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Updated project summary, December 2014

The consortium has developed this summary document to provide a concise overview of the proposed out put from the project, anticipate the impact and to update on progress, as well as providing information on the project collaborators.

Link to NanoChOp Publishable JRP Summary

Below, you can read the text of the publishable summary without having to download the document.

Publishable JRP Summary Report for JRP NEW03 NanoChOp

Background

Materials with external dimensions or internal structures on the nanometre scale develop unique properties not present in their macroscale form. These properties are increasingly being used by companies to overcome scientific and technical challenges and have led to nanomaterials being incorporated into over 1300 commercial products in a global market currently worth €9.6 billion. These nanomaterials now impact all areas of human life from medical devices and solar cells through to paints, sunscreens and cosmetics.

Nanomaterials are usually characterised for their chemical, physical and optical properties in their pure form or in simple idealised matrices. However, when nanomaterials interact with biological systems their properties can change significantly affecting their functionality and behaviour and creating a potential risk to human health. For example, when nanomaterials come into contact with a biological fluid they may become coated with proteins and other biomolecules which influences their interactions with cells (uptake, intracellular trafficking, toxicity etc.). The nanomaterials can also be transported across protective biological barriers, with recent evidence showing that nanoparticles can enter the blood stream after inhalation or ingestion. However, the potential interactions of nanoparticles with biological systems may also be a desirable characteristic, for example, in nanobiotechnology or nanomedical applications. This could involve the coating of nanoparticles with proteins for therapeutic benefit to target specific locations such as the brain (apolipoprotein E coatings) or the coating of fluorescent nanoparticles such as quantum dots with immunoglobulins for diagnostic applications.

It is therefore important to be able to demonstrate that a nanomaterial meets a specified functional demand by characterising the material within an appropriate biological system using quantitative measurements traceable to agreed reference systems. This project will address this by providing comparability and where possible a traceability chain for the measurement of nanomaterial physico-chemical and optical properties in complex biological matrices.

Need for the project

There is a clear concern in the EC and Member States, regarding the widespread use of engineered nanoparticles due to their unknown biological interactions and associated hazard(s). This is exacerbated by a lack of robust methods to characterise nanomaterials in anything other than idealised simple matrices. This project will therefore address this issue by producing measurement techniques that can be used to directly characterise nanomaterials in biological matrices in order to support the general nanotechnology community.

The overall aim of the project is to develop methods to characterise nanomaterials for their physical, chemical and optical properties in biological matrices. This will be achieved by developing a series of nanoparticle reference materials composed of metal oxide materials (to develop methods for physical and chemical characterisation), fluorescently labelled metal oxide materials (to allow nanomaterials tracking within biological systems to be performed) and a quantum dot nanomaterial (to develop methods for the optical characterisation of fluorescent nanomaterials).

Scientific and technical objectives

The project has the following scientific and technical objectives:

  • To produce a series of nanomaterials (for example, oxide and      quantum dot) that are characterised in their native form for size, surface      charge and fluorescence. To characterise a suitable cell based model(s)      for its application as a test system(s) for the interaction of      nanomaterials with biological systems.
  • To validate the use of a range of physical and chemical techniques      for measuring the size and chemical composition of nanomaterials in a      serum based biological system. Measurement techniques will cover chemical      analysis (ICP-MS), light scattering (MALS, DLS, NTA), X-ray technologies      (SAXS), centrifugal sedimentation (DCS) and nanoparticle charge analysis      (SIOS, ELS, Z-NTA).
  • To develop traceable methods for the characterisation of bulk      optical properties of fluorescent nanomaterials, particularly quantum      yield, absorption coefficient and corrected emission spectra. To develop      methods for the characterisation of fluorescent nanomaterials at the single      particle level including intermittency, photo stability and environmental      sensitivity in a serum based biological system.
  • To develop methods for the simultaneous characterisation of      physical and chemical composition of nanomaterials in cell based biological      systems. (SAXS+ASAXS and FFF+MALS+ICP-MS).
  • To develop measurement techniques for biotechnology using      fluorescent nanomaterials.

Expected results and potential impact

The primary beneficiaries of the project are the nanobiotechnology and nanomedicine organisations who will be provided with validated protocols with which to perform their analysis. Secondary beneficiaries will be regulatory bodies and legislators who will be provided with coherent and comparable data recommendations from which to formulate policy. Following this, the tertiary beneficiaries will be the manufacturers of nanomaterials who should be able to operate under a reasonable, rather than an overly stringent, regulatory framework.

To date, Nano ChOp has addressed the scientific and technical objectives as follows:

To produce a series of nanomaterials (for example, oxide and quantum dot) that are characterised in their native form for size, surface charge and fluorescence. To characterise a suitable cell based model(s) for its application as a test system(s) for the interaction of nanomaterials with biological systems

Suitable nanomaterials, biological serum and cell models used for developing methods to characterise nanomaterials for their physical, chemical and optical properties in biological matrices were identified and agreed by the project consortium with input from stakeholders. A series of nanomaterials composed of metalloid oxide nanoparticles for physical and chemical characterisation, fluorescently labelled metalloid oxide materials for monitoring within biological cells and quantum dots for optical characterisation were produced. Plain colloidal silica was sourced commercially, whilst aminated colloidal silica was prepared from the plain colloidal silica material, since a suitable material for meeting the project’s requirements was not commercially available. Quantum dot and fluorescent materials were also produced. As were, protocols for dispersion of the nanomaterials produced in water, buffer and biological media. The liver cell line HepG2 was selected as a suitable cell model to be subsequently exposed to nanoparticles to characterise their properties in biological systems. Cryopreserved master and working cell banks were created and cell culture conditions were optimised and validated. Work to measure the potential cellular toxicity of the nanoparticle materials (colloidal plain silica and quantum dots) to the HepG2 liver cell model and establish appropriate dose ranges for their administration to the cell model has been started and initial results indicate that whilst quantum dots show no significant effect on the liver cell model, colloidal plain silica exhibits dose-response properties, strongly indicative of a biological effect on the cells.

To validate the use of a range of physical and chemical techniques for measuring the size and chemical composition of nanomaterials in a serum based biological system. Measurement techniques will cover chemical analysis (ICP-MS), light scattering (MALS, DLS, NTA), X-ray technologies (SAXS), centrifugal sedimentation (DCS) and nanoparticle charge analysis (SIOS, ELS, Z-NTA)

Work to compare different techniques for measuring the physicochemical and extrinsic properties of the plain colloidal silica and aminated colloidal silica nanomaterials in aqueous and biological media was started and is nearing completion. Through a series of inter-laboratory studies, both nanomaterials have been characterised in terms of their size & size distribution, surface charge, concentration & agglomeration by multiple physical methods; (Small-angle X-ray scattering (SAXS), Field Flow Fractionation coupled with Multi-Angle Light Scattering (FFF/MALS), Differential scanning calorimetry (DCS), Dynamic light scattering (DLS) and Nanoparticle tracking analysis (NTA)). All data has been collated and is currently being analysed.

To develop traceable methods for the characterisation of bulk optical properties of fluorescent nanomaterials, particularly quantum yield, absorption coefficient and corrected emission spectra. To develop methods for the characterisation of fluorescent nanomaterials at the single particle level including intermittency, photo stability and environmental sensitivity in a serum based biological system

A protocol for the traceable determination of relative and absolute quantum yields (QY) using selected spectral fluorescent standards and fluorescent QY standards was developed and used to determine the relative and absolute fluorescence quantum yield of the fluorescent nanomaterials in aqueous media and biological serum. Spectroscopic methods for the determination of parameters affecting the signalling behaviour of fluorescent nanomaterials, such as the number of fluorophores per nanomaterial, the number of selected surface functionalities or the number of proteins adsorbed onto the nanomaterials in biological systems are currently being developed.

To develop methods for the simultaneous characterisation of physical and chemical composition of nanomaterials in cell based biological systems. (SAXS+ASAXS and FFF+MALS+ICP-MS)

Work to develop and validate an isotopic dilution mass spectrometry (IDMS) methodology for total element quantitation of dispersed nanomaterials plain colloidal silica and aminated colloidal silica in biological samples by ICP-MS has been started. Different sample introduction systems were identified and FFF/MALS/ICP-MS working conditions optimised. IDMS spreadsheets for the accurate quantification of silicon in both nanomaterials were also developed and validated. In addition, isotopically enriched silica nanoparticle spikes for nanoparticle quantification using IDMS with FFF-ICP-MS have been produced and characterised by MALS, TEM and FFF-ICP-MS.

To develop measurement techniques for biotechnology using fluorescent nanomaterials

Candidate fluorescent nanoparticles were integrated into an Interleukin 6 (IL-6, an important mediator of fever and of the acute phase response) rapid assay format. This biotechnology assay will allow a better understanding of how the properties of the antibody conjugated nanoparticle affect the resulting assay performance. The use of disc centrifugation (DC) to estimate the relative amounts of agglomerated nanoparticle species associated with chemical antibody conjugation has also been demonstrated. In addition to this, the performance of this assay in aqueous media (mock wound debridement solution) and biological serum has been demonstrated. Suitable levels of precision and bias were obtained over the required measurement range and the LOD was suitable for detection of the IL6 analyte in the indicated matrix.

 

So far the project’s impact and dissemination activities have included:

  • Ongoing contribution to documentary standards development. Specifically input into ISO/TC229 (Nanotechnologies), ISO REMCO (reference materials), CEN TC252 (Nanotechnologies) and ISO/TC24/SC4 (particle characterisation). At such meetings, the project partners contributed actively to discussions on topics such as the need for and design of inter-laboratory comparisons, requirements for in house production of particulate reference materials for quality control and good practice in the use of reference materials.
  • Scientific dissemination via      publications and presentations: 5 scientific publications in peer-reviewed      journals such as Nature Protocols and Analytical and Bioanalytical      Chemistry. 31 Presentations at relevant conferences and international      workshops including: EUROMAT 2013, 15th European Conference on      Surface and Interface Analysis, 2013, Nanosafety, 2013, BiOS SPIE      Photonics West, 2013 & 2014, Meeting of Analytical Division of the Royal      Society of Chemistry, Northern Ireland Region, 2014, the 7th      World Congress on Particle Technology (WCPT7), 2014 and NanoSafe 2014.      Finally, 6 poster exhibits at European conferences such as NANOTECH      Tunisia 2014 and NANOSMAT-USA.
  • Uptake of      methods by end-users. NanoSight has begun using the project’s method for Evaluation      of the performance of a particle tracking analysis method for nanoparticle      sizing and CPS Instruments has had discussion with the project partners      regarding improvements of the centrifugal liquid sedimentation instrument      and calibration procedures.

 

JRP start date and duration: 1st  June 2012 (3   year duration)
JRP-Coordinator:Dr. Heidi Goenaga-Infante, LGC     Tel: +44(0)20 8943 7000       E-mail: Heidi.Goenaga-Infante@LGCGroup.com

JRP website address: http://nanochop.lgcgroup.com/

JRP-Partners:JRP-Partner 1: LGC, UK

JRP-Partner 2: BAM, Germany

JRP-Partner 3: JRC, European Commission

JRP-Partner 4: NPL, UK

JRP-Partner 5: PTB, Germany

 

 
REG-Researcher 1
(associated Home Organisation):
Zoltan VargaRCNS HAS, Hungary
REG-Researcher 2
(associated Home Organisation):
Eric PitkeathlyHWU, UK
REG-Researcher 3
(associated Home Organisation):
Christian SchmidtkeUHAM, Germany
RMG-Researcher
(associated Home Organisation):
Marcell PalmaiRCNS HAS, Hungary

 

The EMRP is jointly funded by the EMRP participating countries within EURAMET and the European Union