all involved stakeholders. In this study, we have compiled information requirements released by regulatory scientists so far and mapped it against available standards that could be of relevance for nanomedicines. Our gap analysis clearly demonstrated that for some endpoints such as drug release/loading and the interaction of nanomedicines with the immune system no standards are available so far. The emerging nanomedicine sector could benefit from cross-sector collaboration and review the suitability of standards that have been developed for nanomaterials used for other industrial applications. Only a concerted action of all parties can lead to a smooth translation of nanomedicines to clinical application and to the market. This is in particular important because nanotechnology-based drug delivery systems are key for the development and implementation of personalized medicine. This article is characterized under: Toxicology and Regulatory Issues in Nanomedicine > Regulatory and Policy Issues in Nanomedicine.

Main trends of immune effects triggered by nanomedicines in preclinical studies.

The application of nanotechnology to emerging medicinal products is a crucial parameter for the implementation of personalized medicine. For example, sophisticated drug delivery systems can target the diseased tissue by recognizing patient-specific biomarkers while carrying pharmacologically active molecules. However, such nanomedicines can be recognized by the immune system as foreign triggering unexpected biological reactions. The anticipation of the immunogenic potential of emerging nanotechnology-based products in the preclinical phase is challenging due to high interspecies variations between the immune systems of laboratory animals and humans. A close monitoring of the scientific literature is required to better understand the relationship between various immune reactions and the diversity of nanomedicines currently in the development pipeline. We have reviewed the most frequent immune reactions induced by the nanomaterials in vivo and have identified the main effects triggered by lipid-based, polymer-based and inorganic nanoparticles, as the main categories of nanomaterials used in medicine. According to our results, almost 50% of the investigated nanomaterials induced effects related to the activation of the immune system. Among them, complement activation-related hypersensitivity reactions and activation of adaptive immune response were the most frequent effects reported for the lipid-based nanoparticles. However, many of these effects are not or are only partially covered by the current regulatory framework applicable for nanomedicines. In addition, we extracted the most relevant nanospecific properties responsible for the observed biological effects. Our analysis led to identification of the most prevalent measurement endpoints relevant for the assessment of the immunotoxic potential of the nanotechnology-based products and will support the smooth and safe translation of the new formulations to clinical applications.

Dispersion Behaviour of Silica Nanoparticles in Biological Media and Its Influence on Cellular Uptake.

Given the increasing variety of manufactured nanomaterials, suitable, robust, standardized in vitro screening methods are needed to study the mechanisms by which they can interact with biological systems. The in vitro evaluation of interactions of nanoparticles (NPs) with living cells is challenging due to the complex behaviour of NPs, which may involve dissolution, aggregation, sedimentation and formation of a protein corona. These variable parameters have an influence on the surface properties and the stability of NPs in the biological environment and therefore also on the interaction of NPs with cells. We present here a study using 30 nm and 80 nm fluorescently-labelled silicon dioxide NPs (Rubipy-SiO2 NPs) to evaluate the NPs dispersion behaviour up to 48 hours in two different cellular media either supplemented with 10% of serum or in serum-free conditions. Size-dependent differences in dispersion behaviour were observed and the influence of the living cells on NPs stability and deposition was determined. Using flow cytometry and fluorescence microscopy techniques we studied the kinetics of the cellular uptake of Rubipy-SiO2 NPs by A549 and CaCo-2 cells and we found a correlation between the NPs characteristics in cell media and the amount of cellular uptake. Our results emphasize how relevant and important it is to evaluate and to monitor the size and agglomeration state of nanoparticles in the biological medium, in order to interpret correctly the results of the in vitro toxicological assays.

Toxicity screenings of nanomaterials: challenges due to interference with assay processes and components of classic in vitro tests.

Given the multiplicity of nanoparticles (NPs), there is a requirement to develop screening strategies to evaluate their toxicity. Within the EU-funded FP7 NanoTEST project, a panel of medically relevant NPs has been used to develop alternative testing strategies of NPs used in medical diagnostics. As conventional toxicity tests cannot necessarily be directly applied to NPs in the same manner as for soluble chemicals and drugs, we determined the extent of interference of NPs with each assay process and components. In this study, we fully characterized the panel of NP suspensions used in this project (poly(lactic-co-glycolic acid)-polyethylene oxide [PLGA-PEO], TiO2, SiO2, and uncoated and oleic-acid coated Fe3O4) and showed that many NP characteristics (composition, size, coatings, and agglomeration) interfere with a range of in vitro cytotoxicity assays (WST-1, MTT, lactate dehydrogenase, neutral red, propidium iodide, (3)H-thymidine incorporation, and cell counting), pro-inflammatory response evaluation (ELISA for GM-CSF, IL-6, and IL-8), and oxidative stress detection (monoBromoBimane, dichlorofluorescein, and NO assays). Interferences were assay specific as well as NP specific. We propose how to integrate and avoid interference with testing systems as a first step of a screening strategy for biomedical NPs.

Transfer of ultrasmall iron oxide nanoparticles from human brain-derived endothelial cells to human glioblastoma cells.

Nanoparticles (NPs) are being used or explored for the development of biomedical applications in diagnosis and therapy, including imaging and drug delivery. Therefore, reliable tools are needed to study the behavior of NPs in biological environment, in particular the transport of NPs across biological barriers, including the blood-brain tumor barrier (BBTB), a challenging question. Previous studies have addressed the translocation of NPs of various compositions across cell layers, mostly using only one type of cells. Using a coculture model of the human BBTB, consisting in human cerebral endothelial cells preloaded with ultrasmall superparamagnetic iron oxide nanoparticles (USPIO NPs) and unloaded human glioblastoma cells grown on each side of newly developed ultrathin permeable silicon nitride supports as a model of the human BBTB, we demonstrate for the first time the transfer of USPIO NPs from human brain-derived endothelial cells to glioblastoma cells. The reduced thickness of the permeable mechanical support compares better than commercially available polymeric supports to the thickness of the basement membrane of the cerebral vascular system. These results are the first report supporting the possibility that USPIO NPs could be directly transferred from endothelial cells to glioblastoma cells across a BBTB. Thus, the use of such ultrathin porous supports provides a new in vitro approach to study the delivery of nanotherapeutics to brain cancers. Our results also suggest a novel possibility for nanoparticles to deliver therapeutics to the brain using endothelial to neural cells transfer.