Engineered Nanomaterials: Impact & Safety Aspects: White Paper

Engineered Nanomaterials:
Impact & Safety Aspects
White Paper
February 2017
2 NRP 64 Opportunities and Risks of Nanomaterials – White Paper
Contents
1 Introduction 1
2. Nanomaterials and the Environment 4
3. Nanomaterials for Human Health Applications 11
4. Safety Aspects and Risk Assessment 15
5. Summary and Recommendations 19

NRP 64 Opportunities and Risks of Nanomaterials – White Paper 1
1. Introduction
Progress in science shows that nanostructured materials can be found practically every-
where in nature, and that natural nanoscale processes at the cellular level are indispensa-
ble for life. Nanotechnology is a key enabling technology used for analysing, for designing
and developing new, bio-inspired and synthetic nanomaterials, each with its own specific
production methods, characteristics and lifecycle. This has been investigated by the 23 re-
search projects within the framework of National Research Programme «Opportunities and
Risks of Nanomaterials» (NRP 64).
Nanomaterials are rapidly being adopted in health-

care and in a broad spectrum of applications in the Nanomaterials and Nanoparticles
consumer and industry sectors, for example in the
field of energy storage. Notable recent developments The International Organisation for Standard-
include intelligent/functional nanomaterials devel- isation (ISO) has defined «nanomaterial» as a
oped in NRP 62, «Smart Materials»1. Nanomedicine «material with any external dimension in the
is an important application of nanoscience with the nanoscale or having internal structure or sur-
goal of exploiting the materials and methods of na- face structure in the nanoscale» (ISO, 2010).
notechnology to the benefit of human health. There is According to the definition of the EU 50 % or
already a broad application in medical in vitro diag- more of the particles need to be in the nano-
nostics, as well as rapidly growing clinical experience
scale range. «Nanoparticle» are defined as a
with nanostructured therapeutics, and nanotechnol-
«nano-object with all three external dimen-
ogy is an enabling technology for personalised med-
sions in the nanoscale» where nanoscale is
icine. Pharmaceutical and MedTech companies are
defined as the size range from approximately
therefore adopting nanotechnologies and nanomate-
1–100 nm (ISO, 2008).
rials as strategic issues for business development and
in order to remain globally competitive.
An important aspect of every innovation is the assessment of risk and the determination of
conditions for safe use. Understanding the potential benefits and risks is important for the
general public, as well as for guiding further research, supporting decision-making in gov-
ernment funding and regulation, for the innovative capacity and competitiveness of indus-
try and for the protection of the environment. In 2009, the Federal Council commissioned
the Swiss National Science Foundation to implement a national five-year programme fo-
cusing on the opportunities and risks of nanomaterials: NRP 64. The total funding amount-
ed to twelve million Swiss francs.
The goal of NRP 64 was to deliver the scientific basis for a deepened understanding of the
potential benefits and undesirable effects of nanomaterials. Risk assessment included elu-
cidation of potentially harmful mechanisms, the probability of exposure to such materials
due to emissions, their potential persistence in the environment and their biological effects.
1 www.nrp62.ch
In the scope of NRP 64, the promise of innovative solutions based on nanomaterials was
analysed, including consideration of their potential risks. For example, the use of nanopar-
ticles in materials for medical implants led to an in-depth study on their biodegradation
carried out by Martin Frenz, University of Bern. Other applications that were analysed in-
depth in terms of their benefits and risks included the use of nanomaterials as immune
modulators (Barbara Rothen-Rutishauser, Adolphe Merkle Institute), for the purification of
blood (Beatrice Beck Schimmer, Institute of Anaesthesiology, University Hospital Zurich),
and for targeted drug delivery (Francesco Stellacci,
Federal Institute of Technology, Lausanne). Several
Risk Assessment: projects focused on the development of methods for
assessing the risks associated with nanomaterials.
The risk of a chemical is defined as the product
of hazard and exposure: risk = hazard (i.e. tox- NRP 64, «Opportunities and Risks of Nanomaterials»,
icity) x exposure (to the hazardous chemical) was thus a timely effort to deepen the understanding
of these important issues. It yielded an array of com-
plementary information in the areas of healthcare,
protection of the environment, energy, food and construction materials. It was also embed-
ded in various past and ongoing national and international initiatives focusing either on
nanoscience innovation, for example, SNSF NCCR Nanoscale Science2 in Switzerland, and
the US Nanoscience initiative, as well as recent major European nano-safety programmes,
including NanoSafety Cluster,3 NanoReg4, NanoImpactNet5 and the global Working Party
on Manufactured Nanomaterials (WPMN, established in 2006).6 WPMN was an initiative of
the Organisation for Economic Cooperation and Development (OECD), where the Mutual
Acceptance of Data in the Assessment of Chemicals7 is an essential agreement.
Switzerland holds a leading position in the field of nanoscience, and NRP 64 supported
the maintenance and further development of this position through risk assessment and
the examination of issues relating to regulatory control. Switzerland aims to maintain its
leading role in responsible and rational risk assessment policies and regulations. In 2008,
the Federal Council adopted its Action Plan for «Synthetic Nanomaterials», which requires
federal authorities to create the legal bases for the safe handling of nanomaterials as an in-
tegral part of technological development. In the scope of this action plan, the Federal Office
of Public Health and the Federal Office for the Environment developed a precautionary ma-
trix intended to help companies to comply with the requirement of due diligence and their
duty to perform self-regulation versus employees, consumers and the environment. This
matrix facilitates the identification of potential risks posed by nanomaterials and the com-
munication of important safety information along the entire production and supply chain.8
A number of experts from NRP 64 were involved in the development of this action plan.
2 http://www.snf.ch/en/researchinFocus/nccr/nccr-nanoscale-science/Pages/default.aspx
3 horizon2020; http://horizon2020projects.com/industrial-leadership/nanosafety-cluster-releases-2016-compendium/
4 www.nanoreg.eu
5 https://empa.ch/web/s506/nanoimpact
6 Rasmussen K, Gonzalez M, Kearns P, Sintes JR, Rossi F, Sayre P. Reg. Toxicol. Pharmacol. 2016; 74, 147-160.
7 OECD. Decision of the Council concerning the Mutual Acceptance of Data in the Assessment of Chemicals. 12 May 1981-C(81)30/
FINAL.
8 Precautionary matrix for synthetic nanomaterials, Version 3.0, Federal Office of Public Health, Division Chemical Products, 2013
Characteristics of Nanomaterials
Every kind of nanoparticle is characterised by its unique physical and chemical prop-
erties which differ from their macroscale counterparts. Nanomaterials can be classified
into five different groups:
• Carbon-based materials: these can take the form of hollow spheres and ellip-
soids (fullerenes), tubes or flakes (e.g. graphene). Carbon-based nanomaterials
are being used in many different applications, e.g. in electronics, in films and
coatings, or they are being incorporated into various materials to improve differ-
ent properties.
• Metal-based materials: these include metals such as gold, silver and platinum,
as well as metal oxides such as titanium dioxide and copper oxide. Quantum dots
also belong to this group. They are characterised by their unique electrical, mag-
netic and optical properties. All materials in this group can be coated with differ-
ent materials such as organic molecules.
• Dendrimers: these nanosized polymers consist of branched units. Their interior

cavities can be used, for example, for drug delivery.
• Self-assembled soft nanoparticles: nanoparticles formed by the process of

self-assembly of individual molecules (unimers) due to specific physicochemical
properties.
• Composites: materials that combine nanoparticles with other nanoparticles or

with other materials. The incorporation of nanomaterials into composites en-
hances, for example, mechanical, thermal and barrier properties of a material.
Source: http://www.azonano.com/article.aspx?ArticleID=1872
Abbreviations
AgNP: Silver nanoparticle
CNT: Carbon nanotube
CBN: Carbon-based nanomaterial
ENM: Engineered nanomaterial
ENP: Engineered nanoparticle
OECD: Organisation for Economic Cooperation and Development
WPMN: Working Party on Manufactured Nanomaterials

This White Paper is one of the synthesis products of NRP 64. Its aim is to summarise the op-
portunities associated with nanomaterials and their safe use in the areas of human health
and protection of the environment based on the activities within the individual research
projects. It also presents conclusions from an integrative point of view, and takes into ac-
count recent progress in international activities in this area. Its purpose is to place the
scientific findings of the research into the broader context of societal progress, healthcare
policy, and environmental relevance in a language that is more easily accessible than the
many individual scientific papers that originated in this research programme. It may serve
as an information source for the formulation of future policies in the areas of research
funding and environmental legislation, and as background information for developing in-
dustrial strategies.
While a detailed description of each project in this programme is beyond the scope of this
paper, a number of exemplary studies are discussed and put into context.
2. Nanomaterials and the Environment

The potential of nanomaterials lies in their new and unique chemical and physical proper-
ties, which differ from their macro-scale counterparts and which, at least in part, originate
from their large surface area per volume. Interesting new properties are also caused by
effects of quantum mechanics. Improved hardness, strength, increased electrical conductiv-
ity, different catalytic efficiency and new thermal properties are typical features that make
engineered nanomaterials (ENMs) beneficial when incorporated into batteries, computers,
cosmetics, sports equipment, building materials, coatings or medical devices. Many such
applications may be of benefit to the environment, such as new environmentally friendly
construction materials and improved energy storage. Yet the potential uses of ENMs, their
diversity and their unique properties, must be evaluated in light of potential risks associated
with the manufacture and use of nanomaterials. The more products containing ENMs that
are developed, the greater the probability of their release into the environment, which can
occur during production, use and (unintentional) disposal of nano-based products. Once
released, the flow of nanomaterials, their fate, their bioavailability and their biodegradation
have to be studied in order to properly evaluate their risk. All these issues were addressed
in NRP 64 in the projects relating to the environment, either from the point of environmen-
tal protection or improved knowledge with regard to the lifecycle of the nanomaterials, from
production to environmental release.
Nanomaterial Developments to Benefit the Environment
Various projects explored the opportunities of nanomaterials with potential to enhance

environmental protection. For example, the group led by Christoph Weder at the Adolphe
Merkle Institute in Fribourg investigated porous cellulose nanocomposites as an environ-
mentally friendly alternative to conventional insulation materials. By applying new proto-
cols for the preparation process and by testing different binders they were able to create
aerogels with improved and very promising properties,9, 10 which could lead to new or im-
proved applications. Another field of innovation in which nanomaterials could offer prom-
ising benefits is energy storage. Lithium-ion batteries provide excellent opportunities for
grid storage of renewable energy, for electric mobility and for energy storage in portable
devices. Within NRP 64, the group led by Katharina Fromm at the University of Fribourg
investigated various nanocomposites as active materials in order to improve currently used
materials and create new materials with improved battery capacity.11 Further, the group led
by Vanessa Wood, Federal Institute of Technology, Zurich, developed an attractive approach
to increasing energy density of nano-based lithium-ion batteries by assembling nanoparti-
cles into microspherical particles.12
Scenarios Relating to the Emission of Nanomaterials into the Environment
Production
During the manufacture and processing of nanomaterials there are several potential sourc-
es leading to environmental and workplace exposure. The most likely route is direct in-
halation of materials released into the air. At the University of Lausanne, the group led
by Michael Riediker conducted inhalation studies with unintentionally produced NPs oc-
curring in welding fumes and revealed an increase in oxidative stress biomarkers in the
blood and urine after such exposure.13 With the nano aerosol chamber for in vitro toxicity
(NACIVT), a method that was improved within the scope of NRP 64 in the project led by
Marianne Geiser at the University of Bern, cell cultures replicating the inner lung surface
can be exposed to a wide range of engineered nanoparticles (ENPs) out of a continuous air-
stream in order to assess the toxic effects of these ENPs on lung cells.14
Emissions during or after Use

Numerous consumer products comprising nanomaterials are already on the market and
their number is growing constantly, increasing the possibility of nanomaterials being unin-
tentionally released into the environment.
9 Mueller S, Sapkota J, Weder C, Foster, EJ J. Appl. Polym. Sci. 2015, Volume 132 (13)
10 Mueller S, Weder C, Foster EJ, Green Materials 2014, 2, 169-182.
11 Kwon NH1, Brog JP2, Maharajan S2, Crochet A2, Fromm KM3. Chimia (Aarau). 2015;69(12):734-6.
12 Nowack LV, Bunjaku T, Wood V, Adv Sci (Weinh), 2015, 2(6)
13 Graczyk H, Lewinski N, Riediker M, Ann Occup Hyg. 2016 Mar;60(2):205-19.
14 Jeannet, N, Fierz, M, Geiser, M, Nanotoxicology, 2015 Feb;9(1):34-42.
Silver nanoparticles (AgNPs) are used in commercial products for their antimicrobial prop-
erties15 and during the life cycle of these products the discharge of AgNPs into the environ-
ment seems inevitable. As dissolved silver is highly toxic to certain organisms, the discharge
of AgNPs is an important field of research and several NRP 64 projects concentrated on the
fate and possible toxicity of released AgNPs (see chapter 4).
Carbon nanotubes (CNTs) are often used in composites in view of their great strength and
stability. There are ongoing debates on the health risks associated with CNTs and whether
their fibrous forms could be as harmful as asbestos if inhaled. The pathogenicity of fibres
greatly depends on their length, thickness and biopersistence. As CNTs come in many com-
binations of sizes, types, purity levels and sources, a comparative study of existing evidence
on the toxicity of CNTs remains difficult. More research is needed in order to determine
whether CNTs can cause fibrosis and cancer in the long term and whether it is possible
to make them safe by design. At the Federal Institute of Technology, Zurich, the project
headed by Jing Wang aimed to develop a technology platform on which the toxicity of CNTs
can be assessed as a function of their release when the composite materials are subjected
to abrasion, weathering processes and elevated temperatures. Amounts of CNTs released
after abrasion, weathering or exposure to elevated temperatures were low, but when carbon
fibre reinforced polymer cables were stretched to breaking point, respirable carbon fibres
were set free.16, 17, 18, 19, 20 The toxicity of the particles was assessed in different cell culture
systems, resulting in no acute negative effects at realistic doses of CNTs (see also chapter
4). The project demonstrated for the first time the cytotoxicity of CNTs incorporated in na-
nocomposites as a direct function of their release.21, 22 Although long-term effects still need
to be studied, this project suggests that it is possible to develop safe consumer products with
CNTs and provides important know-how for their design.23
The platform created in the above-mentioned project was also used in the project led by
Peter Wick at Empa, St. Gallen, where the release of copper was assessed after abrasion of
Scots pine wood that was previously treated with nanocopper-based wood preservatives.
Based on the rationale that nanocopper may penetrate wood more efficiently and provide
a longer-lasting protection against wood-destroying fungi thanks to a reservoir effect, na-
nocopper-based formulations were introduced in 2006 in the USA. With this project it was
demonstrated that the expected beneficial effect of nanobased formulations was not ob-
served in common European wood species, i.e. that nanobased formulations did not pene-
trate wood more efficiently than conventional copper-based preservatives. Copper-loaded
wood dust was released regardless of the treatment with nano-based or conventional pre-
15 Hansen SF, Heggelund LR, Baun A, 2016, Environmental Science: Nano, vol 3, no. 1, pp. 169-180
16 Schlagenhauf L, Kuo YY, Wang J. J Occup Environ Hyg. 2015;12(8):D178-83.
17 Schlagenhauf L, Nüesch F, Wang J, 2014, Fibers 2, 108-127
18 Schlagenhauf L, Chu BTT, Wang J, 2012 Environ. Sci. Technol, 46, 7366 – 7372
19 Schlagenhauf, L, Kuo YY, Wang J, 2015, Journal of Nanoparticle Research, 17:440
20 Wang J, Schlagenhauf L, Setyan A, J Nanobiotechnology, 2017 Feb 20;15(1)
21 Schlagenhauf L, Kianfar B, Wang J, NanoScale, 2015, 7 18524-36
22 Schlagenhauf L, Buerki-Thurnherr T, Wang J, EST, 2015, 49,10616-23
23 Schlagenhauf L, Kianfar B, Wang J, Nanoscale. 2015 Nov 28;7(44)
servatives.24 This could pose a health risk as previously recognised.25, 26 Further, the project
demonstrated that, over time, substantial amounts of copper are released into the environ-
ment via dissemination of spores. Mechanisms of copper release via wood dust or spores
are not specifically related to nanocopper formulations, but rather generally apply to cop-
per-based systems. The findings obtained from this project indicate that while nanocopper
may be applied successfully in North America, where timber is readily treatable, in Europe
the preservative is not superior to conventional wood preservatives.27 This finding is of
importance for European regulatory bodies when approving or rejecting nanocopper as a
wood preservative.
A reliable assessment of environmental emissions and concentrations of various nanoma-

terials is possible on the basis of a modelling tool developed by the research group led
by Bernd Nowack at Empa, St. Gallen.28 Based on the production, use and disposal of im-
portant nanomaterials such as nanosilver, nano-titanium dioxide, nano-zinc oxide, carbon
nanotubes, fullerenes and nano-gold and their behaviour in technical systems, a dynamic
probabilistic mass-flow model was developed which estimates the former, current and fu-
ture flows of these ENMs into the environment for Switzerland and the EU.29 Estimated
concentrations of these nanomaterials in sewage effluent, surface water, sediments and
sludge treated soils are several orders of magnitude lower than those of conventional ma-
terials.30 The combination of the mass-flow model with an environmental fate model (see
section on environmental fate) and with hazard data taken from the ecotoxicological lit-
erature facilitates a probabilistic environmental risk assessment of the above-mentioned
nanomaterials.31
Detection and Characterisation of Nanomaterials in the Environment
Due to the diversity of types of materials available and being developed, together with the
fact that each nanomaterial has its unique properties, their detection, evaluation of their
fate in the environment and assessment of their potential risk are very challenging tasks.
Before NRP 64 was initiated, it was not yet possible to detect and trace concentrations of
nanomaterials in natural systems such as bodies of water, sediments or soils. Substantial
progress has been made in the past few years and various projects within the scope of
NRP 64 have made a valuable contribution. For example, in order to quantify and detect
carbon-based nanomaterials (CBNs) and to identify possible biotransformation products,
various new methods and techniques were developed and applied by the group led by
Hans-Peter Kohler at Eawag, Dubendorf. The same research group contributed to a crit-
24 Civardi C, Van den Bulcke J, Schwarze FWMR, PLOS One, 2016:11 (9)
25 Civardi C, Schlagenhauf L, Schwarze FWMR, Journal of Nanobiotechnology, 2016 14:77
26 Civardi C, Schwarze FWMR, Wick P. Env Poll. 2015. May; 200:126–132.
27 Habicht J, 26. Holzschut-Tagung. Neue Normen, neue Erkenntnisse; 2010 Apr 22-23; Göttingen, DE. Münster: Self published;
2010. p. 161-188 German
28 Sun TY, Gottschalk F, Nowack B. Environ Pollut. 2014, Feb;185:69-76.
29 Bornhöft NA, Sun TY, Nowack B, Environmental Modeling and Software, 2016; 76: 69-80.
30 Sun TY, Bornhöft NA, Nowack B, Environ Sci Technol, 2016, 3;50(9):4701-1
31 Coll C, Notter D, Nowack B, Nanotoxicology, 2016;10(4):436-44.
ical review of methods and techniques used in carbon nanotube (CNT) research.32, 33 Jing
Wang’s group at the Federal Institute of Technology, Zurich, implemented new methods
for quantifying CNTs after abrasion processes. Further, the project led by Thomas Bucheli
(Agroscope, Zurich), contributed tools and methods for monitoring nanomaterials in water
and in soil34, 35, 36, while the group headed by Ralf Kaegi (Eawag, Dubendorf) contributed
to an increased understanding of sulphidation kinetics and reaction mechanisms of silver
nanoparticles in wastewater systems.
Environmental Fate of Nanomaterials
Predictions based on Computational Modelling

Once released into the environment, the flow of ENMs within the environment, as well as
their potential for transformation and their possible interactions with other substances,
determine their potential environmental risks and govern their accumulation within the
ecosystem. In order to model such processes, the group led by Bernd Nowack at Empa, St.
Gallen, developed an environmental fate model in collaboration with the Federal Institute
of Technology, Zurich. Concepts of corresponding models for organic pollutants were re-de-
signed to take account of ENM-specific properties and fate behaviour such as aggregation,
sedimentation, dissolution and other surface transformations.37 The results show that ENMs
aggregate with naturally occurring suspended particles, which determines their transport
in surface waters.38 Also, the studies confirm that sediments act as sinks for ENMs. This
highlights the need to consider regional and site-specific conditions of soils and sediments
when assessing the spatial and temporal variability of ENM concentrations and their po-
tential risks for the environment and human health. The mass-flow model described in the
previous section was combined with the environmental fate model in order to predict the
fate of ENMs across a network of all Switzerland’s major rivers.
Processes in Wastewater Systems

The fate of silver nanoparticles (AgNPs) in (waste) water systems was studied by the re-
search group led by Ralf Kaegi at Eawag, Dubendorf. The use of silver nanoparticles in
commercial products such as textiles and cosmetics is increasing, and their discharge into
wastewater is inevitable. Due to the well-known antimicrobial effects of silver, AgNPs may
impair biological processes that are vital to sewage treatment (e.g. nitrification during the
activated sludge process). Furthermore, AgNPs may be released into the environment via
the effluents of these treatment plants or the use of digested sludge as a fertiliser. AgNPs
32 Petersen EJ, Flores-Cervantes DX, Winchester MR, Environ Sci Technol. 2016; 3;50(9):4587-605.
33 Flores-Cervantes DX, Maes HM, Kohler HPE, Environ Sci Technol, 2014; 48: 4826-4834.
34 Mortimer M, Gogos A, Slaveykova VI, Environ. Sci. Technol., 2014, 48, 8760-8767.
35 Gogos A, Kaegi R, Bucheli TD, Environ. Sci. Nano, 2014. 1, 584-594.
36 Gogos A, Moll J, Bucheli TD, J. Nanobiotechnol., 2016, 14:40.
37 Praetorius A, Scheringer M, Hungerbühler K, Environ Sci Technol. 2012;19;46(12):6705-1
38 Praetorius A, Labille J, Bottero JY. Environ Sci Technol. 2014; 16;48(18)
were spiked into a sewage system and their changes inside a 5-kilometre-long sewer pipe
were studied. Based on total silver concentrations measured in samples collected along the
sewer pipeline at increasing time intervals, complemented by results from transmission
electron microscopy analyses, an efficient transport of the AgNPs along the sewer chan-
nel was shown without substantial losses to the sewer biofilm.39 Synchrotron based X-ray
absorption spectroscopy revealed that AgNPs become efficiently sulphidised during sew-
age treatment, which reduces their toxicity by several orders of magnitude.40 Experiments
where AgNP reacted with bisulfides in the presence of various amounts of humic acid sug-
gested that the sulfidation of AgNP in wastewater proceeds very fast with half-life times
of a few minutes to hours.41 AgNPs accumulate in the sewage sludge, which results in their
very efficient removal from the wastewater (up to 95 percent). Depending on applicable lo-
cal policies, sewage sludge is used as a fertiliser in agriculture, which represents a possible
route for the release of AgNPs into the environment. However, for Switzerland this release
route is not relevant, as sewage sludge is combusted. Together, the findings indicate that
only a small amount (< 5 percent) of AgNPs are released from wastewater treatment plants
and reach surface waters. Experiments where AgNP reacted with other metal sulfides pres-
ent in urban surface waters (e.g. CuS and ZnS) resulted in the sulfidation of the AgNP and
the dissolution of the metal sulfides. Half live times for the sulfidation reaction ranged from
hours to days and depended on the AgNP size and the type and concentration of the metal
sulfides.42 Treated sewage can be ozonated in order to degrade remaining micropollutants,
whereby sulphidised AgNPs are oxidised, which results in the release of dissolved silver
into the effluent (see chapter 4).43 These effects may become relevant at elevated AgNP
concentrations and should be taken into consideration when evaluating ozone treatments
of wastewater plants, explicitly in Switzerland where several such plants are planned to be
upgraded by ozone treatment. Experiments conducted with different kinds of particles (Ag,
Au) of different sizes and with different coatings indicate that sewage treatment plants act
as a very efficient barrier for different kinds of (metal) nanomaterials.
Agriculture
Titanium dioxide and multi-walled CNTs are considered as active ingredients or additives
of pesticides and fertilisers. The project led by Thomas Bucheli at Agroscope, Zurich, an-
alysed the mobility of such ENMs in soil, together with their uptake by soil microbes and
the effects on the ecological function of these microbes.44, 45, 46 Both types of nanomaterials
show limited mobility due to strong interactions with soil particles.47 To assess the potential
benefits and risks associated with nanomaterials used in plant protection and fertilisation,
a detailed review of the current status, foreseeable applications and research priorities was
39 Kaegi R, Voegelin A, Mueller E, Water Research, 2013, 47(12), 3866–3877.

40 Kaegi R, Voegelin A, Siegrist H, Environmental Science & Technology, 2011, 45, 3902–3908.
41 Thalmann B, Voegelin A, Kaegi R, Environ. Sci.:, 2016, Nano 3, 203–212.
42 Thalmann B, Voegelin A, Kaegi, R, Environmental Science & Technology, 2014, 48, 4885–4892.
43 Thalmann B, Voegelin A, Kaegi R, Environmental Science & Technology, 2015 ; 49(18), 10911–10919
44 Moll J, Gogos A, Bucheli TD, van der Heijden MGA, J. Nanobiotechnol., 2016, 14:36
45 Moll J, Okupnik A, Bucheli, TD, Widmer F, PLoS ONE. 2016, 11(5)
46 Moll J, Gogos A, Bucheli TD, van der Heijden MGA, J. Nanobiotechnol., 2016, 14:36.
carried out within the scope of this project. This review was highly recognised by inter-
national experts in the field and was discussed at international meetings, workshops and
conferences.48
Biodegradation
Biodegradation of nanoparticles may occur via routes typically observed in the biodegrada-
tion of organic molecules. However, it may also result in changes in the physical structure
or surface characteristics of the material, depending strongly on the chemical and physical
nature of the particle. When NRP 64 was initiated, there was a great need to find evidence
of possible biotransformation mechanisms, including potential structural defects of CBNs
acting as reaction sites. Familiarity with such processes is of great importance as the pro-
duction of CBNs steadily increases, with an estimated worldwide production of more than
300 tonnes per annum of CNTs and fullerenes alone. CBNs are known for their very high
thermal, chemical and mechanical durability. Based on these characteristics it is expected
that CBNs are very persistent in the environment and undergo biotransformation at very
slow rates. The project led by Hans-Peter Kohler (Eawag, Dubendorf) systematically ana-
lysed various CNT materials and developed methods to quantify and assess the extent of
enzymatic transformations of CNTs. In this in-vitro setup, peroxidase mediated degradation
was very slow.49 In contrast, in biological systems such as macrophages, neutrophilic and
eosinophil leucocytes, much faster degradation is reported in the literature.50, 51, 52 However,
CNT characteristics that make materials more or less resistant to biological transformation
could be identified – a finding with implications for CNT design. Also, the findings provided
information about enzymes that could play a key role in degradation processes and there-
fore might be used for the long-term treatment of CNTs in water or soils. The key outcome
of this project emphasised the importance of the systematic characterisation of CBNs in
various settings in order to draw reliable conclusions with regard to transformation pro-
cesses. A review was therefore carried out regarding the status of techniques for the quali-
tative and quantitative analysis of CBNs and their applications.53
48 Gogos A, Knauer K, Bucheli TD, J. Agric. Food Chem, 2012, 60, 9791-9792
49 Flores-C ervantes DX, Maes HM, Kohler HPE, Environ Sci Technol, 2014; 48: 4826-4834
50 Andón FT, Kapralov AA, Kagan VE. Small 9.16 (2013): 2721-2729.
51 Farrera C, Bhattacharya, Fadeel B. Nanoscale 2014,6 (12), 6974-6983.
52 Kotchey GP, Zhao Y, Kagan VE, Star A. Advanced drug delivery reviews (2013), 65 (15), 1921-1932.
53 Petersen EJ, Flores-Cervantes DX, Bucheli TD, Winchester MR, Environ Sci Technol., 2016; 3;50(9):4587-605.
3. Nanomaterials for Human Health

Applications
The development and study of nanomaterials for medical applications is a key focus of
nanomedicine research and development (where «nanomedicine» denotes the use of nano-
science methods, materials and tools to the benefit of human health). The design of ENPs is
seen as one of the most promising applications of nanomaterials, because such innovative
materials open the door to new ways to protect human health and prevent disease by al-
lowing the monitoring of health and the early detection of diseases. Furthermore, they are
suitable for delivering therapies that overcome problems with conventional drugs.54 This
was also successfully demonstrated by projects within the scope of NRP 64.
Various projects focused on specific types of nanoparticles, including magnetic, gold and
polystyrene nanoparticles. Magnetic nanoparticles are currently used together with a mag-
netic force for cell sorting,55 e.g. to gather circulating stem cells that might then be used
after leukaemia therapy to replenish blood cells. Such nanomagnets are also used in med-
ical imaging and permit the new therapeutic modality of targeted hyperthermia in can-
cer.56 By combining diagnostic and therapeutic capabilities, they are an enabling technology
for theranostics, e.g. in simultaneous detection and treatment of a disease such as cancer.57
Gold nanoparticles are already widely used in rapid diagnostic tests in clinical practice
worldwide due to their characteristic optical properties. Multifunctional gold-based nano-
composites have the potential to improve theranostics of cancer and other diseases.58 Other
types of nanoparticles include polystyrene nanoparticles used as biosensors in rapid tests,
nanoparticle vaccines59, 60 and nanoparticles for targeted drug delivery.61
Research results can contribute to our understanding of the world around us, but they can
also contribute towards societal goals such as public health, medical applications and in-
dustrial progress. In order for a scientific finding to have an impact, the process of indus-
trial «translation» is of the utmost importance. This transition from theory to practice in
medicine often requires 4 to 6 years in diagnostic applications and devices, and 6 to 12 years
(or even longer) for applications for new therapies. Current concepts for the development
of new therapies are highly regulated in order to assure safety and ethics when human
participants are involved. While NRP 64 was not designed as a product development pro-
gramme, but rather for elucidating the scientific bases for the safe use of ENMs in human
54 Kagan CR, Fernandez LE, Weiss PS, ACS-Nano, 2016, 10 (10), 9093–9103.
55 Plouffe BD, Murthy SK, Lewis LH, Rep Prog Phys., 2015, 78(1)
56 Hayashi K, Nakamura M, Ishimura K. Theranostics. 2013;3:366-76
57 Gobbo OL, Sjaastad K, Prina-Mello A. Theranostics; 2015; 5(11): 1249–1263.
58 Dykman LA, Khlebstov NG. Biomaterials; 2016, 108, 13-34
59 Zhao L, Seth A, Middelberg AP. Vaccine; 2014, 32 (3), 327-337
60 Powles L, Xiang SD, Plebanski M, Vaccines, 2015, 3(4): 894–929
61 Masood F. Materials Science and Engineering, C, 2016; 60, 569 - 578
health and the environment, it nevertheless resulted in project outcomes that approximate
medical applications.
The following projects focusing on health applications demonstrate the benefits of ANMs
and raise awareness of the associated risks, and illustrate the multidisciplinary character
and translational aspects of this field. The majority of projects used nanoparticles, except
the last one listed here, which used cellulose nanofibres and was the result of an unexpect-
ed discovery.
The first project presented here focused on the development of a new extracorporeal blood
purification system by exploiting biocompatible carbon-coated magnetic nanoparticles de-
veloped by Beatrice Beck Schimmer at the Institute of Anaesthesiology, University Hospital
Zurich. It offers the possibility of target molecule isolation and removal from the body,
e.g. in a detoxification procedure used after a drug overdose or in case of an imbalance in
inflammatory mediators. This was demonstrated by the removal of the drug, digoxin, and
the toxic heavy metal, lead, from the blood of living rats62 and by the rapid elimination of
endotoxins from human blood in vitro.63 Potential risks of this technique were examined by
studying the possible interaction of the nanoparticles with blood cells and plasma, includ-
ing inflammation reactions and toxicity. Coagulation was not affected to a clinically relevant
degree and no inflammatory reaction could be shown during the experiments. Long-term
exposure that could, for example, result from a device failure of the magnetic trap while
eliminating toxins in extracorporeal circulation, did not reveal any sign of inflammation,
necrosis or induction of malignancy in liver and lung tissues. This blood purification system
based on carbon-coated magnetic nanoparticles developed in this NRP64 project has the
potential to be translated to clinical medicine. The nanoparticles were extensively tested
in the extracorporeal blood compartment, thus permitting the next step towards a clinical
phase I study.
The immune-modulatory potential of gold nanoparticles with different surface coatings

was explored in the project led by Barbara Rothen-Rutishauser at the Adolphe Merkle In-
stitute by using a sophisticated 3D lung model that emulates the human airway tissue bar-
rier (in vitro) and a mouse model (in vivo). The findings indicated that specific surface
properties, including charge and composition of surface modified gold nanoparticles, can
modulate the uptake of the nanoparticles by human monocyte-derived dendritic cells, but
this does not affect the phenotype, cytotoxicity or cytokine secretion.64 The influence of the
surface charge on the interaction between the nanoparticles and antigen presenting cells
in different respiratory tract compartments was then further investigated in vitro as well
as in vivo.65, 66 Positively charged particles coated with a poly-vinyl-alcohol polymer were
taken up to a larger degree in comparison with particles with negative surface charge in
both models. In addition it was demonstrated that the positively charged gold nanoparti-
62 Hermann IK, Schlegel A, Beck-Schimmer B, Nanoscale 2013; 5: 8718-23

63 Hermann IK, Urner M,, Beck-Schimmer B, Adv Healthc Mater, 2013, 6: 829-35
64 Fytianos K, Rodriguez-Lorenzo L, Rothen-Rutishauser B. NBM, 2015, 11(3):633-44.
65 Fytianos K, Chortarea S, Rothen-Rutishauser B, ACS Nano. 2017 Jan 24;11(1):375-383
66 Seydoux E, Rodriguez-Lorenzo L,, von Garnier C. NBM; 2016, 12 (7), 1815 - 1826
cles induced T-cell activation in draining lymph nodes of the respiratory tract in vivo. Thus,
surface charge determines the uptake by antigen presenting cell population in different
respiratory tracts and plays a role in the modulation of downstream immune responses
such as the proliferation of CD4+ T cells in lung draining lymph nodes. These findings are
examples of particle-specific properties, which are important for the development of, for
example, immune-modulatory nanoparticles.
A Federal Institute of Technology, Lausanne, project led by Francesco Stellacci explored

new composite nanoparticles that combine a metallic core with a coating of organic mol-
ecules. Remarkably, these are able to cross the cell wall.67 This involves an energy inde-
pendent interaction of the ligand of the metallic nanoparticle with the plasma membranes
in diverse biological systems. Proteins are able to adsorb in a corona-like structure on the
particles’ surface. Modification of this surface results in changes in the corona and clev-
er design could therefore optimise the interaction of the particles with, for example, cell
membranes, proteins or viruses68 when such an interaction is required. While the design
of such nanoparticles contributes towards progress in science, the effective and safe use
of such materials for targeted drug delivery will require more research. Nevertheless, such
achievements in early phase innovative projects require a wise research funding policy,
which ensures that academic innovations can ultimately lead to competitive applications to
the benefit of patients, as well as provide impulses for Swiss industry.
In the project led by Peter Wick at Empa, St. Gallen, the translocation mechanism of nan-
oparticles across the human placenta barrier was studied using the ex-vivo placenta per-
fusion model. It was demonstrated that unmodified polystyrene nanoparticles are able to
cross this barrier in a size dependent manner69 and that their surface modification plays a
significant role in the translocation rate.70 Understanding such quantitative structure-ef-
fect relations opens the door to «Safety-by-Design» approaches for future products. Safety
studies demonstrated that specific particles were accumulated in placental tissue without
affecting the viability and functionality of the placenta within the period of observation.71,
72
Such nanoparticle-based transport systems will require further study in order to identify
detailed mechanisms and could also open up another possible route for future nanomedical
applications.
A project led by Martin Frenz at the University of Bern studied the use of nanoparticles
for suture-less blood-vessel repair in surgery by laser. Dye-enhanced nanoparticles were
incorporated into a biodegradable implant for the purpose of transforming laser light into
localised heat for the suture-less laser soldering of blood vessels, in particular in the brain.
Two types of nanoparticles (gold and silica) with different surface properties were devel-
oped and investigated. Thorough safety studies were carried out and included the study of
67 Carney RP, Carney TM, Stellacci F. Biointerphases;, 2012, 7, 17

68 Huang R, Carney RP, Stellacci F, Lau BL. Nanoscale; 2013, 5 (15), 6928-35
69 Grafmüller S, Manser P, von Mandach U, J. Vis. Exp. 2013, 76, e50401
70 Grafmueller S, Manser P, Wick P, Environ Health Persp, 2015 123:12 1280-1286
71 Grafmueller S, Manser P, WickP, Sci Technol adv Mat, 2015(16) 044602
72 Grafmueller S, Manser P, Wick P, Environ Health Persp, 2015 123:12 1280-1286
biodegradation of the implants containing nanoparticles. The effects of the nanomaterials

on enzymes, cell lines and primary cells ex-vivo were examined and an organotypic model
was used to emulate an in-vivo situation.73 The nanoparticles were mostly absorbed in a
concentration- and time-dependent manner by the immunoreactive cells of the brain. Con-
firming the findings by others in NRP 64, less negatively charged nanoparticles were taken
up more rapidly. Among several involved endocytosis pathways, macropinocytosis was the
most prominent. Studies to determine the safety of the material indicated that cell viability
was not affected, but at the highest tested nanoparticle concentration a brief increase in ox-
idative stress was detected. Among several tested particle types, gold nanoparticles proved
to be the best candidates for laser assisted vascular soldering in the brain. In addition, sev-
eral particle types, including polycapsulated nanoparticles for laser tissue soldering, were
also developed.74
The group led by Christoph Weder at the Adolphe Merkle Institute focused on the study of
new high-performance polymer nanomaterials based on natural cellulose nanofibres iso-
lated from renewable sources such as plants or wood. Here, one of the goals was to assess
the potential risks of these materials following inhalation,75 while another was to gain con-
trol over the synthesis of morphologies by understanding and exploiting nanofibre-to-na-
nofibre or nanofibre-to-polymer interactions. Such architectures could be key materials for
an abundance of applications including tissue engineering. Multi-zonal polymer / nanofi-
bre composite scaffolds were developed, emulating the structure, chemical cues and me-
chanical characteristics of mature articular cartilage.76, 77 Thes e scaffolds bear phosphate
moieties at the surface that nucleate and promote the formation of hydroxyapatite, which
is required to integrate with the subchondral bone. Further studies led to the production
of poly (D,L-lactide) / nanofibre composites. In vitro studies showed that such multi-zonal
scaffolds promote the growth of neo-cartilage. Most importantly, the characteristics are sim-
ilar to mature native tissue. Even though the findings were extremely promising, significant
additional research and reliable in vivo data will be required in order for this method to be
translated from research into practical application.
73 Koch F, Möller AM, Mevissen M, Toxicology in Vitro, 2013,, 28(5), 990 - 98

74 Schönbächler A, Andereggen L, Reinert M, J Neurol Surg A Cent Eur Neurosurg, 2012, 73 - P048
75 Endes C, Schmid O, Clift MJ, Part Fibre Toxicol, 2014 Sep 23;11:40
76 Camarero Espinosa S, Rothen-Rutishauser B, Foster EJ. Biomaterials, 2016, 42 - 52.
77 Camarero Espinosa S, Kuhnt T, Weder C, Biomacromolecules, 2013, 14, 1223-30
4. Safety Aspects and Risk Assessment

Gaining greater insight into nano safety in the context of applications was a declared goal
of NRP 64. It is well known that nanomaterials may lose their specific properties after their
release into the environment due to the simultaneous occurrence of numerous potential
transformation processes.78 Life cycle considerations are an important aspect for the safe
use of new materials in general, including nanomaterials. The rapidly increasing number
of newly discovered and synthesised nanomaterials in various fields of application, and the
growing number of scientific methods that are suitable for studying the safety and risks
associated with a given material, are producing a large body of knowledge in this field.79, 80
However, not every available analytical method can, should or will be applied to every ma-
terial. As this is a rapidly evolving field, standardisation and development of testing guide-
lines for nanomaterials is an ongoing activity in international organisations such as the ISO
and OECD. In this context the main aim of the NRP 64 studies is to provide fundamental,
mechanistically based scientific insight that in the long term will be of great value for the
development of assessment methods for existing or newly developed materials.
Impacts of Nanomaterials on Freshwater and Soil Organisms
Due to their small size and high surface reactivity, engineered nanomaterials (ENMs) have
the potential to interact with organisms in different ways compared with the correspond-
ing bulk substances or dissolved forms of the same material. This interaction depends on
a combination of factors such as the chemical composition of the material, the size and
surface properties that can influence how the material behaves in the environment, with
possible implications for biological impacts.81 Biological effects caused by ENMs include ox-
idative stress due to formation of reactive oxygen species (ROS) and inflammatory cytokine
generation, alteration of membrane integrity, composition and permeability, and effects due
to the toxic metal ions as a result of dissolution of metal and metalbased ENMs.
In the scope of NRP 64, a new, highly sensitive bio-sensing platform was developed by
Olivier Martin at the Federal Institute of Technology, Lausanne, that enables the real-time
and non-invasive measurement of several oxidative stress markers when aquatic microor-
ganisms are exposed to ENMs.82 This project focused on the fact that the characterisation
of ENPs as well as realistic exposure conditions is crucial in order to derive meaningful
information from ecotoxicological studies. Also, it became clear that the environmental im-
pacts of ENMs strongly depend on the environment in which they are measured, including
environmental conditions that are usually neglected, such as illumination. The developed
78 Lowry GV, Gregory KB, Apte SC, Lead J.R. Environ. Sci.Technol., 2012, 46, 6893-6899
79 Nel AE, Parak WJ, Weiss PS, ACS Nano, 2015; 23;9(6):5627-30.
80 Kagan CR, Fernandez LE, Weiss PS, ACS Nano, 2016 (Epub ahead of print
81 Lundqvist M, Stigler J, Dawson KA, Proc Natl Acad Sci U S A, 2008; 23;105(38): 14265-70.
82 Koman VB, Santschi C, Martin OJ, Biosens Bioelectron, 2015 Jun 15;68:245-52.
platform facilitates dynamic toxicity assessments in model organisms for different types of
ENPs under various realistic environmental conditions.
It is the antimicrobial properties of nanosilver (AgNPs) that pose the greatest environ-
mental risk for systems dominated by microorganisms. Renata Behra (Eawag, Dubendorf)
focused on the effects of AgNPs on food webs and key ecosystem functions. Two systems
were studied that depend on healthy microorganism communities. Firstly, microbial bio-
films dominated by algae and various other microorganisms, and secondly, a system dom-
inated by leaf-decomposing fungi that interact with bacteria and aquatic fauna such as
shrimps. In short-term exposure, AgNPs and ionic silver showed both the potential to dis-
rupt important functions for both systems 83, while long-term exposures also resulted in
changes of community composition, with a disruption of key stream functions driven by
either periphyton or by microbial litter-decomposers84. The efficient AgNP affected the be-
havior of organisms feeding on these biofilms corroborating their role of algal biofilms as
an important entry of nanomaterials and metals in the trophic chain. The results indicate
that AgNPs can have direct and indirect effects on aquatic communities and thus challenge
the use of simple toxicity assays as a sole basis for risk assessment.84, 85 The fact that some
effects were found to be caused specifically by AgNPs and cannot be explained by the pres-
ence of ionic silver implies the need to reconsider the adequacy of current regulations for
water-quality with regard to nanoparticles. Finally, within this project it was demonstrated
that ozone treatment of waste water, a process used in sewage treatment plants to degrade
residual micropollutants, increases the toxicity of wastewater plant effluents through the
release of toxic Ag ions.86
Whereas the project described above concentrated on the effects of AgNPs on microor-
ganisms, the group led by Kristin Schirmer at Eawag, Dubendorf, set out to understand
how AgNPs specifically interact with cells of aquatic organisms. This project studied algal
cells, which are protected by a cell wall, and fish cells, which only have a cell membrane.
It was found that algal cells do not internalise the nanoparticles, and the toxicity of AgNPs
was explained by the ionic silver that was dissolved from the particles.87 In contrast, fish
cells take up AgNPs quickly via endocytosis mechanisms..88, 89, 90 In a unique approach, the
group characterised the proteins in the protein corona of the particle and demonstrated
that the corona comprises a footprint of the destination of a particle within a living cell and
reveals mechanisms underlying the particle toxicity.91 This method will be of great value
for the identification of protein-particle interactions, not only in the field of environmental
protection, but also in the areas of human health and biomedical sciences. Together, the
findings demonstrated the dynamic nature of nanoparticle-cell interaction, underlined the
83 Gil-Allué C., Schirmer K., Behra R, Environ Sci Technol, 2015; 49, (2), 1165-1172.
84 Tlili A, Jabiol J, Gessner MO, Environmental Science & Technology, 2017: 51 (4), pp 2447–2455.
85 Tlili A, Cornut J, Gessner MO, Nanotoxicology, 2016;10(6):728-35.
86 Thalmann B, Voegelin A, Kaegi R, Environ Sci Technol., 2015 15; 49(18):10911-9.
87 Li X, Schirmer K, Behra R, Environmental Science, 2015, Nano. ,2, 594-602.
88 Yue Y, Behra R, Schirmer K, Nanotoxicology, 2015, 9(1):54-63.
89 Yue Y, Behra R, Schirmer K, Environmental Science, 2016, Nano 3 (5), 1174-1185
90 Yue Yang Y, Li X, Schirmer K, Journal of Nanobiotechnology, 2017, accepted.
91 Yue Y, Behra R, Schirmer K, Environmental Science, 2016, Nano 3 (5), 1174-1185
importance of studying particle characteristics as closely as possible in the environment

they truly encounter, and highlighted the importance of considering potential long-term
effects. Using a newly developed fish cell model that is suitable for long-term experiments
the group found that AgNPs inhibit cell proliferation.92 Additional research will be required
in order to better understand the long-term effects. However, a suitable in-vitro model is
now available.93, 94
Possible adverse effects of ENMs on soil bacteria and crops were investigated by the group
led by Thomas Bucheli at Agroscope, Zurich. In this project, titanium dioxide and mul-
ti-walled CNTs both indicated limited mobility in soil due to strong interactions with soil
particles.95 In liquid culture experiments, the growth rate of Rhizobium trifolii, a soil bac-
terium which is able to fix nitrogen in symbiosis with clover, was decreased significant-
ly when exposed to high concentrations of titanium dioxide.96 In soil systems however,
high concentrations of titanium dioxide did not affect symbiosis of R. trifolii with clover or
wheat.97 Although the experiments conducted here concentrated on specific ENPs and did
not legitimate an extrapolation of results to other agricultural systems, the results indicated
that the low mobility and availability of NPs in soil reduces their risk for soil organisms.
Overall, the project provided important exposure and effect data as well as methods for a
risk assessment of ENP applications in the agro-environmental context. However, there are
still important knowledge gaps. Taking into consideration the fact that research and devel-
opment activities in the field of ENP-containing pesticides and fertilisers are constantly
progressing, it is highly important to further investigate fate, exposure and mechanisms of
toxicity of various ENPs in the soil.
Impacts of Nanomaterials on Human Health
Nanomaterials may have direct or indirect (e.g. silica particles via food) impacts on hu-
man health and could therefore pose risks. Risk depends on multiple factors, including the
produced quantity of a given material, the exposure of individuals (duration of exposure,
time after exposure), uptake by the body, interaction with the organism, stability of a ma-
terial and its biodegradation, excretion from the body and distribution and accumulation
in the environment. In order to develop nanomaterials for applications in human health, a
proactive approach to mechanistic understanding of nano-bio interaction is advisable be-
cause it allows a more reliable assessment of risk and opens the door to a «safe-by-design»
approach in which materials are designed so that they cannot lead to undesirable effects
outside their location of application and the time window of choice.
The project led by Hanspeter Nägeli (University of Zurich) focused on the development
of an in-vitro test to assess the biological activity of food-borne nanomaterials and their
92 Yue Y, Behra R, Schirmer K, Nanotoxicology, 2016, 10:8, 1075-1083

93 Schirmer K, Nanoscience and the Environment, 2014, Vol 7, FNS, UK: Elsevier, 195-222.
94 Schirmer K, Behra R, Zweck A, Pan Stanford Publishing Pte. Ltd. Singapore, 137-158
96 Moll J, Okupnik A, Widmer F, PLoS ONE, 2016, 11(5):e0155111.
97 Moll J, Gogos A, van der Heijden MGA, J. Nanobiotechnol., 2016, 14:36.
capabilities to disrupt gut-associated immunity activity of food-borne nanomaterials. Den-

dritic cells embedded in the intestinal mucosa play a critical role in provoking an immune
reaction against invading pathogens and need to maintain tolerance towards normal innoc-
uous luminal commensals and nutrients. An in-vitro system developed in NRP 64 for the
risk assessment of nanomaterials comprises the two main components, namely well-char-
acterised nanoparticles and primary steady state dendritic cells. Analytic methods in this
system included flow cytometry, electron microscopy, enzyme linked immuno assays and
Western blotting. It was demonstrated that synthetic amorphous silica nanoparticles ac-
tivate dendritic cells that mediate pro-inflammatory reactions in the gut. No comparable
pro-inflammatory activities were observed in response to titanium or iron nanoparticles.
It was concluded that nanoparticles do not pose a completely new threat, thus supporting
similar findings by other groups. The potential risk of such materials can be assessed by
following established procedures for conventional chemical hazards.98 Nevertheless, some
fundamental properties of food-borne nanoparticles should be further investigated by us-
ing in-vitro tests with decision-making cells of the immune system, in order to conduct
in-vivo studies in the future.
A related project led by Michael Bruce Zimmermann at the Federal Institute of Technol-
ogy, Zurich, focused on gastrointestinal exposure to nanoscale iron compounds in foods:
absorptive pathways and potential toxicity. Similar to the above project, the nanoparticles
were well characterised, and in order to investigate the mechanism of uptake from the
gastrointestinal tract, three different non-cancerous gastrointestinal cell lines and animal
studies were performed. The non-cancerous cells used were epithelial cells derived from
biopsy of healthy epithelium. In contrast to many commercial standard cell lines, such cells
are not tumorigenic, and therefore are not expected to have any genetic mutations in hot
spot genes and they express both epithelial as well as stem cell markers. The administration
of diet enriched nanoparticles in two dose ranges did not result in adverse excess accu-
mulation of tissue iron or other significant effects. In addition, direct toxicity or oxidative
stress did not occur in the cell lines. In collaboration with the above project, the potential of
the nanoparticles to induce an inflammation reaction by dendritic cells was assessed. Even
though so far results point to an absence of toxicity in food, the researchers recommend
further investigation. The developed methodology in NRP 64 is suitable for assessing the
toxicity of any nanoparticle and non-nano materials that might come into contact with the
gastrointestinal tract.
The group led by Jing Wang at the Federal Institute of Technology, Zurich, set out to deter-
mine the potential health and environmental risks associated with carbon nanotube com-
posites. Nanotubes are long, thin fibres, typically of pure carbon, that are thought to behave
very differently than spherical particles, and can convey strong mechanical properties,99
e.g. for more durable medical implants. A new measurement method was developed in this
project in which multi-walled CNTs were labelled with lead ions allowing quantification
despite their tiny size. This method involved the production of the nanocomposites, abra-
98 Winkler HC, Suter M, Naegeli H, J. Nanobiotechnol. 2016; 14; 44

99 Wang J, Schlagenhauf L, Setyan A, J Nanobiotechnology, 2017, 20;15(1):15.
sion and the collection of the inhalable particle fraction. The protruding and freestanding
nanotubes were subsequently quantified by measuring the concentration of released lead
ions. Assessment of the safety of the nanotubes was performed with two different cell lines:
human alveolar epithelial cells and blood monocyte-derived macrophages.100, 101 The cell vi-
ability, genotoxicity and formation of reactive oxygen species were investigated and did not
reveal that the abraded particles induce any acute cytotoxic effects.102, 103
5. Summary and Recommendations

Engineered nanomaterials are high-performance, high-value materials. They have become
a key technology platform in research and industry resulting in a broad spectrum of inno-
vative applications in the healthcare, industry and consumer sectors.
More specifically, the key sectors of application for new, engineered

nanomaterial-based products are as follows:
1) In medical technology, engineered nanomaterials are widely used for in vitro diagnostics,
in particular for point-of-care diagnostic tests.
2) In the pharmaceuticals industry, nanomaterials have led to targeted therapies for severe
diseases.104 Nanomaterials offer improved efficacy and reduced toxicity, and are enabling
clinical molecular imaging for earlier and more accurate diagnosis.
3) In personalised medicine, nanotechnology is the key enabling technology for molecular
diagnostics and for personalised, molecularly targeted therapies.
4) In environmental applications, engineered nanomaterial-based technologies offer
great potential for innovations that could be beneficial for the environment, such as
photocatalytic surfaces that degrade environmental contaminants, efficient water
cleaning technologies and nanoparticles for remediation of contaminated soil and
groundwater, and may also have an indirect beneficial effect on the environment by
optimising product lifecycle or efficiency.
5) In energy applications, engineered nanomaterials play a fundamental role and will
become a key technology for enabling grid storage of renewable energy.

101 Schlagenhauf L, Kianfar B, Wang J, NanoScale, 2015, 7 18524-36
103 Schlagenhauf L, Buerki-Thurnherr T, Wang J, Environ. Sci. Technol., 2015, 49, 10616 - 10623
104 Lehner R, Wang X, Hunziker P. Nanomedicine 2013, 9:742–57.
Highlights: New applications or materials studied within the scope of NRP 64:
1) A blood purification system based on carbon-coated magnetic nanoparticles has the
potential to be translated to clinical medicine and pave the way for a clinical phase I
study.
2) New composite nanoparticles that combine a metallic core with a coat of organic
molecules can be designed to optimise the interaction of particles with, for example,
cell membranes, proteins or viruses, providing an enabling technology for targeted drug
delivery.
3) New high-performance polymer nanomaterials based on natural cellulose nanofibres
offer new opportunities for tissue engineering. Very promising results for neo-cartilage
have to be upscaled for future application, though this is still in the planning stage and
its continuation has not been secured.
4) In the energy sector, synthetic nanomaterials could open up a significant
opportunity for energy storage.
Switzerland’s Impact on Nanotechnology
Academic and industrial researchers from Switzerland were instrumental in triggering the
era of nanoscience, and there is now an active community of academics in the field of na-
nomaterials, encompassing the two Federal Institutes of Technology (in Zurich and Lau
sanne), Empa and the majority of cantonal universities. Switzerland’s cooperation between
the regulatory authorities, universities and industry has been a pioneering approach aimed
at securing the safe industrial translation of engineered nanomaterials.
Switzerland has various internationally relevant organisations (e.g. the International Soci-
ety of Nanomedicine) and organises special events (e.g. the CLINAM conference for nano-
medicine, SwissNanoconvention), and Swiss research is contributing to major international
initiatives such as the NanoSafety Cluster, the OECD Working Party on Manufactured Na-
nomaterials and numerous EU FP7 Horizon 2020 projects, including NanoImpactNet, SUN,
NANoREG, ProSafe and Nanocalibrate.
Risks and Potential Environmental Impacts of Nanomaterials
Technological innovations always carry intrinsic risks that need to be identified and as-
sessed, and this also applies to engineered nanomaterials. Quantification of risk is based on
the inherent toxicity of the material, the level of exposure, the probability of release and the
stability or reactivity of the material after its release. The potential risks of nanomaterials
do not only depend on their size, but are mainly determined by specific material charac-
teristics, i.e. biocompatibility, biodegradability, interaction with the complex biological en-
vironment, stability and accumulation in biological organisms and the environment, all of
which are highly material-dependent. Therefore a widespread consensus has evolved both
in Switzerland and abroad that the existing principles that apply to risk assessment and
risk management in the field of industrial products in the macro range are also applica-
ble to nanomaterials. However, some of the guidelines require specific adaptation because
materials in the nano range do not (always) behave in the same way as the bulk material.
In addition to chemical differences, this also applies for interactions of the material with
biological systems.
In 2008, the Swiss government, universities and industry jointly developed the Action Plan
for «Synthetic Nanomaterials», which represents a pioneering approach to assessing the
potential risks associated with nanomaterials.
Key facts that determine the risks associated with nanomaterials

1) Many nanomaterials can be biodegraded, although the kinetics of this process is highly
variable between specific materials.
2) Long-term safety depends on the potential for persistence and bioaccumulation of a
material in living organisms or in the environment.
3) Some nanomaterials can be designed to be low-risk: biocompatible, biodegradable,
soluble (thus increasing their propensity to agglomerate), consisting of non-toxic
building blocks, tested for known toxicity pathways, controlled quantity, controlled
exposures.
In this respect, the most important findings of NRP 64 are as follows:

1) The creation of new mass-flow and environmental fate models enables a realistic
estimation of concentrations of nanomaterials in the environment.
2) Carbon nanotubes vary strongly in their potential for persistence in the environment,
depending on their specific characteristics. Data from NRP64 showing slow degradation
in certain setups, combined with recent reports in the scientific literature documenting
the capability of biological cells to degrade them, have implications for the rational
design of future carbon nanotube applications.
3) Sewage treatment plants act as a very efficient barrier to the emission of silver
nanoparticles and only a small fraction of sulphidised silver nanoparticles are
discharged into surface waters. The ozonation of treated waste water oxidises the
sulphidised AgNPs which results in the release of silver ions into sewage treatment
plants where effluents are ozonated. These findings should be taken into account
when evaluating different options for further improving the quality of treated
wastewater.
4) The characterisation of the protein corona of nanoparticles provides information on the
destination of the particles within living organisms and reveals mechanisms underlying
the particles’ toxicity.
5) Adverse effects on microorganisms were only detected at concentrations of AgNP
about one magnitude higher than those expected to be found in nature. However, long-
term experiments are still needed, as well as further studies on the mechanisms of
bioaccumulation in microorganisms and along food webs.
6) Possible risks to human health caused by nanoparticles should not be limited to

cytotoxicity, but should include end points such as immunoreactivity and genotoxicity.
7) Surface characteristics such as surface charge play an important role in the modulation
of immune responses. These particle-specific properties are important for the
development of, for example, immune-modulatory nanoparticles.
8) Multi-walled carbon nanotubes released from nanocomposites due to abrasion show no
acute cytotoxic effects on human lung cells or macrophages in vitro.
9) The anticipated beneficial effects of nanobased wood preservatives were less pronounced
than hypothesised in common European wood species, because they did not penetrate
wood more efficiently than conventional preservatives – a key finding for regulatory
purposes.
10) Synthetic silica nanoparticles activate dendritic cells and mediate inflammatory
reactions in the gut. The next steps are in vivo studies and comparison with human
exposure data, which in turn could result in a new definition of food toxicity affecting
the digestive system. However, results of this project suggest that concentrations of
silica nanoparticles in food additives should be reduced.
General findings of NRP 64:

In addition to the specific results from individual projects listed above, the NRP 64 led to
various overarching scientific and sociopolitical findings:
1) Creation of an “early warning” group with a sentinel function for rapidly evolving
potential threats in the field of synthetic nanomaterials.
2) Enhancement of our knowledge base concerning properties, techniques, benefits and
risks of synthetic nanomaterials.
3) Resulting implementation of a number of devices and methods that allow the
quantification and characterisation of nanomaterials in living organisms and the
environment.
4) Pioneering of a number of promising nanomaterials for progress in medicine with
documented low potential toxicity.
5) Contribution to international efforts aimed at assessing the risks associated with
nanomaterials.
6) Contribution by participants in NRP 64 towards the standardisation of methods in
the context of international organisations such as the International Organisation for
Standardisation (ISO) and the Organisation for Economic Cooperation and Development
(OECD).
Research carried out within the scope of NRP 64 also highlighted a number of
limitations:
1) The development of synthetic nanomaterials is a rapidly progressing field. The body of
knowledge is still increasing and was not finalised at the time of completion of NRP 64.
2) In contrast to other industrialised nations, in Switzerland there is still only limited
recognition of the role of synthetic nanomaterials as a strategic platform technology.
3) Switzerland does not have an institution that is dedicated, committed, and funded
to maintain and advance excellence in the assessment and characterisation of
nanomaterials in a manner that is independent of short-term academic projects and
industry.
4) There is a need for strongly quality-oriented institutions that are not subject to publication
pressure and commercial considerations, which can deliver reliable, reproducible and
certifiable results concerning specific materials, and identify new methods for research
and industry to develop material innovations in a timely manner.
Recommendations:
1) In the sense of a service for the economy, an excellence laboratory for the characterisation
and evaluation of nanomaterials should be established, for testing and certifying their
quality in applications, and to promote their use in a safe manner, particularly in the
areas of healthcare and protection of the environment.
2) To establish a contact point making the knowledge gained in NRP 64 accessible for
smaller and bigger Swiss companies.
3) To institutionalise the «Issues Monitoring» of the NRP 64 which observes new develop
ments in the sector of nanotechnology to a permanent task force.
4) To maintain the exemplary cooperation between universities, government and industry
in the action plan and the «Vorsorgeraster für synthetische Nanomaterialien» through
periodical reviews and reports.
Publisher
Swiss National Science Foundation SNSF

National Research Programme NRP 64
Wildhainweg, P. O. Box 8232
CH-3001 Berne
Telephone: + 41 (0)31 308 22 22
www.snf.ch
Authors
Dr. Ing. Georgette B. Salieb-Beugelaar, Nanomedicine Research Lab CLINAM, University Hospital Basel
Dr. Fabienne Schöpfer, Life Science Communication AG, Zurich
EMSc Mark Bächer, Head of Knowledge Transfer, Life Science Communication AG, Zurich
Dr. Marjory Hunt, Programme Coordinator NRP 64, SNSF, Bern
Prof. em. Peter Gehr, President of NRP 64, Institute of Anatomy, University of Bern
Prof. Dr. Med Patrick Hunziker, Nanomedicine Research Lab CLINAM, University Hospital Basel
© February 2017, Swiss National Science Foundation, Bern

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Engineered Nanomaterials:

Impact & Safety Aspects

2. Nanomaterials and the Environment 4

3. Nanomaterials for Human Health Applications 11

4. Safety Aspects and Risk Assessment 15

5. Summary and Recommendations 19

Nanomaterials are rapidly being adopted in health-

• Dendrimers: these nanosized polymers consist of branched units. Their interior

• Self-assembled soft nanoparticles: nanoparticles formed by the process of

• Composites: materials that combine nanoparticles with other nanoparticles or

AgNP: Silver nanoparticle

CNT: Carbon nanotube

CBN: Carbon-based nanomaterial

ENM: Engineered nanomaterial

ENP: Engineered nanoparticle

OECD: Organisation for Economic Cooperation and Development

WPMN: Working Party on Manufactured Nanomaterials

2. Nanomaterials and the Environment

Nanomaterial Developments to Benefit the Environment

Various projects explored the opportunities of nanomaterials with potential to enhance

Scenarios Relating to the Emission of Nanomaterials into the ­Environment

Emissions during or after Use

A reliable assessment of environmental emissions and concentrations of various nanoma-

Detection and Characterisation of Nanomaterials in the Environment

Environmental Fate of Nanomaterials

Predictions based on Computational Modelling

Processes in Wastewater Systems

39 Kaegi R, Voegelin A, Mueller E, Water Research, 2013, 47(12), 3866–3877.

3. Nanomaterials for Human Health

The immune-modulatory potential of gold nanoparticles with different surface coatings

62 Hermann IK, Schlegel A, Beck-Schimmer B, Nanoscale 2013; 5: 8718-23

A Federal Institute of Technology, Lausanne, project led by Francesco Stellacci explored

67 Carney RP, Carney TM, Stellacci F. Biointerphases;, 2012, 7, 17

biodegradation of the implants containing nanoparticles. The effects of the nanomaterials

73 Koch F, Möller AM, Mevissen M, Toxicology in Vitro, 2013,, 28(5), 990 - 98

4. Safety Aspects and Risk Assessment

Impacts of Nanomaterials on Freshwater and Soil Organisms

importance of studying particle characteristics as closely as possible in the environment

Impacts of Nanomaterials on Human Health

92 Yue Y, Behra R, Schirmer K, Nanotoxicology, 2016, 10:8, 1075-1083

capabilities to disrupt gut-associated immunity activity of food-borne nanomaterials. Den-

98 Winkler HC, Suter M, Naegeli H, J. Nanobiotechnol. 2016; 14; 44

5. Summary and Recommendations

More specifically, the key sectors of application for new, engineered

100 Schlagenhauf L, Buerki-Thurnherr T, Wang J, EST, 2015, 49,10616-23

Switzerland’s Impact on Nanotechnology

Risks and Potential Environmental Impacts of Nanomaterials

Key facts that determine the risks associated with nanomaterials

In this respect, the most important findings of NRP 64 are as follows:

6) Possible risks to human health caused by nanoparticles should not be limited to

General findings of NRP 64:

Swiss National Science Foundation SNSF

© February 2017, Swiss National Science Foundation, Bern

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