Multimethod approach for the detection and characterisation of food-grade synthetic amorphous silica nanoparticles

AbstractSynthetic amorphous silica (SAS) has been used as food additive under the code E551 for decades and the agrifood sector is considered a main exposure vector for humans and environment. However, there is still a lack of detailed methodologies for the determination of SAS’ particle size and concentration. This work presents the detection and characterization of NPs in eleven different food-grade SAS samples, following a reasoned and detailed sequential methodology. Dynamic Light Scattering (DLS), Multiangle Light Scattering (MALS), Asymmetric Flow-Field Flow Fractionation (AF4), Inductively Coupled Plasma Mass Spectrometry (ICPMS) and Transmission Electron Microscopy (TEM) were used. The suitability and limitations, information derived from each type of analytical technique and implications related to current EC Regulation 1169/2011 on the provision of food information to consumers are deeply discussed. In general the z-average, AF4 hydrodynamic diameters and root mean square (rms) radii measured were in good agreement. AF4-ICPMS coupling and pre channel calibration with silica NPs standards allowed the reliable detection of NPs below 100nm for ten of eleven samples (AF4 diameters between 20.6 and 39.8nm) and to quantify the mass concentration in seven different samples (at mgL−1 concentration level). TEM characterisation included the determination of the minimum detectable size and subsequent measurement of the equivalent circle diameter (ECD) of primary particles and small aggregates, which were between 10.3 and 20.3nm. Because of the dynamic size application range is limited by the minimum detectable size, all the techniques in this work can be used only as positive tests

Physicochemical characterisation of gold, silica and silver nanoparticles in water and in serum-containing cell culture media

This report presents the results from a study organised under the coordination of JRC as part of a project aiming at the adaptation of the in vitro micronucleus test (Test Guideline 487) for the assessment of manufactured NMs. The aim of the first step of the project was to evaluate the physicochemical characterisation of selected representative nanomaterials (5 nm gold, 30 nm gold, 22 nm silica, 30 nm citrate and 30 nm PVP stabilised silver nanoparticles) in pure water and in different complete culture media. The results of the study show that using a combination of different characterisation techniques is important to providing reliable information about the agglomeration behaviour of the tested nanoparticles in complete cell culture media (CCM). Most of the materials exhibited mild agglomeration in serum containing CCM. Only the PVP functionalised silver nanoparticles showed a size distribution change in all of the culture media that is so small that it could be attributed to solely protein adsorption without notable agglomeration. Silica nanoparticles were found to be the most sensitive to interaction with serum containing CCM, showing massive concentration and time dependent agglomeration strongly affected by the CCM composition. Extensive agglomeration might lead also to the accelerated sedimentation of the particles changing drastically the true, effective dose that the cells will receive under in vitro conditions1, 2. Thus, it has to be investigated in more detail and taken in account when designing in vitro experiments in the next phase of the project.JRC.F.2-Consumer Products Safet

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.JRC.I.4-Nanobioscience

Highly specific targeting of human acute myeloid leukaemia cells using pharmacologically active nanoconjugates

In this study we used 5 nm gold nanoparticles as delivery platforms to target cancer cells expressing the immune receptor Tim-3 using single chain antibodies. Gold surfaces were also covered with the cytotoxic drug rapamycin which was immobilised using a glutathione linker. These nanoconjugates allowed highly specific and efficient delivery of cytotoxic rapamycin into human malignant blood cells

Interlaboratory comparison study of the Colony Forming Efficiency assay for assessing cytotoxicity of nanomaterials

Nanotechnology has gained importance in the past years as it provides opportunities for industrial growth and innovation. However, the increasing use of manufactured nanomaterials (NMs) in a number of commercial applications and consumer products raises also safety concerns and questions regarding potential unintended risks to humans and the environment. Since several years the European Commission’s Joint Research Centre (JRC) is putting effort in the development, optimisation and harmonisation of in vitro test methods suitable for screening and hazard assessment of NMs. Work is done in collaboration with international partners, in particular the Organisation for Economic Co-operation and Development (OECD). This report presents the results from an interlaboratory comparison study of the in vitro Colony Forming Efficiency (CFE) cytotoxicity assay performed in the frame of OECD's Working Party of Manufactured Nanomaterials (WPMN). Twelve laboratories from European Commission, France, Italy, Japan, Poland, Republic of Korea, South Africa and Switzerland participated in the study coordinated by JRC. The results show that the CFE assay is a suitable and robust in vitro method to assess cytotoxicity of NMs. The assay protocol is well defined and is easily and reliably transferable to other laboratories. The results obtained show good intra and interlaboratory reproducibility of the assay for both the positive control and the tested nanomaterials. In conclusion the CFE assay can be recommended as a building block of an in vitro testing battery for NMs toxicity assessment. It could be used as a first choice method to define dose-effect relationships for other in vitro assays.JRC.I.4-Nanobioscience

Workshop on Regulatory Preparedness for Innovation in Nanotechnology

This report summarises the presentations and discussions at the first NanoReg2 Workshop on Regulatory Preparedness for Innovation in Nanotechnology held in Ispra, Italy 5 to 6 October 2017 and attended by approximately 60 regulators, industry representatives and other stakeholders. NanoReg2 is a European Union (EU) Horizon 2020 project. At the workshop, Regulatory Preparedness was defined as the regulators' timely awareness of innovations and the regulator's actions to check whether present legislation covers all safety aspects of each innovation, including initiating revision of the legislation as appropriate. Regulatory Preparedness, and Safe-by-Design (SbD) jointly constitute the NanoReg2 Safe Innovation Approach (SIA) for developing innovative products based on nanotechnology. The workshop aimed to gather views and identify current practices in regulatory work on safety of innovative products, tools already in use or needed, and potential difficulties in implementing Regulatory Preparedness in the EU. Presentations addressed the current state of the safety of nanotechnology innovation. The viewpoints included the regulatory framework, the principles behind it and the agencies and authorities enforcing it; nanosafety research projects and their support system (e.g. the current EU Horizon 2020 Framework Programme); national nanosafety initiatives; and the development of tools, such as foresight tools and harmonised test guidelines by the OECD for data generation. The workshop served to generate ideas for achieving Regulatory Preparedness. The participants recognised that while regulators deal with the safety of innovations, only few systematic approaches to this work exist. Some innovative products may reach the market before their safety has been appropriately assessed, as illustrated by RAPEX, the Rapid Exchange of Information System. A continuous and proactive combination of interconnected activities was considered to be required for ensuring Regulatory Preparedness. Thus, anticipation, e.g. horizon scanning, was seen as important, as was communication between regulators, innovators (industry) and other stakeholders. Regulators need to become aware of innovative products under development to ensure that the legislation and methods for safety assessment are available and adequate. Innovators must be aware of regulatory requirements and their likely development. This mutual awareness helps to develop safe products and to avoid delays or other problems in obtaining market approval. Awareness can be achieved through communication, which requires trust, e.g. promoted via "trusted environments" for confidential inquiries and information sharing. Furthermore, regulators need early access to the existing information and data relevant to safety assessment of innovative products to provide timely guidance and advice to Industry as well as to develop strategies for dealing with uncertainty, e.g. by applying the precautionary principle. Regulatory Preparedness was discussed as part of the SIA, and a "road map" of actions was suggested and outlined. The workshop has thus contributed towards acceptance of implementing Regulatory Preparedness for innovation in nanotechnology through the participation of a variety of stakeholders. This paves the way for a better dialogue among stakeholders in a fast economic development cycle, where it is even more important to quickly identify emerging needs for new approaches to regulatory issues for innovationJRC.F.2-Consumer Products Safet

A random walk approach to estimate the confinement of α-particle emitters in nanoparticles for targeted radionuclide therapy

Abstract Background Targeted radionuclide therapy is a highly efficient but still underused treatment modality for various types of cancers that uses so far mainly readily available β-emitting radionuclides. By using α-particle emitters several shortcomings due to hypoxia, cell proliferation and in the selected treatment of small volumes such as micrometastasis could be overcome. To enable efficient targeting longer-lived α-particle emitters are required. These are the starting point of decay chains emitting several α-particles delivering extremely high radiation doses into small treatment volumes. However, as a consequence of the α-decay the daughter nuclides receive high recoil energies that cannot be managed by chemical radiolabelling techniques. By safe encapsulation of all α-emitters in the decay chain in properly sized nanocarriers their release may be avoided. Results The encapsulation of small core nanoparticles loaded with the radionuclide in a shell structure that safely confines the recoiling daughter nuclides promises good tumour targeting, penetration and uptake, provided these nanostructures can be kept small enough. A model for spherical nanoparticles is proposed that allows an estimate of the fraction of recoiling α-particle emitters that may escape from the nanoparticles as a function of their size. The model treats the recoil ranges of the daughter nuclides as approximately equidistant steps with arbitrary orientation in a three-dimensional random walk model. Conclusions The presented model allows an estimate of the fraction of α-particles that are emitted from outside the nanoparticle when its size is reduced below the radius that guarantees complete confinement of all radioactive daughter nuclides. Smaller nanoparticle size with reduced retention of daughter radionuclides might be tolerated when the effects can be compensated by fast internalisation of the nanoparticles by the target cells

A random walk approach to estimate the confinement of α-particle emitters in nanoparticles for targeted radionuclide therapy

Background: Targeted radionuclide therapy is a highly efficient but still underused treatment modality for various types of cancers that uses so far mainly readily available β-emitting radionuclides. By using α-particle emitters several shortcomings due to hypoxia, cell proliferation and in the selected treatment of small volumes such as micrometastasis could be overcome. To enable efficient targeting longer-lived α-particle emitters are required. These are the starting point of decay chains emitting several α-particles delivering extremely high radiation doses into small treatment volumes. However, as a consequence of the α-decay the daughter nuclides receive high recoil energies that cannot be managed by chemical radiolabelling techniques. By safe encapsulation of all α-emitters in the decay chain in properly sized nanocarriers their release may be avoided. Results: The encapsulation of small core nanoparticles loaded with the radionuclide in a shell structure that safely confines the recoiling daughter nuclides promises good tumour targeting, penetration and uptake, provided these nanostructures can be kept small enough. A model for spherical nanoparticles is proposed that allows an estimate of the fraction of recoiling α-particle emitters that may escape from the nanoparticles as a function of their size. The model treats the recoil ranges of the daughter nuclides as approximately equidistant steps with arbitrary orientation in a three-dimensional random walk model. Conclusions: The presented model allows an estimate of the fraction of α-particles that are emitted from outside the nanoparticle when its size is reduced below the radius that guarantees complete confinement of all radioactive daughter nuclides. Smaller nanoparticle size with reduced retention of daughter radionuclides might be tolerated when the effects can be compensated by fast internalisation of the nanoparticles by the target cells.JRC.F.2-Consumer Products Safet

Influence of different cleaning processes on the surface chemistry of gold nanoparticles

In this paper we have investigated the effects of different cleaning methods (centrifugation and dialysis) on the surface chemistry and composition of 15 nm citrate stabilized gold nanoparticles (AuNPs). The Nuclear Magnetic Resonance (NMR) results indicates that three centrifugation cycles are sufficient to remove most of the citrate molecules, whilst CLS and DLS data reveal some nanoparticles aggregation when three centrifugation cycles are exceeded. Regarding the dialysis procedure, NMR analysis demonstrated that after nine cleaning cycles the citrate concentration is comparable to that measured after the first centrifugation (about 6E-04 mM) with an increase of the solution polydispersivity index. X-ray Photoelectron Spectroscopy results support the NMR findings and revealed a major hydrocarbon contamination after the nanoparticles cleaning process. Moreover, functionalization with 1H,1H,2H,2H-Perfluorodecanethiol hydrophobic thiols up to about 0.9 of a monolayer (ML) was achieved after two centrifugation cycles, whilst less of 0.6 ML was functionalized by simple thiol-citrate substitution in the pristine gold nanoparticle solution and after a 3 dialysis cyclesJRC.F.2-Consumer Products Safet

Multimethod approach for the detection and characterisation of food-grade synthetic amorphous silica nanoparticles

Synthetic amorphous silica (SAS) has been used as food additive under the code E551 for decades and the agrifood sector is considered a main exposure vector for humans and environment. However, there is still a lack of detailed methodologies for the determination of SAS' particle size and concentration. This work presents the size characterisation of NPs in eleven different food-grade SAS samples, following a reasoned and detailed sequential methodology. Dynamic Light Scattering (DLS), Multiangle Light Scattering (MALS), Asymmetric Flow-Field Flow Fractionation (AF4), Inductively Coupled Plasma Mass Spectrometry (ICPMS) and Transmission Electron Microscopy (TEM) were used. The suitability and limitations, information derived from each type of analytical technique and implications related to current EC Regulation 1169/2011 on the provision of food information to consumers are deeply discussed. In general the z-average, AF4 hydrodynamic diameters and root mean square (rms) radii measured were in good agreement. AF4-ICPMS coupling and pre channel calibration with silica NPs standards allowed the reliable detection of NPs below 100 nm for ten of eleven samples (AF4 diameters between 20.6 and 39.8 nm) and to quantify the mass concentration in seven different samples (at mg L-1 concentration level). TEM characterisation included the determination of the minimum detectable size and subsequent measurement of the equivalent circle diameter (ECD) of primary particles and small aggregates, which were between 10.3 and 20.3 nm. Because of the dynamic size application range is limited by the minimum detectable size, all the techniques in this work can be used only as positive tests.JRC.I.1-Chemical Assessment and Testin