Editorial
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Testing strategies for the safety of nanoparticles used
in medical applications
“A better understanding of how properties of NPs define their interactions with cells,
tissues and organs in exposed humans is a considerable scientific challenge, but one
that must be addressed if there is to be safe and responsible use of biomedical NPs.”
Nanoparticles (NPs) have unique physicochemical properties that are different from
those of the bulk material. Properties, such as
small size (<100 nm), relatively high surfaceto-volume ratio, quantum dot effect and reactivity, allow a wide range of different applications in modern industry. Nanotechnology is
considered as a technology of the future, with
great potential in biomedical applications. It
is becoming increasingly important in nanodiagnostics (medical imaging, biosensors,
contrast agents and in vitro ‘lab-on-a-chip’),
drug-delivery devices (for gene delivery and
targeting drugs) and regenerative medicine
(prostheses and implants). NPs have already
been used for treating a variety of human diseases including infections, genetic disorders
and tumors.
The unique properties of NPs, whilst likely
to benefit many aspects of our lives, are also
a cause of concern as their possible impact
on human health is not known. With some
NP-based products already in use in nanomedicine and many more under development,
it is critically important that the potential risks
are properly assessed. Nanomedicine brings
humans into direct contact with NPs and it is
therefore essential for both public confidence
and nanotechnology companies that appropriate risk assessments are undertaken in relation
to health and safety. Clearly, there is a pressing
need to understand how engineered NPs interact with the human body following exposure of
patients or consumers, in the workplace or from
the environment. The European Commission
has adopted the Action Plan ‘Nanosciences and
nanotechnologies: an action plan for Europe
2005–2009’ [101] , defining actions for the
‘immediate implementation of a safe, integrated
and responsible strategy for nanosciences and
nanotechnologies’. In addition, there are ongoing discussions within European Commission
committees [102] , and within REACH (the
Registration, Evaluation, Authorization and
Restriction of Chemicals)-competent authorities and the subgroup on nanomaterials as to
hazard and risk assessment of nanomaterials
[103,104] , although originally, nanotechnologies
and nanomaterials were not clearly included in
the scope of REACH.
With respect to human health, the current
methodologies described in the Technical
Guidance Documents [105] are generally likely
to be able to identify the hazards associated with
the use of NPs. However, the specific features
and properties of NPs that may contribute to
their potential toxicity are not fully addressed.
Special attention should be given to the NPs’
physicochemical characteristics and the changes
that may occur under local environmental
conditions. Such properties and their changes
include size, surface, chemical composition,
bioavailability, solubility, agglomeration, dissociation and adsorption of environmental substances, all of which may have an impact on the
ultimate toxicity of the NPs.
One of the main problems encountered in
testing NPs for human toxicity has been the
lack of appropriate standardized protocols.
Expressing the NP dose as a mass concentration is probably not the best description for the
determination of the dose–response relationship
(as it is for conventional chemicals or drugs).
Other metrics such as number of NPs or surface area are likely to be more appropriate. With
respect to exposure to NPs, the validity and
appropriateness of existing technologies are not
always clear. The environmental effects of NPs
need to be evaluated through the establishment
of scenarios reflecting their typical production
and use. Exposure and dose–effect models may
need to be adapted, taking into account their
changing physicochemical properties over time,
and their slow degradation.
10.2217/NNM.09.47 © 2009 Future Medicine Ltd
Nanomedicine (2009) 4(6), 605–607
Maria Dusinska† &
NanoTEST consortium*
Author for correspondence:
Health Effects Group,
Norwegian Institute for Air
Research (NILU), Centre for
Ecology and Economics,
POB 100, Instituttvn. 18,
N-2027 Kjeller, Norway
Tel.: +47 6389 8157;
Fax: +47 6389 8050;
maria.dusinska@nilu.no
*NanoTEST consortium:
M Dusinska, LM Fjellsbø,
Z Magdolenova, A Rinna,
E Runden Pran, A Bartonova,
ES Heimstad, M Harju, L Tran,
B Ross, L Juillerat,
B Halamoda Kenzaui,
F Marano, S Boland,
R Guadaginini, M Saunders,
L Cartwright, S Carreira,
M Whelan, CH Kelin, A Worth,
T Palosaari, E Burello,
C Housiadas, M Pilou,
K Volkovova, J Tulinska,
A Kazimirova, M Barancokova,
K Sebekova, M Hurbankova,
Z Kovacikova, L Knudsen,
MS Poulsen, T Mose, M Vilà,
L Gombau, B Fernandez,
J Castell, A Marcomini,
G Pojana, D Bilanicova
& D Vallotto
†
ISSN 1743-5889
605
Editorial
Dusinska
Improvements to the methodologies should
take into account the following factors:
n Tests for exposure should be designed that
express dose in terms of number concentration
and surface area, rather than mass concentration;
n
NPs may agglomerate and disagglomerate in
different environments, which may affect their
properties and these need to be characterized;
n
Impurities within, and adsorbed species on the
surface of, NPs may have significant effects on
risks and these possibilities should be taken
into account;
n
Biological processes involving NPs, including
translocation, cellular uptake and toxicological
mechanisms, are still largely unknown and
testing methodologies have to address these
possible pathways of effect.
It should also be noted that reference materials for the evaluation of NPs have not yet been
identified. With respect to specific concrete
suggestions, there is a clear need for validated
in vitro assays for NP evaluation. In vitro tests
should address key properties of the NPs, their
biopersistence and possible mechanisms such
as free-radical generation, cellular toxicity, cell
activation, genotoxicity and other generic end
points. In vitro tests should also provide target cell-specific end points addressing specific
organs or tissues such as effects on nerve cells or
the phagocytic capacity of macrophages.
“With some NP-based products already in
use in nanomedicine and many more under
development, it is critically important that
the potential risks are properly assessed.”
The FP7 project NanoTEST [106] addresses
these requirements in relation to the toxicological
profile of NPs used in medical diagnostics. The
overall aim of this project is to develop alternative testing strategies and high-throughput toxicity testing protocols using in vitro and in silico
methods that are essential for the risk assessment
of these NPs. To be able to achieve this ambitious
goal, the specific aims of NanoTEST are:
606
n
To carry out a detailed characterization of
selected NPs, in order to define their main
physicochemical properties;
n
To study specific and nonspecific interactions
of NPs with molecules, cells and organs and
to develop in vitro methods that can identify
the toxicological potential of NPs;
Nanomedicine (2009) 4(6)
n
To validate in vitro findings in short-term
in vivo models, to study manifestation of particle effects in animals and humans, and to
assess individual susceptibility in the response
to NPs;
n
To perform both structure–activity modeling
and physiologically based pharmacokinetic
modeling of NPs;
n
To adapt the most advanced and promising
assays for high-throughput automated systems;
n
To prepare for validation by the European
Centre for the Validation of Alternative
Methods (ECVAM).
All pertinent physicochemical properties of
concern, for example size distribution, shape,
specific surface area, porosity, chemical composition, purity, impurities of concern, surface
chemistry, surface charge and crystal structure are being determined in order to ensure
high-quality results. Dispersion and stability of selected NPs in aqueous and biological
media, as well as their interactions with culture
media components, are also being investigated.
Eventually, specific analytical protocols will be
developed for the quantitative determination
of the uptake and distribution of selected NPs
in biological tissues after in vitro and in vivo
exposure experiments.
This project will address many key elements
in the evaluation of NP exposure, cellular
uptake, transport and toxicological effects using
different biological systems. The mechanism of
action of particles is likely to involve oxidative
stress and inflammation. We will therefore focus
on the cross-cutting areas of cellular toxicity,
oxidative stress, inflammation, immunotoxicity,
genotoxicity and related end points. We aim to
propose relevant in vitro toxicological models
based on reference cell lines of these organs, in
order to identify reference biological markers
and end points that can be used to test the possible toxicity of medical NPs. To identify these
biomarkers, we will utilize different cell cultures (in vitro models), organotypic cell culture
and small fragments of organs (ex vivo models),
exposing them to NPs in clinical use. This will
allow us to determine the mechanism of action
and to predict the biological and physiological
response of exposure to medical NPs and finally
to evaluate the potential health hazards of NPs.
Whilst it is not possible to consider all organs
that may potentially be affected by exposure to
medical NPs, we intend to use a range of representative organs and to define representative
future science group
Testing strategies for the safety of nanoparticles used in medical applications
cell lines of these organs. We will focus on cells
derived from eight different biological target
systems: blood, vascular, liver, kidney, lung, placenta, digestive and CNS. All in vitro studies
will be coordinated and harmonized as much as
possible by using the same batch of NPs, handling and safety procedures, exposure time,
concentration and protocols with similar end
points, in order to be able to compare results
and to evaluate the most relevant biomarkers
and target cell types. Experimental protocols
(standard operating procedures) for biomarker
detection to be used across the cell systems are
being developed. The toxicological profile of
NPs in vitro will be validated in an experimental
in vivo model addressing the same toxicological
end points and organs.
“Nanotechnology is considered as a
technology of the future, with great potential
in biomedical applications.”
A better understanding of how properties of
NPs define their interactions with cells, tissues
and organs in exposed humans is a considerable scientific challenge, but one that must be
addressed if there is to be safe and responsible
use of biomedical NPs. NanoTEST will evaluate toxic effects and interactions of NPs used
in nanomedicine. Testing strategy and a battery of in vitro and in silico assays to evaluate
the toxic effects of NPs used in nanomedicine
will be developed, but these methodologies, we
hope, have a broader application to the safety of
other nanomaterials. Following development of
standard operating procedures and generation
of a common database, and in parallel with
in silico assays (quantitative structure activity
Websites
101 European Commission (2004). Towards a
European strategy for nanotechnology.
http://cordis.europa.eu/nanotechnology/
actionplan.htm
102 European Commission (2007). SCENIHR
(EU Scientific Committee on Emerging and
Newly Identified Health Risks) report
‘Opinion on the appropriateness of the risk
assessment methodology in accordance with
the technical guidance documents for new
and existing substances for assessing the risks
of nanomaterials’.
future science group
Editorial
relationship and physiologically based pharmacokinetic modeling), NanoTEST will evaluate
the toxic effects and interactions of NPs used
in nanomedicine. Results will be validated in
an experimental, ethically approved in vivo
model. The most advanced and standardized
techniques will be adapted for automation and
prepared for validation by the Joint Research
Centre (ECVAM). Finally, we will propose
recommendations for evaluating the potential
risks associated with new medical NPs, which
will be communicated to the scientific and
industrial community.
Financial & competing interests disclosure
The NanoTEST consortium is supported by the European
Commission FP7 (Health-2007-2001.3-4), contract no:
201335. The NanTEST consortium also receives contributions from Norwegian Institute for Air Research (NILU),
Kjeller, Norway; Institute of Occupational Medicine (IOM),
UK; Centre Hospitalier Universitaire Vaudois (HospicesCHUV), Switzerland; University Paris Diderot Parts7
(UP7), France; University Hospitals Bristol NHS
Foundation Trust (UBHT), UK; Commission of the
European Communities – Directorate General Joint Research
Centre (JRC), Italy; National Centre for Scientific Research
Demokritos (DEMOKRITOS), Greece; Slovak Medical
University (SMU), Slovakia; University of Copenhagen
(UCPH), Denmark; Advanced In Vitro Cell Technolgoies
SL (ADVANCELL), Spain; and Università Ca’ Foscari di
Venezia (University of Venice) (UNIVE), Italy.
The authors have no other relevant affiliations or financial involvement with any organization or entity with a
financial interest in or financial conflict with the subject
matter or materials discussed in the manuscript apart from
those disclosed.
No writing assistance was utilized in the production of
this manuscript.
http://ec.europa.eu/health/ph_risk/
committees/04_scenihr/docs/
scenihr_o_004c.pdf
103 European Parliament: Regulation (EC) no
1907/2006 of the European Parliament and of
the council of 18 December 2006 concerning
the registration, evaluation, authorisation and
restriction of chemicals (REACH), (OJ L 396,
30.12.2006) (2006).
http://eur-lex.europa.eu/LexUriServ/
LexUriServ.do?uri=OJ:L:2006:396:SOM:en:
HTML
www.futuremedicine.com
104 European Commission (2008). Follow-up to
the 6th meeting of the REACH competent
authorities for the implementation of
regulation (EC) 1907/2006; (REACH).
http://ec.europa.eu/environment/chemicals/
reach/pdf/nanomaterials.pdf
105 Technical Guidance Documents.
http://guidance.echa.europa.eu/guidance_
en.htm
106 NanoTEST.
www.nanotest-fp7.eu
607