Nanotoxicology, 2011; Early Online, 1–11©2011 Informa UK, Ltd.ISSN: 1743-5390 print / 1743-5404 onlineDOI: 10.3109/17435390.2011.604439

A novel platform for pulmonary and cardiovascular toxicologicalcharacterization of inhaled engineered nanomaterials

Georgios A. Sotiriou1, Edgar Diaz2, Mark S. Long2, John Godleski2, Joseph Brain2, Sotiris E. Pratsinis1, &Philip Demokritou2

1Department of Mechanical and Process Engineering, Particle Technology Laboratory, Institute of Process Engineering, SwissFederal Institute of Technology Zurich (ETH Zurich), Sonneggstrasse 3, CH-8092, Zurich, Switzerland and 2Department ofEnvironmental Health, Harvard School of Public Health, Harvard University, 401 Park Drive, Boston, MA 02215, USA

AbstractA novel method is presented which is suitable for assessingin vivo the link between the physicochemical properties ofengineered nanomaterials (ENM) and their biological outcomes.The ability of the technique to generate a variety of industry-relevant, property-controlled ENM exposure atmospheres forinhalation studies was systematically investigated. The primaryparticle size for Fe2O3, SiO2, Ag and Ag/SiO2 was controlled from4 to 25 nm, while the corresponding agglomerate mobilitydiameter of the aerosol was also controlled and varied from40 to 120 nm. The suitability of the technique to characterize thepulmonary and cardiovascular effects of inhaled ENMs in intactanimal models is also demonstrated using in vivochemiluminescence (IVCL). The IVCL technique is a highlysensitive method for identifying cardiopulmonary responses toinhaled ENMs under relatively small doses and acute exposures.It is shown that moderate and acute exposures to inhalednanostructured Fe2O3 can cause both pulmonary andcardiovascular effects.

Keywords: engineered nanomaterials, in vivo inhalation,nanotoxicology, cardiovascular and pulmonary effects,nanotoxicology, iron oxide

Introduction

The use of engineered nanomaterials (ENMs) in householdproducts, textiles, industrial processes, medical devices andtherapeutics is widespread and increasing exponentially.Environmental and occupational exposures are consideredby experts to be ‘inevitable’ (National Research Council2008; National Science and Technology Council Comiteeon Technology 2008; Powell et al. 2010). Preliminary evi-dence demonstrates the potential for ENM to cause adversebiological effects (Colvin 2003; Nel et al. 2009; Service 2000).Indeed, the potential of nanoparticles and nanofibres to

translocate through the air–blood barriers, and thus to reachthe pulmonary connective tissues, lymphatic system, or evento reach the circulating blood and thus have access to othercritical organs, is of concern. Nano-sized particles may entercells, and be more biologically active than their micro-sized counterparts due to their small size and large surfaceto volume ratio (Hamilton et al. 2009; Larsen et al. 2010;Li et al. 2010; Muller et al. 2005; Poland et al. 2008; Rossi et al.2010; Service 2004; Witzmann & Monteiro-Riviere 2006) andincreased release of ions (Sotiriou & Pratsinis 2010).

Many in vitro assays and novel aerosol generation meth-ods suitable for inhalation studies have been developed andcontributed significantly to improving our understanding ofbiological mechanisms related to atmospheric particlehealth effects (Demokritou et al. 2002; Demokritou et al.2003; Gupta et al. 2004). However, these currently availableparticle exposure methods cannot be applied in the nano-toxicology field, primarily because of the unique propertiesof ENM. For in vivo ENM inhalation studies, an importantlimiting factor is the difficulty in aerosolizing and dispersingcommercially available nanopowder ENM down to thenano-size level, as they would exist in many relevant inha-lation exposures (Schmoll et al. 2009). Some common aero-sol generators used in the past to disperse nanopowders forinhalation studies, including nebulizers, fluidized beds andother venturi aspirator-type systems, have limited applica-bility for nanotoxicology (Schmoll et al. 2009). For example,such nanopowder aerosol-generation systems have difficultyin producing consistent nano-sized aerosol distributionsand, more importantly, fail to control physicochemicalproperties in order to elucidate the link between propertiesand toxic biological responses (Fischer & Chan 2007;Schmoll et al. 2009).

As a result of the challenges and limitations, the majorityof nanotoxicity research has focused on in vitro cellular orin vivo instillation studies using commercially available ENMnanopowders and nanopowder liquid suspensions; however,

Correspondence: Philip Demokritou, Department of Environmental Health, Harvard School of Public Health, Harvard University, 401 Park Drive, Boston,MA 02215, USA. E-mail: pdemokri@hsph.harvard.edu

(Received 21 February 2011; accepted 6 July 2011)

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there is a consensus among scientists that data will requireverification from animal inhalation experiments (NationalScience and Technology Council Comitee on Technology2008; Schmoll et al. 2009). There are also serious short-comings related to the physiological relevance of this particleto cell delivery approach using liquid ENM suspensions. Theshortcomings of these in vitro and instillation approaches aretwofold: First, commercial ENM are limited in diversity ofphysicochemical and morphological properties – usually to afew sizes for a given composition – making it impossible toperform comparative parametric toxicological studies ofENM properties (size, surface, composition, shape, charge,etc.). Second, nanoparticles suspended in culture mediaflocculate or dissolve, and interact with serum components(Fadeel & Garcia-Bennett 2010; Jones & Grainger 2009;Verma and Stellacci 2010), which can alter their biologicalproperties and effects. Furthermore, comparison of particledoses delivered in suspension to those administered byinhalation is difficult, which can result in large differencesin effective dosage between in vivo and in vitro studies.These limitations may explain some of the disparitiesreported in the literature between in vivo and in vitroENM studies (Fadeel & Garcia-Bennett 2010; Fischer &Chan 2007; Sayes et al. 2007). It is apparent that newmethods and systems suitable for both in vitro andin vivo inhalation toxicological characterization need to bedeveloped.

Here, a novel technique is presented that is suitable forboth ENM in vivo inhalation and in vitro toxicologicalcharacterization studies. The ability of this technique togenerate a variety of industry-relevant, property-controlled

exposure atmospheres for inhalation studies was systemat-ically investigated. The suitability of the technique to char-acterize the pulmonary and cardiovascular effects of inhaledENM in intact animal models was also demonstrated in anin vivo study involving Sprague–Dawley rats, using freshlygenerated nano-iron oxide (Fe2O3) as a test aerosol. Wedemonstrated both pulmonary and systemic toxicity usingin vivo chemiluminescence (IVCL) of heart and lung(Bartoli et al. 2009; Clarke et al. 1999; Clarke et al. 2000;Godleski et al. 2002; Gurgueira et al. 2002; Rhoden et al.2004; Saldiva et al. 2002). This novel platform will makeit possible for toxicologists to link physicochemical proper-ties of inhaled ENM to biological outcomes and help theindustry to develop safer ENM.

Methods

Versatile Engineered Nanomaterial Generation SystemFigure 1 illustrates the Versatile Engineered NanomaterialGeneration System (VENGES) (Demokritou et al. 2010). Thesystem consists of four main components: ENM synthesis,particle sampling and collection system, animal exposuresystem and exposure monitoring system.

ENM synthesisThe synthesis of ENM is based on the industry-relevant flame spray pyrolysis (FSP) method, as most oftoday’s ENMs are made by gas-phase techniques (Pratsinis2010). An inflammable liquid precursor, containing dis-solved organometallic compounds, is pumped through astainless-steel capillary tube (inner diameter = 0.41 mm;

1 Laerosol

container

ENM sampling/collection

Filter

HE

PA

HE

PA

Qs

QR

T1

QD

QA QP

Flame synthesis Animal exposuresystem

Animal exposurechamber

HEPA

FMPS P-TRAK

Exposure monitoring equipment

O2dispersion Liquid

precursor

CH4/O2supportflame

50 c

m CO2, CO,RH, T2

NO2

Figure 1. Overview of VENGES. All different parts of VENGES are presented. Engineered nanomaterials (ENMs) are generated by flame synthesis.The nanoparticles are collected on a filter for further ex situ characterization. Directly above the flame, in situ sampling of the test aerosol isperformed (QS). Immediately after, the ENM aerosol stream can be further diluted with HEPA-filtered room air (QD) and directed into the exposureanimal chambers (QA) for in vivo toxicological testing. Simultaneously, the test ENM aerosol is in situ real-time characterized (QP). The total particlenumber concentration is measured, as well as the mobility particle-size distribution. Finally, the CO2, CO, NO2, relative humidity (RH) andtemperature (T2) are continuously monitored.

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outer diameter = 0.71 mm). Immediately surrounding thecapillary tube is a narrow annular gap of adjustable cross-sectional area that supplies oxygen, which is used to dis-perse the liquid precursor into fine droplets. A small pilotflamelet (premixed CH4 and O2 at 1 and 2 l/min, respec-tively) ignites the droplets so that the organic parts of theprecursor combust, while the metals are oxidized, and formparticles that grow by coagulation and sintering in the flame.The precursor liquid feed is metered by a mass rate-controlled syringe pump, and all gas flows are controlledby mass flow controllers (Madler et al. 2002; Teleki et al.2008).

Sampling and off-line characterization of generated ENMThe flame-generated particles are collected using a water-cooled, stainless-steel filter housing supporting a glass fibrefilter (Whatman GF6, 25.7 cm in diameter, Switzerland), withexhaust gases drawn by a vacuum pump (Busch, Seco SV1040C, Netherlands). Collected particles can be extractedfrom the filter as nanopowder, and used for off-line physicochemical and morphological characterization,as well as for in vitro or in vivo instillation studies. Off-line ENM characterization included N2 adsorption (five-point isotherm; Micromeritic Tristar, Switzerland). ENMsamples were degassed under N2 at 150�C for 1.5 h. Theaverage primary particle diameter dBET was calculated fromtheir specific surface area, SSA: dBET = 6000/(r �SSA), wherer is the material’s density. X-ray diffraction (XRD, BrukerD8 Advance, Cu Ka, Switzerland) was also performed overthe scan range 2q = 20�–70�. Morphology of the generatedENM was also obtained using transmission electron micros-copy and scanning transmission electron microscopy(STEM, instrument for both: Tecnai F30, Switzerland). Thesamples were dispersed in ethanol and then placed on acarbon-coated copper grid.

Animal exposure systemThe continuously produced (in the gas phase) nanoparticlesare sampled in situ over the flame (QS, Figure 1), andtransferred to inhalation chambers for animal studiesthrough a 1-l aerosol container to stabilize the flow rate.The sampled aerosol can be further diluted with high-efficiency particulate air (HEPA)-filtered dry air (QD). TheENMs remain airborne, with a very low level of agglomer-ation, and are drawn directly through the animal exposurechambers (QR, Figure 1). For this study, each animal washoused in its own whole-body exposure chamber (10 cm indiameter, 18 cm in length). Chambers were made of clearpolycarbonate to allow visualization of the animals during

the exposure, and used a modified rubber stopper as a cap.Flow through each chamber was maintained constant at1.5 l/min.

Exposure monitoring systemThe animal exposure atmosphere was monitored continu-ously, using a number of real-time instruments. Exposureparameters monitored included total particle counts(P-Track; TSI, MA, USA), aerosol-size distribution (FMPS;TSI, MA, USA), NO2, CO, CO2, temperature and relativehumidity (Figure 1).

Performance characterization experimentsThe versatility and ability of VENGES to generate an array ofindustrially relevant ENM while controlling important nano-particle properties (i.e. primary particle size, agglomerationstate, surface properties, shape) as well as its suitability forin vivo inhalation studies were demonstrated in a number ofperformance characterization experiments, as follows.

ENM-generated nanopanelFour different ENM, silicon dioxide (silica, SiO2), iron oxide(Fe2O3), silver (Ag, nanosilver) and composite nanosilversupported on silica particles (Ag/SiO2), were generated here.Table I summarizes the ENMs used in these experiments andtheir respective liquid precursor characteristics. It is worthpointing out that the FSP synthesis method used in VENGESallows for the generation of a large universe of industriallyrelevant ENM (Strobel & Pratsinis 2007).

Controlling both the aerosol size (airborne phase)and primary particle sizeIn order to investigate whether VENGES can successfullyvary both the size of the generated aerosol as well as theprimary particle size, a study was performed by varying twoimportant VENGES process parameters: (1) liquid precursorfeed rate (from now on: x) over the O2 dispersion flow rate(from now on: y) ratio (x/y); and (2) liquid precursor molar-ity, M (mol/l). Table I summarizes for each ENM the x/yratios and the variable molarity experiments, used in theexperiments. For all experiments, the properties of the ENMVENGES-generated aerosol were characterized in situ in realtime. In addition, the physicochemical and morphologicalcharacterization of the ex situ collected nanoparticles wasalso performed off-line as previously described.

Reproducibility of the generated test aerosols over timeFor inhalation toxicological studies, it is important to: (1) gen-erate in a reproducible manner test aerosols with constantconcentration and size distribution for the duration of the

Table I. Synthesized ENM, their corresponding precursors and solvent, as well as their process parameters (precursor molarity and x/y ratio).

ENM Metal precursor SolventPrecursor

molarity (M)

Liquid feed rate(x)/O2 dispersion

flow rate (y)

SiO2 Hexamethyl disiloxane Ethanol 0.1–1 2/7 to 6/4

Fe2O3 Iron acetyloacetonate Acetonitrile/2-ethylhexanoic acid (1:1) 0.01–0.5 2/7 to 6/4

Ag Silver nitrate Ethanol/diethylene glycol monobutyl ether (1:1) 0.06–0.31 2/7

Ag/SiO2 Silver nitrate/Hexamethyl disiloxane Ethanol/diethylene glycol monobutyl ether (1:1) 0.1 2/7

In vivo toxicological characterization of inhaled ENM

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exposure (hours); and (2) for dose/response toxicologicalstudies, the number concentration should be able to becontrolled. The ability of VENGES to fulfil both requirementswas explored by varying the ratio of the sampling to thedilution flow rate (QS/QD). ENMs were synthesized at con-stant precursor molarity and x/y ratio was kept constant. TheQS/QD ratio varied in a stepwise manner over a period ofmore than 5 h of continuous ENM aerosol generation. Themass concentrations were calculated from the FMPS dataassuming Fe2O3 density of 5.24 g/cm

3 for the test aerosol and1 g/cm3 for indoor particles.

ENM surface modification experimentThe VENGES system also allows for the generation ofsurface-modified test aerosols in the gas phase (Madleret al. 2003). At the nanoscale level, surface properties andchemistry may affect the biological properties of ENM andare among the important parameters for toxicological inves-tigation. Surface modification is also one of the emergingmethods for safer nanomaterial formulation. Surface mod-ification may result in a less toxic ENM while its importantelectrical or structural properties of interest remainunchanged. Here, VENGES was used to generate SiO2 nano-particles and their surface was modified in situ by addingnanosilver particles on their surface (Demokritou et al.2010). The total particle number concentration and sizedistributions of both the basic SiO2 and the surface-modifiedAg/SiO2 aerosol properties were measured in situ. Further-more, ex situ characterization of the collected ENM was alsoperformed as described above.

Acute pulmonary and cardiovascular effects of inhalednanostructured Fe2O3 using the VENGES platform andIVCL assayThe suitability of VENGES platform for in vivo animalinhalation studies was demonstrated here. The pulmonaryand cardiovascular effects of inhaled Fe2O3 nanoparticleswere assessed. Fe2O3 nanoparticles have been used widely ina number of applications including drug delivery, bio-imaging and nutrition (Hilty et al. 2010; Lu et al. 2007;Teleki et al. 2009). The toxicological assay used to assesshealth outcomes in this study was IVCL of the lung and heartsurface immediately after exposure; this technique has beenwidely used in our laboratory for ambient particle toxicitystudies and is a highly sensitive method for identifyingpulmonary and cardiovascular responses to inhaled particlesunder relatively small doses and acute exposures (Godleski2006; Gurgueira et al. 2002).

ProtocolMale Sprague–Dawley rats (200–250 g) were obtained fromTaconic Laboratories (Rensselaer, NY, USA), housed andmanaged according to the NIH guidelines for the care anduse of laboratory animals. Upon arrival, animals wereassigned a unique identification number, which determinedthe exposure date and exposure group (aerosol or filteredair) for the animal. Rats were allowed to acclimate to theanimal facility for 4–5 days prior to the start of experiments.The Harvard Medical Area’s Animal Use Committee

approved the animal protocols used in this study. Eachday two animals were exposed to Fe2O3 test aerosol andtwo animals to filtered air (sham) in individual exposurechambers for 5 h. Four repetitions (days) with a new set ofanimals each day were carried out. At the end of each 5-hexposure, two animals from each group had IVCL (Boveriset al. 1980).

Results

Performance characterization experimentsEffect of x/y ratio on both the aerosol size (airborne phase)and primary particle sizeFigure 2a illustrates the particle number concentration as afunction of mobility diameter for pure SiO2. It is worthmentioning that the mobility diameter is correlated to theaerodynamic diameter and volume equivalent (thermody-namic) diameter (Hofmann et al. 2009; Kasper 1982;Peters et al. 1993). Particles that are sized based on theirmobility in an electric field are considered for practicalpurposes classified by their thermodynamic diameter(McCawley et al. 2001; Schiller et al. 1988). Thermodynamicdiameter is an important determinant of nanoparticle lungdeposition (ICRP 1994). As shown in the Figure, theVENGES-generated aerosol has a unimodal and narrow-size distribution. As inset in Figure 2a, the mode, mean,median diameters and the geometric standard deviations arepresented showing the statistical analysis of the obtainedparticle-size distributions (Hinds 1999). Additionally, themodal diameter increases with increasing x/y ratios, asthe SiO2 aerosol concentration increases. It is clear thatVENGES not only has the ability to generate aerosols withunimodal-size distributions but also has the ability to adjustthe aerosol size. This can be seen more clearly in Figure 2b,where greater modal diameters (circles, left axis) were indi-cated for larger x/y ratios. This is attributed to the higherparticle concentration with higher x/y ratio during theirflame formation that leads to larger aggregates/agglomer-ates. It is noteworthy that all particle-size distributions arestable over time, as it will be discussed later on (see Figures5a and 6b).

Figure 2b compares the average primary particle diameterof the collected ENM as obtained by ex situ N2 adsorptionwith their airborne phase modal diameter. The average SiO2

primary particle size of the collected ENM decreases withincreasing x/y ratios (Demokritou et al. 2010; Madler et al.2002). However, there is a difference between the primaryparticle size of the collected ENM determined by N2 adsorp-tion and the modal diameter of the same particles in theirairborne phase. This discordance between the mobility(airborne phase) and the primary particle diameter (nano-powder form) indicates, that the latter is not necessarily agood predictor of the ENM behaviour when it becomesairborne. In other words, the nanopowder-derived primaryparticle diameter is not a good predictor of its fate, transportand deposition in the environment and in human or animallungs. This finding may explain some of the discrepancies ofthe toxicological results reported in the literature betweenin vivo and in vitro ENM studies (Fadeel & Garcia-Bennett

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2010; Fischer & Chan 2007; Sayes et al. 2007). The in vivovalidation of in vitro (cellular) studies is considered animportant element of the proposed Environmental Healthand Safety strategy recently released by the NationalNanotechnology Initiative (NNI).

Similarly, Figure 2d show the effect of x/y ratio on boththe primary particle and the mobility diameter of Fe2O3.Figure 2c also shows the XRD patterns of the ex situ collectedand characterized nanoparticles as a function of the x/y ratio.This indicates that larger Fe2O3 particles are formed withhigher x/y ratio, in agreement with literature (Demokritouet al. 2010; Teleki et al. 2009). This is due to their largerresidence time in higher temperature zones that resultin larger crystals. It is worth pointing out that theXRD-estimated average crystal particle sizes (filled triangles,

Figure 2d) are consistent with their average primary particlesizes (open triangles, Figure 2d), with the latter havingslightly larger values indicating polycrystalline or aggregatednanoparticles. The modal mobility diameter for the Fe2O3

aerosol (Figure 2d, circles, left axis) is not significantlyaffected by the x/y ratio, as for most ratios it is fairly constant.Furthermore, as it is shown above in the case of SiO2, there isa difference between mobility diameter (airborne phase)and the primary particle diameter (nanopowder form).

Effect of precursor molarity on both the aerosol size andprimary nanopowder particle sizeFigure 3 indicates the effect of another important processparameter of VENGES, the metal precursor molarity, onboth the aerosol size (mobility diameter) and primary

6000SiO2, 0.5 M SiO2, 0.5 M

Fe2O3, 0.5 M

2/7 80.693.193.1107.1124.0

71.881.991.6101.1112.2

76.3 1.551.551.521.451.40

85.494.4106.2116.0

Mode(nm)

Mode mobility diameter

Primary particle ” , dBET

Mode mobility diameterPrimary particle ” , dBET

Crystal ” , dXRD

Median(nm)

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3/74/65/56/4

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100 1000

Mobility diameter dm, nm Liquid feed rate/Dispersion O2 flow rate

Par

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/cm

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SiO

2F

e 2O

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Mo

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, nm

Pri

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arti

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ET),

nm

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a

Figure 2. Controlling particle-size distribution by precursor liquid feed rate (x) over O2 dispersion flow rate (y) ratio (x/y). (a) Mobility-size distributions of SiO2 for a varying x/y ratio and the mode, median, mean diameters and the geometric standard deviation are shown. (b)The mode mobility (circles, left axis) and primary particle diameter (triangles, right axis) as a function of the x/y ratio. (c) XRD patterns of Fe2O3

nanoparticles for different x/y ratios. (d) Mode mobility (circles, left axis), primary particle (open triangles, right axis) and crystal (filled triangles,right axis) diameters as a function of the x/y ratio. The precursor molarity was 0.5 M and the total particle number concentration (CT) 2� 105 #/cm3

for both SiO2 and Fe2O3 samples.

In vivo toxicological characterization of inhaled ENM

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4000

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1 10 100 1000

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1 10 100 1000

Mobility diameter dm, nm

Metal precursor molarity, M

Metal precursor molarity, M

Par

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/cm

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/cm

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60.469.880.693.1

58.064.471.890.3

53.167.476.394.5

1.731.551.551.50

0.10 M

SiO2, 2/7

CT = 2*105 #/cm

3

CT = 2*105 #/cm

3

CT = 2*105 #/cm

3

0.35 M0.50 M1.00 M

Mode(nm)

Median(nm)

Mean(nm)

σg

Mode(nm)

Mode(nm)

39.269.869.880.693.1

38.061.264.278.286.5

44.4

29.434.052.3

26.1 24.829.646.3

1.611.601.57

0.06 M0.22 ”0.31 ”

Ag, 2/7

31.151.8

1.921.601.521.511.50

0.01 M0.02 ”0.10 ”0.35 ”0.50 ”

Fe2O3, 2/7

66.370.383.182.5

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a b

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dc

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Figure 3. Controlling particle size by precursor molarity. Mobility (a,c,f) of SiO2 (a), Fe2O3 (c) and Ag (nanosilver, f), for different precursormolarities. The mode, median, mean diameters and the geometric standard deviation are also shown. (b,d) Mode mobility (circles, left axis),primary particle (open triangles, right axis) and crystal (filled triangles, right axis) diameters for SiO2 (b) and Fe2O3 (d). (e) TEM image of Fe2O3

nanoparticles for precursor molarity 0.5 M and 2/7 ratio.

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nanopowder particle size for SiO2, Fe2O3 and Ag ENM.Figure 3a shows for the SiO2 aerosol, the particle numberconcentration as a function of mobility diameter (for aconstant 2/7 ratio) for different metal precursor molaritieswith their corresponding diameters and geometric standarddeviation as inset. It can be seen that the mobility-size distribution shifts to larger sizes with an increasedprecursor molarity. It is worth mentioning that there is anoverlapping among the different particle-size distributionspresented in the figure. However, the aerosol-size distribu-tions were randomly selected to prove the point that aerosolproperties can be adjusted. Whether these size distributiondifferences are clinically relevant remains to be seen infuture toxicological studies.

Similarly, this pattern can be seen also in Figure 3b, wherethe mode mobility diameter (circles, left axis) is also increas-ing for higher precursor molarity. This could be attributed tothe higher concentration of the particles at an early stageduring their synthesis that would lead to the formation oflarger agglomerates. In contrast to the behaviour of the SiO2

particles in airborne phase, the average primary particlediameter (dBET, triangles, right axis) does not significantlyvary with precursor molarity (Jossen et al. 2005) as sinteringrather than coagulation determines the primary particle sizein contrast to mobility diameter that is determined bycoagulation and particle concentration levels.

Similarly for Fe2O3 nanoparticles, the mobility-size dis-tributions shift to larger sizes for higher precursor molarities(Figures 3c and 3d). The average primary particle (Figure 3d,dBET, open triangles, right axis) and the crystal (Figure 3d,dXRD, filled triangles, right axis) diameter seem to slightlyincrease for higher precursor molarity, forming polycrystal-line nanoparticles, as with SiO2. The TEM image of Fe2O3

nanoparticles shown in Figure 3e shows that there are manyagglomerates/aggregates composed of smaller primary par-ticles, in agreement with the results obtained by XRD, N2

adsorption and mobility-size distribution analysis. Finally,Figure 3f indicates that the mobility-size distribution of

nanosilver (Ag) can also be shifted to larger values for higherprecursor molarity.

Control of concentration/reproducibility of the generatedtest aerosols over timeWe have demonstrated earlier that VENGES has the ability tocontrol both the aerosol or agglomerate size (airbornephase) and the primary particle or crystal size (nanopowderform), in a highly reproducible way. However, for inhalationtoxicological studies, aerosol-generation methods need to beable to (1) adjust the concentration level (dose/responsestudies); and (2) deliver test aerosols with constant proper-ties for hours. Figure 4a shows the total particle numberconcentration CT (solid line) and the modal (broken line)and median (dotted line) mobility diameters of the nano-silver aerosol (2/7, 0.02 M precursor concentration) as afunction of exposure time. The dilution of the sampledaerosol (QS/QD, Figure 1) was periodically adjusted in astepwise manner during the experiment to control the con-centration level. As expected, for higher QS/QD ratio, higherCT is reached. Most importantly, when a constant QS/QD

ratio is used, both the aerosol total particle concentration(CT) and the size distribution indicated by the modal andmedian (dotted line) mobility diameters remain constant.This is clearly an indication that VENGES can control theaerosol concentration levels and deliver test aerosols withconstant size distributions over time.

It is noteworthy that when the QS/QD ratio is adjustedback to its initial value, the aerosol-size distribution returnsto its initial one, a clear indication of the reproducibility ofthe method. Similarly, for Fe2O3, Figure 4b shows thisconstant aerosol particle concentration and size distributionover time for different QS/QD ratios.

ENM surface modificationThe surface of ENMs can be modified in order to add desiredattributes (Teleki et al. 2008) such as dispersibility (e.g. SiO2-coated Ag; Sotiriou et al. 2010) or antibacterial activity (e.g.

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Figure 4. Controlling total particle number concentration and mobility-size distribution by dilution. Total particle number concentration (CT, solidlines, left axis), mode (broken lines, right axis) andmedian (dotted lines, right axis) mobility diameters of nanosilver (a) and Fe2O3 (b) nanoparticlesfor different QS/QD ratios.

In vivo toxicological characterization of inhaled ENM

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SiO2 supported nanosilver; Sotiriou & Pratsinis 2010). It mayalso be a useful concept for the formulation of safer ENM(Demokritou et al. 2010; Sotiriou et al. 2010). Recently, it wasshown that doping ZnO nanoparticles with Fe can reduce therelease of Zn ions (George et al. 2010). Figure 5a illustratesthe particle number concentration as a function of mobilitydiameter for both pure SiO2 and the surface-modified nano-composite Ag/SiO2 (Ag content: 10 wt%). Both size distribu-tions are almost identical (Figure 5a) as verified also by themode, median, mean diameters and the geometric standarddeviations (inset). This indicates that VENGES has the abilityto generate surface-modified nanoparticles while maintain-ing the intended size distribution. This will be useful whenstudying the comparative toxicity of surface modified nano-particles in vivo. By controlling the size, we can also controlthe particle deposition in the lungs. In addition, ENMs canalso be used as a ‘vehicle’ for delivery of drugs in vivo.Figure 5b shows a STEM image of the composite Ag/SiO2

nanoparticles. The nanosilver particles (bright spots) arehomogeneously dispersed on the amorphous SiO2 (diffusegrey) surface (Demokritou et al. 2010;Sotiriou & Pratsinis2010).

Pulmonary and cardiovascular effects of inhalednanostructured Fe2O3 using the VENGES platformFigure 6a indicates the total particle number concentrationas a function of time in the 5-h exposure period for the Fe2O3

aerosol (2/7 ratio, 0.02 M precursor molarity). As shown inthis figure, the concentration levels remained fairly constantfor the whole animal exposure (the 5-min stops indicated inthe figure were necessary in order to replace the syringe withliquid precursor). For this exposure scenario, VENGESwas tuned to generate a test aerosol of a total numberconcentration of 2–3 ��105 particles/cm3 (this would

correspond to approximately 100–200 mg/m3), approxi-mately 10–20 times higher concentration of indoor condi-tions (10 mg/m3). The temperature, relative humidity, CO2,CO and NO2 concentrations of the test aerosol wereidentical to the room conditions, ensuring no interferencewith the in vivo toxicological results. Additionally,Figure 6b shows the averaged mobility-size distributionof the test aerosol. The error bars correspond to thestandard deviation of the particle concentration for eachparticle size, over the exposure period. This clearly showsthat VENGES can generate a highly reproducible exposureaerosol with constant properties (size, size distributionand concentration) for toxicological inhalation studies.

Figure 7 shows the IVCL difference [cps (counts persecond)/cm2] of lungs and hearts, which corresponds tothe relative reactive oxygen species (ROS) concentration inthose organs of both exposed (red) and unexposed (blue)groups of animals. The error bars correspond to the standarddeviation of the data points, and the significance level of themeasured data is p < 0.001. The IVCL measurements in thelungs of the exposed animals were about 60 times higherthan for the unexposed animals, indicating that the Fe2O3

test aerosol increased ROS in the lungs. In addition, oxidativestress was present in the heart of the animals (11-foldincrease in the chemiluminescence of the heart).

Discussion

A promising technological platform suitable for both in vitroand in vivo toxicological characterization of ENMs, withemphasis on the cardiovascular and pulmonary effects ofinhaled ENM, is presented in this study. ENMs are producedcontinuously in the gas phase allowing their continuoustransfer to inhalation chambers, with minimal alterations

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Figure 5. Surface modification of SiO2 with nanosilver. (a) Mobility-size distributions of SiO2 (triangles) and composite Ag/SiO2 nanoparticles(circles, Ag content: 10 wt%) for 0.10 M precursor molarity and 2/7 x/y ratio for a total particle number concentration CT = 2 � 105 #/cm3. Thesize distribution remains the same, as seen also in the inset, where the mode, median, mean diameters and the geometric standard deviation arealso shown. The error bars correspond to the standard deviation of the particle concentration corresponding to each particle size over time.(b) STEM image of the Ag/SiO2 nanoparticles. The nanosilver particles (bright spots) are homogeneously dispersed on the amorphous SiO2

matrix (diffuse grey).

G. A. Sotiriou et al.

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in their state of agglomeration. Defining properties of thegenerated aerosols (i.e. primary and aerosol particle size,concentration, shape, state of agglomeration, surface chemis-try) can be easily modified by adjusting simple process para-meters allowing for both in vitro and in vivo investigations oftoxicity. The ability of the developed technique to generate avariety of industry relevant, property-controlled exposureatmospheres for inhalation studies was systematicallyinvestigated and documented in the previous section.

The suitability of the technique to characterize thepulmonary and cardiovascular effects of inhaled ENM in

intact animal models was also demonstrated here using thehighly sensitive IVCL assay. IVCL measures the ROS gener-ation. ROS and free radical generation is considered one ofthe primary mechanisms of nanoparticle toxicity; it wasshown in many ambient particle health effect studies thatROS generation may result in oxidative stress, inflammation,and damage to proteins, membranes and DNA (Bartoli et al.2009; Clarke et al. 1999; Clarke et al. 2000; Godleski et al.2002; Gurgueira et al. 2002; Rhoden et al. 2004; Saldiva et al.2002). ROS generation has also been found in many ENMsincluding carbon-based ENMs (fullerenes, carbon nano-tubes) and metal oxides (Nel et al. 2006). Our results indicatethat moderate and acute exposures to inhaled nanostruc-tured Fe2O3 can generate ROS and oxidative stress and causeboth pulmonary and cardiovascular effects. The IVCL mea-surements in the lungs of the exposed animals were about60 times higher than for the unexposed animals, indicatingthat the Fe2O3 test aerosol increased ROS in the lungs(Figure 7). This oxidative stress was also present in the heartof the animals showing that the inhalation of ENM influencesnot only the respiratory but also the cardiovascular systemwith a 11-fold increase in the chemiluminescence of theheart. This substantial effect was found with a moderatemass exposure concentration of 200 mg/m3, a concentrationapproximately 20 times the fine-particle concentration ofroom air. The substantial IVCL response observed in ourstudy indicates that these particles reached deep in the lungand evoked a toxicological effect. The increased IVCLresponse of the heart may not only indicate a direct effecton the heart (Godleski 2006) but also may be a manifestationof indirect effects via the autonomic nervous system(Ghelfi et al. 2008; Rhoden et al. 2005). It should be noted,however, that the proposed platform is not only limited tothe evaluation of pulmonary and cardiovascular effects, but itcan also be used to assess other biological outcomes related

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Figure 6. (a) Total particle number concentration (CT) of the test ENM aerosol containing Fe2O3 nanoparticles monitored continuously over thewhole period of exposure for in vivo toxicological characterization. The CT remains constant at approximately 2–3�105 #/cm3. Every 25min there wasa 5-min break during the system was filled up (grey area). (b) Mobility-size distribution averaged over the periods that the CT was constant. Themode, median, mean diameters and the geometric standard deviation are also shown. The error bars correspond to the standard deviation of theparticle concentration corresponding to each particle size over time.

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Figure 7. In vivo chemiluminescence of the lungs and hearts of theexposed to the test aerosol for 5-h rats (red) and of the ones to filteredroom air (blue). The error bars correspond to the standard deviation ofthe data and the significance level p is also shown.

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to inhaled ENM . Therefore, this platform can be a powerfultool to understand the link between certain ENM propertiesand their bioavailability and toxicity.

Conclusions

In conclusion, this novel approach enables us to generateindustry-relevant, property-controlled ENM exposure atmo-spheres suitable for inhalation toxicological studies andassess the link between ENM physicochemical propertiesand specific biological outcomes. In addition, ability of thetechnique to alter in situ ENM surface properties can be oneof the ways to further explore the formulation of safer ENM.Furthermore, this technological platform can be a powerfultool for the validation of in vitro screening assays withadverse biological effects in intact animals, an importantelement of the strategy recently proposed by the NNI onEnvironmental Health and Safety of ENM. Its future use willhelp to assess the cardiovascular, pulmonary and othertoxicological effects of inhaled ENM and improve our under-standing on our central hypothesis that physical and chem-ical characteristics of ENM determine their bioavailability,redistribution, and toxicity in the lungs and elsewhere.

Acknowledgements

This research project was supported by NIEHS Grant(ES-0000002), the Swiss National Science Foundation (Grant200020-126694) and the European Research Council. Theauthors also thank Dr Frank Krumeich (Electron MicroscopyCenter, ETH Zurich) for the electron microscopy analysis.

Declaration of interest

The authors report no conflicts of interest. The authors aloneare responsible for the content and writing of the paper.

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