Protein Corona Formation in Bronchoalveolar Fluid Enhances Diesel

Exhaust Nanoparticle Uptake and Pro-inflammatory Responses in

Macrophages

Catherine A. Shaw*1, Gysell Mortimer3, Zhou J. Deng3, Edwin Carter1, Shea P Connell1, Mark R. Miller1, Rodger Duffin2, David E. Newby1, Patrick W.F. Hadoke1

& Rodney F. Minchin3

1 BHF/ University of Edinburgh Centre for Cardiovascular Science, The Queen’s Medical Research Institute, University of Edinburgh, Edinburgh, U.K.

2 MRC/University of Edinburgh Center for Inflammation Research, The Queen’s Medical Research Institute, University of Edinburgh, Edinburgh, U.K.

3 Laboratory for Molecular and Cellular Pharmacology, School of Biomedical Sciences, University of Queensland, Brisbane, Australia

*Corresponding author

Address for correspondence:Dr Catherine A. ShawCentre for Cardiovascular Science,University of Edinburgh,The Queen’s Medical Research Institute,47 Little France CrescentEdinburgh, EH16 4TJUnited Kingdom

Telephone: +44 (0)131 242 9334Fax: +44 (0)131 242 9215E-mail: Catherine.Shaw@ed.ac.uk

1

1

2

3

4

5678

910111213141516171819202122232425262728293031323334

35

36

37

38

39

ABSTRACT

In biological fluids nanoparticles bind a range of molecules, particularly proteins, on

their surface. The resulting protein corona influences biological activity and

nanoparticle fate in vivo. Corona composition is often determined by the biological

milieu encountered at the entry portal into the body, and, can therefore, depend on the

route of exposure to the nanoparticle. For environmental nanoparticles where

exposure is by inhalation, this will be lung lining fluid.

This study examined plasma and bronchoalveolar fluid (BALF) protein binding to

engineered and environmental nanoparticles. We hypothesized that protein corona on

nanoparticles would influence nanoparticle uptake and subsequent pro-inflammatory

biological response in macrophages.

All nanoparticles bound plasma and BALF proteins, but the profile of bound proteins

varied between nanoparticles. Focusing on diesel exhaust nanoparticles (DENP), we

identified proteins bound from plasma to include fibrinogen, and those bound from

BALF to include albumin and surfactant proteins A and D. The presence on DENP of

a plasma-derived corona or one of purified fibrinogen failed to evoke an inflammatory

response in macrophages. However, coronae formed in BALF increased DENP

uptake into macrophages two fold, and increased nanoparticulate carbon black

(NanoCB) uptake fivefold. Furthermore, a BALF-derived corona increased IL-8

release from macrophages in response to DENP from 1720±850pg/mL to

5560±1380pg/mL (p=0.014). These results demonstrate that the unique protein

corona formed on nanoparticles plays an important role in determining biological

reactivity and nanoparticle fate in vivo. Importantly, these findings have implications

for the mechanism of detrimental properties of environmental nanoparticles since the

principle route of exposure to such particles is via the lung.

2

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

INTRODUCTION

Human exposure to nanoparticles, both manufactured and environmental, is

increasing. Advances in nanotechnology are resulting in many applications for

nanoparticles, for example in medical imaging (Minchin and Martin, 2010) and novel

biomaterials and therapeutics (Thorley and Tetley, 2013). In addition, traffic exhaust

represents a major environmental exposure to carbon-based, combustion-derived

nanoparticles (Donaldson et al., 2013). There is increasing evidence that exposure to

nanoparticles contributes to adverse health effects (Bakand et al., 2012). For

example, air pollution is an independent risk factor for cardiovascular disease

(Mustafic et al., 2012). Air pollution composition varies greatly (Miller et al., 2012),

but epidemiological studies indicate the strongest associations between air pollution

and ill health occur with the PM2.5 fraction (particles with diameter <2.5µM), and are

specifically linked to combustion-derived particles (Laden et al., 2000). In urban

areas diesel exhaust nanoparticles (DENP) represent a significant component of the

particulate fraction of air pollution (Zheng et al., 2007). Exposure to DENP leads to

endothelial dysfunction (Mills et al., 2007), altered fibrinolytic function (Lucking et

al., 2008) and accelerated atherosclerotic plaque formation (Miller et al., 2013). The

mechanisms of adverse health effects in vivo are not yet fully elucidated, but many

studies have implicated a central role for inflammation.

It is increasingly evident that the bio-reactivity and fate of nanoparticles in vivo is

influenced by adsorption of biological molecules, particularly proteins, onto the

nanoparticle surface, forming a protein corona surrounding the nanoparticle core

(Kondej and Sosnowski, 2013, Saptarshi et al., 2013). This is a highly dynamic,

complex process due to the vast array of proteins with differing binding affinities

3

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

present in the biological environment, and the physicochemical characteristics of the

nanoparticle such as size, shape and surface charge, that can influence protein binding

kinetics (Kreyling et al., 2014a, Lundqvist et al., 2008). The corona is often

considered to consist of a ‘hard’ corona, a monolayer of tightly bound proteins, and a

‘soft’ corona containing proteins more loosely associated with the nanoparticle and

likely to be rapidly exchanging with the biological milieu (reviewed by (Monopoli et

al., 2012)). Thus, the protein corona composition, including that of the ‘hard’ corona,

can change over time affording the nanoparticle different biological identities as it

passes through biological fluids of varying composition (Monopoli et al., 2011).

Additionally, binding to the nanoparticle can alter the protein structure, for example

conformational changes in bound proteins can drive biological responses to

nanoparticles including phagocytosis and inflammation (Mortimer et al., 2014, Deng

et al., 2011, Monopoli et al., 2012).

Many nanoparticle-protein interaction studies are performed in plasma and serum

derived from blood (Lesniak et al., 2012, Ashby et al., 2014). Whilst this is relevant

for nanoparticles that ultimately become blood-borne in vivo, such as those developed

for pharmaceutical and therapeutic applications, it is less relevant for environmental

exposure to nanoparticles, where the principal route of exposure is via the lung.

Nanoparticles inhaled in the lung deposit in the distal alveolar regions where the

biological milieu they encounter is the bronchoalveolar fluid (BALF) and pulmonary

surfactant system. Pulmonary surfactant consists of approximately 90% lipids and

10% surfactant associated proteins (surfactant proteins A, B, C and D) (Perez-Gil,

2008). Although some studies have examined the impact of nanoparticles on the

biophysical function of lung surfactant (Kondej and Sosnowski, 2013) and on the

4

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

protein profile of BALF (Lewis et al., 2007, Kendall et al., 2004), few have

investigated the opposite relationship; that of the role of BALF constituents on the

biological reactivity of the nanoparticle.

Our laboratory has previously demonstrated that interactions between nanoparticles

and plasma proteins result in enhanced uptake of nanoparticles and inflammatory

cytokine release from macrophages (Mortimer et al., 2014, Deng et al., 2011). Since

inflammation underlies many of the adverse health conditions linked to environmental

nanoparticle exposure, we hypothesised that protein coronae formed on environmental

nanoparticles would increase nanoparticle internalisation and subsequent

proinflammatory responses in macrophages. Furthermore, because the route of

exposure to environmental nanoparticles is via the lungs, we sought to compare the

effects of protein coronae formed in plasma with those formed in lung-derived BALF.

5

1

2

3

4

5

6

7

8

9

10

11

12

13

METHODS

Particle Dispersion and Characterisation

Suspensions of DENP (Standard Reference Material 2975; National Institute of

Standards and Technology, USA), NanoCB (Printex 90; Evonik Degussa, Germany),

synthetic layered silicate nanoparticles (Lucentite-SWN (LSN); CBC Co Ltd, Tokyo,

Japan), titanium dioxide (Degussa P25; Evonik Degussa, Germany), silica dioxide

(Sigma Aldrich, Australia), and zinc oxide (Nanosun™ 99/30; kindly supplied by

Microniser, Australia) were suspended (all 1 mg/mL except LSN; 0.5mg/mL ) in

ultrapure water and dispersed by probe sonicator (3x 15 second cycles; 70% power;

250W Branson Sonifier ultrasonic probe). Particle size distribution and zeta potential

were determined by dynamic light scattering (DLS) and laser Doppler electrophoresis

(Nanosizer Nano ZS, Malvern Instruments, United Kingdom).

Gel Electrophoresis

Human plasma samples (combined from eight healthy volunteers) were obtained

according to University of Queensland institutional ethics. Whole blood was

collected into sodium citrate and centrifuged (5min; 800g) to pellet the red blood

cells. Resulting plasma supernatants were collected, combined and stored (-80 °C).

Immediately prior to use plasma samples were centrifuged for 2 min at 18000g. Rat

BALF samples (combined from five healthy adult male Wister rats) were collected

immediately following euthanasia by intraperitoneal administration of sodium

pentobarbital (100mg/kg). Briefly, the trachea was exposed and cannulated with

plastic tubing allowing the lungs to be lavaged with sterile H2O. The lungs were

gently aspirated and the resultant BALF from all five rats was combined, centrifuged

6

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

(5min; 1800g) to remove contaminating cells and stored (-80 °C). All animal work

was performed in accordance with the guidelines of the University of Queensland

Animal Ethics Committee.

Nanoparticles (1mg/mL) were dispersed by probe sonication in phosphate buffered

saline (150 mM sodium chloride, 10 mM phosphate; PBS) prior to incubation (37°C;

30 mins) with 1% human plasma or rat BALF (final nanoparticle concentration

0.5mg/mL). Nanoparticle-free plasma, BALF or H2O served as controls. Following

incubation, nanoparticles plus bound proteins were pelleted by ultracentrifugation

(200,000g; 1hr; 4°C). Resulting nanoparticle-protein pellets were washed 3 times

(PBS) before being re-suspended in sodium dodecyl sulfate (SDS) -buffer to give a

final concentration of 1 mg/mL nanoparticles plus 2% SDS, 5% β-mercaptoethanol,

10% glyercol, and 62.5 mM Tris-HCL. Bound proteins were desorbed from the

nanoparticle pellet by heating (5 min; 95°C). Samples were separated by one-

dimensional (12% SDS-PAGE gel; constant voltage of 200V for 35 min) and two-

dimensional electrophoresis according to the methods of Deng et al. (Deng et al.,

2011). Gels were stained overnight with SyproRuby stain (BioRad; one-dimensional

gels) or silver stain solution (2% potassium carbonate, 0.04% sodium hydroxide,

0.007% formaldehyde, 0.002% sodium thiosulfate; two-dimensional gels) and

visualized by ultraviolet or white light transilluminator, respectively.

For DENP, molecular weights of proteins separated by one-dimensional

electrophoresis were calculated by measuring the separation distance from the top of

the gel to the mid-point of each band. Non-linear regression was used to calculate the

molecular weight on a plot of separation distance versus known molecular weights of

a protein ladder maker (PageRuler™ Prestained Protein Ladder; Fermentas, USA).

The range of molecular weights represented by individual bands on one-dimensional

7

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

gels was estimated by calculating the molecular weight represented by the top of the

band and that at the bottom of the band. These values were used to estimate the range

of molecular weights of all proteins resolving in that band. Protein bands from one-

dimensional gels and protein spots from two-dimensional gels were excised and in-gel

digested with trypsin prior to peptide analysis by liquid chromatography tandem mass

spectrophotometry (LC-MS/MS). Protein identification was performed by the Protein

Pilot 2.0.1 (Applied Biosystem) database. The UniProt Protein knowledgebase

(www.Uniprot.org) was used to cross reference the molecular weight of proteins

based on the accession number identified by Protein Pilot. For one-dimensional gels,

the upper two bands (indicated by arrow heads on Figure 3A) were estimated to span

a range of approximately 3.6 ± 0.3 kDa and 1.3 ± 0.02 kDa respectively. Whilst the

lower two, narrower bands spanned a range of < 1 kDa. Therefore any proteins that

were not rat in origin or those that were more than 10 kDa (for the larger, upper two

protein bands on figure 3A) or 5 kDa (for narrower, lower two bands on figure 3A)

out with the molecular weight calculated for the relevant gel band were excluded.

Gel images were manipulated using Adobe Photoshop software to adjust the

brightness and contrast in order to achieve a grey background. Manipulations were

applied uniformly across the entire gel. No other manipulations were performed on

the images.

Fibrinogen Binding Assay

Binding of purified human fibrinogen to DENP was determined by protein pull down

procedure as described previously (Deng et al., 2011). Briefly, 1µg of fluorescently-

labelled fibrinogen was incubated (37°C; 5min) with increasing concentrations of

DENP. DENP-fibrinogen aggregates were pelleted by centrifugation (50 000g; 30

8

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

min) and the amount of fibrinogen remaining in the supernatant was determined by

fluorescent spectroscopy (POLARstar, BMG Labtech). Protein-only controls showed

that unbound fibrinogen did not pellet during centrifugation and that nonspecific

binding of fibrinogen to the incubation tubes did not occur. Appropriate background

fluorescence levels were subtracted from all measurements prior to calculation of the

amount of bound fibrinogen, which was determined by subtracting the amount of

fibrinogen remaining in the supernatant following centrifugation from total

fibrinogen. All experiments were performed in triplicate.

Cell Culture

THP-1 cells (American Type Culture Collection, USA) were maintained (37°C; 5%

CO2) in suspension in RPMI medium containing 5% foetal bovine serum (FCS) and

1% penicillin/streptomycin. THP-1 cells are a monocytic cell line and, when

differentiated with an appropriate stimulus, are used commonly as an in vitro model

for macrophages (Daigneault et al., 2010).

Quantification of Cytokines in THP-1 Cells

Nanoparticles (2mg/mL) were suspended in ultrapure H2O, dispersed by probe

sonicator and then diluted in PBS plus human plasma (1%) or fibrinogen (10µg/mL)

to give a final nanoparticle concentration of 0.5mg/mL. Nanoparticle-protein

suspensions were incubated for 30 min in a waterbath (37°C) incubator to allow

protein coronae to form on the nanoparticle surface. Nanoparticle suspensions and

nanoparticle-protein complexes were prepared freshly prior to each experiment. THP-

1 cells were aliquoted into 96-well V-bottom plates (2x104 cells/well) and incubated in

triplicate with nanoparticle-protein complexes (final nanoparticle concentration

9

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

100µg/ml) for 6 hours. Following incubation, supernatants were harvested by swing

bucket rotor centrifugation (500g; 10min) and cytokine quantification determined by

cytometric bead array (Human Inflammatory Cytokine Kit; BD Biosciences, USA).

Data were captured by FACSCanto flow cytometer equipped with FACSDiva

software (both BD Biosciences) and analysed by BD FCAP Array™ software

(Softflow Ltd., Hungary). Experiments to determine cytokine release were conducted

in undifferentiated THP-1 as the agents used to differentiate these cells are often pro-

inflammatory which can complicate interpretation of the results. The concentration

of individual cytokines was calculated by comparison with a standard curve (0-5000

pg/mL) generated for each experiment. The limit of detection for each cytokine was

20 pg/mL.

Particle Up-Take in THP-1 Cells

For particle uptake studies, THP-1 cells were differentiated for 3 days into

macrophage-like cells using 100ng/mL phorbol 12-myristate 13- acetate (PMA) to

represent a model for alveolar macrophages. Resident alveolar macrophages are likely

to be the first line of cellular defense for any particle depositing in the distal alveolar

regions and commonly phagocytose particles after in vivo instillation (Robertson et

al., 2012). Nanoparticles (2 mg/mL) were suspended in ultrapure H2O, dispersed by

probe sonicator and diluted in PBS plus rat BALF (final nanoparticle concentration

0.5 mg/mL). Nanoparticle-protein suspensions were incubated (37 °C) for 30 min in

a waterbath incubator to allow protein coronae to form on the nanoparticle surface.

Differentiated THP-1 cells (dTHP-1) were incubated for 24 hours with rat BALF-

DENP or BALF-NanoCB complexes (final nanoparticle concentration 10µg/mL).

Following treatment, cells were gently washed with PBS to remove extracellular

10

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

nanoparticles and recovered from tissue culture plasticware by trypsin-EDTA.

Recovered cells were cyto-centrifuged (300rpm; 3min) onto glass microscope slides.

The resultant slides were fixed in methanol (100%; 1min) and stained using Diff-

Quik™ physiological stain (Reagena, Finland) prior to observation by light

microscopy (x40 magnification). Images of five randomly selected fields of view

were captured from each slide. The black colour of DENP and NanoCB particles was

exploited to assess nanoparticle uptake, which was quantified using Adobe Photoshop

software to enumerate the number of intracellular black pixels present in a minimum

of 15 cells per slide. The number of black pixels was used as a surrogate marker of

nanoparticle uptake. Extracellular particles present in background (acellular) areas of

the slide were excluded from the analysis. To prevent observer bias slides were

analysed in a random order with the analyst blinded to experimental conditions.

Human Peripheral Blood Mononuclear Cells Isolation

For final experiments investigating the effects of a corona consisting of human BALF,

human peripheral blood mononuclear cells (PBMC) were isolated from healthy

volunteers. Use of human PBMC avoids issues of species cross-reactivity when using

human BALF, and does not require use of a large pro-inflammatory stimulus to

differentiate the cells into a macrophage phenotype. PBMC were isolated according

to Lothian Research Ethics Committee approvals and isolated by dextran

sedimentation followed by centrifugation through a discontinuous Percoll Percoll®

gradient (Pharamcia, UK) according to method previously described in detail

elsewhere (Shaw et al., 2011). PBMC were suspended in Iscove’s modified

Dulbecco’s medium (IMDM) containing penicillin and streptomycin (both 100

U/mL). Enrichment for monocytes was performed by selective adherence to 48-well

11

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

(2x106 cells/well) tissue culture plates for 1hr (37°C; 5% CO2). Adherent monocytes

were differentiated into monocyte-derived macrophages (MDM) for 5 days in IMDM

containing 10% autologous serum (prepared by recalcification of platelet rich

plasma). Medium was replaced every 2-3 day throughout the differentiation period.

Quantification of Cytokines in Human MDM Cells

Human BALF was obtained from consenting healthy adult never-smokers by a

previously described method (Roos-Engstrand et al., 2011) as part of a study

approved by the local Ethics Review Board at Umea University, Sweden. Subjects

underwent bronchoscopy in a supine position by use of a flexible video bronchoscope

(Olympus BF IT160, Tokyo, Japan). Lavage samples were filtered through a nylon

filter (pore diameter 100µm), centrifuged (400g; 15min) to remove contaminating

cells, and the supernatants stored (-70°C) until experimentation.

Nanoparticles (2mg/mL) were suspended in ultrapure H2O, dispersed by probe

sonicator and then diluted in PBS plus human BALF (final nanoparticle concentration

0.5mg/ml). Suspensions were incubated (37°C waterbath) as described above. MDM

were incubated for 24 hours with Human BALF-DENP or BALF-NanoCB complexes

(final nanoparticle concentration 100µg/mL).

Cell culture supernatants were harvested, clarified by centrifugation to remove any

contaminating nanoparticles (13,000g; 10min) and IL-8 concentration determined by

ELISA (BD OptEIA™ Human IL-8 ELISA kit; BD Biosciences, USA) according to

the manufacturer’s instructions. Optical densities were recorded by Dynatech MXR

microplate reader (Dynatech Laboratories; USA), and the concentration of IL-8 in

each sample calculated from a standard curve produced from serial dilutions of the IL-

8 standard supplied by the manufacturer. All samples were diluted 1:50 with the

12

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

appropriate assay diluent to bring the optical densities within the range of the standard

curve. All standards and samples were assayed in duplicate.

Statistical Analysis

Statistical analyses were performed by GraphPad Prism (version 6.0) software. All

experiments were performed a minimum of three times and results are expressed as

mean ± SEM. Results were compared by two-tailed t-test where relevant. P < 0.05

was regarded as statistically significant.

13

1

2

3

4

5

6

7

8

9

Results

Particle Characterisation

Primary particle diameters for dry particles are 11nm and 14nm for DENP and

nanoCB respectively (values quoted from manufacturer’s Certificates of Analyses).

Particle hydrodynamic diameters for DENP and nanoCB (both 10µg/mL) prepared in

aqueous suspension, in PBS plus human plasma, human BALF or rat BALF, and

RPMI tissue culture medium as used in subsequent experiments are shown in Table 1.

Zeta potentials were -26.8mV for DENP and -21.8mV for nanoCB. Particle

characterisation for metal oxide and LSN nanoparticles have been published

elsewhere by our group (Mortimer et al., 2014, Deng et al., 2009)

Diesel Exhaust Nanoparticles Bind Plasma Proteins But Do Not Illicit A Pro-

Inflammatory Response in THP-1 Cells

One-dimensional gel electrophoresis revealed DENP (0.5mg/mL) bound multiple

plasma proteins (Figure 1A). A large band was observed at approximately 65kDa

(presumed to be albumin based on molecular weight) but this was predominantly low

affinity binding as one wash with PBS was sufficient to remove most of this protein

(Figure 1A). The remaining bands represent high affinity protein binding as the

adsorption profile remained consistent after three washes with PBS.

Further separation of DENP-bound plasma proteins by two-dimensional gel

electrophoresis yielded at least seven clear protein spots (Figure 1B) that were

identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and

included fibrinogen (Table 2). The ability of DENP to bind fibrinogen specifically

14

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

was confirmed by protein pull down experiment that demonstrated approximately

12µg DENP bound 1.15µg fibrinogen (Figure 1C).

Our laboratory has previously demonstrated that fibrinogen unfolds on the surface of

gold nanoparticles to cause MAC-1 receptor activation and inflammatory cytokine

release (Deng et al., 2011). Since DENP also bound fibrinogen, we investigated

whether the presence of a protein corona formed of human plasma proteins or one

formed of purified human fibrinogen on the surface of DENP would affect cytokine

release from THP-1 cells. The concentration of IL-8, IL-1β and TNFα present in the

supernatant of control, untreated, THP-1 cells was below the limit of detection of the

assay (<20pg/mL; Figure 2). DENP (100µg/mL) increased the supernatant IL-8

concentration to 536±230pg/mL (Figure 2A) and the IL-1β concentration to 249

±37pg/mL (Figure 2B). These values represent approximately 30–40% of the

concentration of IL-1β and IL-8 released by LPS (1µg/mL), which served as a

positive control. DENP caused a very slight increase in TNFα concentration

(39.4±12.7pg/mL), but this was considered to be biologically insignificant in

comparison to the response to LPS (734±484pg/mL; Figure 2C). DENP-induced

increases in all cytokines were abolished when DENP had been pre-incubated with

either plasma or fibrinogen (all <20pg/mL; Figure 2).

Nanoparticles Bind Proteins in Bronchoalveolar Fluid

Because exposure to many nanoparticles occurs via inhalation of airborne

environmental particulate, we investigated whether nanoparticles bind proteins

present in lung BALF. For these experiments, we selected a panel of nanoparticles of

differing composition: two carbon based nanoparticles (DENP and NanoCB), one

15

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

synthetic layered silicate nanoparticle (LSN (Mortimer et al., 2014)) and three metal

oxide nanoparticles (titanium dioxide, TiO2; silica dioxide, SiO2; and zinc oxide, ZNO

(Deng et al., 2009). All nanoparticles bound BALF proteins but the profile of bound

proteins varied across the nanoparticle panel (Figure 3).

One-dimensional gel electrophoresis revealed each nanoparticle bound a unique

profile of BALF proteins that remained consistent after 3 washes with PBS, indicating

high affinity protein binding (Figure 3). For example, LSN bound one clear major

protein band of approximately 32kDa whilst the metal oxide nanoparticles bound

multiple proteins. Interestingly, the metal oxide nanoparticles, TiO2, SiO2 and ZnO,

bound a similar profile of proteins, whilst the two carbon-based nanoparticles, DENP

and NanoCB, bound strikingly different proteins, with NanoCB binding many more

proteins than DENP. DENP bound four major protein bands of approximate

molecular weight 63, 44, 32, and 25kDa (indicated by arrow heads on Figure 3).

These bands were excised for further analysis by LC-MS/MS. Multiple proteins were

identified in each band as detailed in Table 3.

Bronchoaleveolar Lavage Fluid Increases Nanoparticle Uptake by Macrophages

We next investigated the impact of BALF-derived corona formation on nanoparticle

uptake by differentiated THP-1 (macrophage-like) cells. The presence of a BALF-

derived corona increased the internalisation of both DENP and nanoCB nanoparticles

(Figure 4). Cytospin preparations allowed visualisation of black nanoparticles (and

their aggregates) in the cytoplasm of cells (Figure 4A). The nanoparticles were

largely excluded from the nucleus indicating an intracellular localization rather than

extracellular surface attachment. No gross morphological changes in cell structure

16

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

were observed after particle internalization and we have previously demonstrated

these nanoparticles are not cytotoxic at this concentration (Shaw et al., 2011).

Using the number of black pixels in the cytoplasm as a surrogate marker of particle

up-take, we found that pre-incubation of DENP and NanoCB with BALF significantly

increased nanoparticle internalization. No black pixels were detected in cells exposed

to control media without particles. The number of intracellular black pixels was

increased two-fold by the presence of a pre-formed BALF corona on DENP and 3

fold for NanoCB (Figure 4B).

Bronchoalevolar Fluid Corona on Nanoparticles Increases Pro-Inflammatory Effects

of Diesel Exhaust Nanoparticles

To investigate the biological significance of BALF-derived corona on nanoparticles,

we measured IL-8 concentrations elaborated from macrophages treated with DENP-

BALF complexes and compared the response to untreated DENP (Figure 5). In order

to avoid cross-reaction between species and having to differentiate THP-1 cells with a

large pro-inflammatory stimulus (PMA), these experiments were performed in freshly

isolated human monocyte-derived macrophage (HuMDM) cells using human BALF

obtained by bronchoscopy.

Control experiments demonstrated human BALF alone (in the absence of particles)

was not pro-inflammatory in HuMDM. In control, untreated cells (particle and BALF

free), the concentration of IL-8 present in the supernatant of HuMDM was

929±97pg/mL. The presence of human BALF (particle-free) did not significantly alter

this (1240±787pg/mL; p = 0.73; data not shown graphically).

17

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

The concentration of IL-8 released from macrophages was greater in response to

DENP-BALF complexes compared to untreated DENP, although the result only

reached statistical significance for the higher dose of DENP-BALF (100µg/mL

DENP-BALF). At this dose, the presence of BALF increased the IL-8 concentration

from 1720±850pg/mL (untreated DENP) to 5560±1380pg/mL (p=0.014; Figure 5).

DISCUSSION

In this study we have investigated protein corona formation on nanoparticles and

compared the effects of corona formed in plasma with those formed in BALF.

Focusing on the environmental nanoparticle, DENP, we have demonstrated that

nanoparticles bind proteins from both plasma and BALF, but only coronae formed in

BALF increase the biological response to DENP, driving nanoparticle uptake into

macrophages and significantly increasing the inflammatory response observed

following macrophage exposure to DENP. This is the first study to demonstrate that a

corona formed in human BALF is pro-inflammatory.

Interestingly, we found that whilst DENP bound plasma proteins including fibrinogen,

the formation of DENP-fibrinogen complexes did not cause the release of

inflammatory cytokines from THP-1 cells but rather appeared to abolish the response

to DENP alone. Further experimental work is required to investigate if this is a

genuine anti-inflammatory effect, however these results appear to be in contrast to a

previous study demonstrating fibrinogen binding and subsequent conformational

change in bound protein structure is critical to elaborating an inflammatory response

to gold nanoparticles (Deng et al., 2011). It may be that the surface of DENP does not

18

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

cause the same structural changes in the fibrinogen protein structure necessary to

elicit a pro-inflammatory response. Since fibrinogen did not increase cytokine release

in response to DENP, we investigated whether other plasma proteins may play a role

in the biological response to DENP. Incubating DENP with whole plasma rather than

purified fibrinogen similarly abolished the cytokine release from THP-1 cells in

response to DENP alone, indicating that plasma-based protein coronae do not enhance

the pro-inflammatory response to this nanoparticle. The reasons for the abolition of

the pro-inflammatory response to DENP by plasma proteins and fibrinogen have not

been further investigated in this study so it is not clear whether this is a genuine anti-

inflammatory effect, or merely a bystander result of protein-induced particle

agglomeration, as has been previously reported (Kendall et al., 2011). Particle

aggregation results in a reduction of overall particle surface area, so although micro-

sized particles are well phagocytosed by macrophages (Geiser, 2010), the resulting

intracellular dose, as expressed by nanoparticle surface area, may be reduced

(Monteiller et al., 2007, Park et al., 2011). However, our DLS results indicate that the

hydrodynamic diameters of DENP and nanoCB are similar in the presence of serum,

plasma and BALF proteins; therefore size variations alone cannot fully account for

these differences. This highlights an important challenge in nanoparticle research and

nanotoxicology: the characteristics of nanoparticles are unique and so variable that it

is often not possible to draw parallels between different types of nanoparticles in order

to accurately predict biological response.

Because the formation of a plasma-derived protein corona was unable to account for

the well-documented pro-inflammatory effects of DENP (Schwarze et al., 2013), we

hypothesized that a corona formed in lung lining fluid may be more relevant for this

19

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

type of environmental nanoparticle, where the principal route of entry to the body is

via the lungs. For these experiments, we selected a nanoparticle panel with three

broad groupings: metal oxide nanoparticles (TiO2, SiO2, ZnO), synthetic layered

silicate nanoparticles (LSN) and carbon-based nanoparticles (combustion-derived;

DENP, and non-combusted; NanoCB). An environmental exposure is relevant to all

of these nanoparticles, either as an occupational exposure to workers during

manufacture of the bulk material, or to the general public as an atmospheric pollutant.

All nanoparticles investigated bound proteins from BALF, but the profile of bound

proteins varied uniquely across the panel.

We selected the four major protein bands bound by DENP for further analysis and

found them to include (of note) albumin, surfactant protein D (SP-D) and surfactant

protein A (SP-A). Albumin is known to bind a variety of nanoparticles (Zhang et al.,

2014), and a recent study from our laboratory has found a critical role for albumin

during uptake of nanoparticles via scavenger receptors (Mortimer et al., 2014). SP-A

is the most abundant of the surfactant proteins and, together with SP-D, has a central

role in the opsinization of pathogens and inhaled particles to ensure successful

phagocytic clearance during the immune response (Seaton et al., 2010). SP-A has

also been demonstrated to bind to a range of nanoparticles (Schulze et al., 2011) and,

together with SP-D, to enhance nanoparticle uptake by macrophages (Ruge et al.,

2012). These reported effects of albumin, SP-A and SP-D are in keeping with the

data presented in this study demonstrating a corona formed in BALF increases uptake

of DENP and NanoCB nanoparticles by macrophages. Although Vranic et al.

recently reported the opposite finding, that lung surfactant down-regulated

20

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

nanoparticle uptake, their study was performed using the commercially available lung

surfactant substitute Curosurf that lacks SP-A (Vranic et al., 2013).

Whilst our study has focused on protein binding, we cannot exclude a role for lipids.

Protein binding is affected by nanoparticle hydrophobicity (Gessner et al., 2000) and

BALF is composed of approximately 90% phospholipid. It, therefore, seems highly

likely that some lipid will be present in the corona formed in lung lining fluid.

Indeed, Ruge et al. found that the presence of lung lining fluid lipids significantly

modulated the effects of SP-A and SP-D when investigating macrophage uptake of

nanoparticles (Ruge et al., 2012). The specificity of the biological fluid in

determining the corona composition is further highlighted by differences in the profile

of proteins bound by LSN in lung lining fluid compared to those bound by this

nanoparticle in blood-derived plasma and serum. When incubated with human

plasma, the major protein bound by LSN is albumin (Mortimer et al., 2014). Whilst

we have not specifically identified the proteins bound in lung lining fluid by LSN,

there is no evidence on the one-dimensional gel of a protein band resolving at the

appropriate molecular weight of albumin (68 kDa). The possibility that albumin is

not present in the lung lining fluid is excluded as albumin was identified in the DENP-

bound proteins. These differences further underline the importance of using the

appropriate biological fluid relevant to the most applicable route of exposure when

studying nanoparticle corona formation.

Our observation that the corona composition can influence internalization and pro-

inflammatory effects of nanoparticles has implications for in vivo studies using intra-

tracheal instillation as the dosing method. Some studies utilize either distilled water

21

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

or PBS as a particle vehicle (Ma et al., 2015, Robertson et al., 2012) whilst others add

a small percentage of serum or BALF to the particle vehicle (Bourdon et al., 2012,

Poulsen et al., 2015). Use of serum or BALF in the vehicle preparation will allow a

protein corona to form prior to dosing, which could result in enhanced macrophage

phagocytosis and pro-inflammatory effects in vivo. However, whether this will cause

a change in the overall interpretation of such studies is debatable since the corona

would likely form immediately in vivo regardless of the vehicle preparation of

particles.

Quantifying phagocytosis of nanoparticles can be technically challenging as many of

the available techniques, such as flow cytometry and fluorescence microscopy, rely

either on intrinsic fluorescence of the nanoparticle or on the addition of fluorescent

labels or tags. Such additions may actually alter the characteristics of the

nanoparticle, for example, increasing the particle size, causing agglomeration or

altering the zeta potential. In an effort to avoid these technical issues, we selected the

carbon-based nanoparticles for our uptake studies and exploited their black colour to

quantify uptake. Using computer software to enumerate intracellular black pixels as a

surrogate marker of uptake allowed us to quantify phagocytosis. Because this

technique relies on black coloration, it was only applicable to DENP and NanoCB

because the other nanoparticles in the panel were either white or colourless. Although

we only identified proteins bound by DENP, scrutiny of the profile of proteins bound

by the metal oxides, NanoCB and LSN reveals protein bands resolving at the same

molecular weight as the bands found to contain SP-D and SP-A (labeled by arrows 2

and 4 on Figure 3). For the metal oxides and NanoCB both bands are present, whilst

for LSN only a band of equal molecular weight to SP-A is present. Metal oxide

22

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

nanoparticles have been demonstrated to bind SP-A (Schulze et al., 2011) and both

metal oxides and LSN are known to be phagocytosed by macrophages (Mortimer et

al., 2014, James et al., 2013). So, although we were unable to use our technique to

examine uptake of the metal oxides and LSN, we can speculate that a similar uptake

mechanism may be applicable to these particles.

Having demonstrated corona formation facilitated nanoparticle uptake by

macrophages, we next investigated the macrophage response to nanoparticle

internalisation. Inhalation of DENP is linked to adverse cardiovascular events and

although the exact mechanism of this link remains unknown, inflammation emanating

from the lungs is a key hypothesis (Mills et al., 2009). We therefore investigated

whether a corona formed in BALF led to elevated cytokine elaboration from

macrophages in response to DENP exposure. In contrast to a plasma-derived corona,

a human BALF-derived corona on DENP was proinflammatory, increasing the

concentration of IL-8 released from human macrophages compared to DENP without

a pre-formed BALF corona. In the current study we have not attempted to elucidate

the mechanism of this pro-inflammatory effect. Increased cytokine release could

result from the enhanced nanoparticle uptake that effectively results in a higher

intracellular dose. Oxidative stress, a shift in the balance of pro- and anti-oxidants in

the cell in favour of a pro-oxidant environment, has been linked to exposure to

nanoparticles, including DENP (Miller et al., 2012, Miller, 2014). Increased reactive

oxygen species (ROS) generation by nanoparticles has been reported in the literature

when nanoparticles are prepared in components of lung lining fluid (Foucaud et al.,

2007, Herzog et al., 2009). Transcription of many pro-inflammatory genes, including

IL-8, is under the control of redox sensitive transcription factors, such as nuclear

factor-κB (NF-κB), that are up-regulated by exposure to nanoparticle-derived ROS

23

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

(Brown et al., 2004). Therefore, a BALF-derived corona could result in enhanced

intracellular ROS leading to increased transcription and translation of pro-

inflammatory cytokines. Alternatively, structural changes in the bound proteins may

be exposing novel, as yet unidentified, ligands for pro-inflammatory receptors, as was

recently demonstrated for nanoparticle bound fibrinogen and albumin (Mortimer et

al., 2014, Deng et al., 2011). This is the first study to demonstrate that the presence of

a human BALF-derived corona can increase the inflammatory response to DENP and

supports recent findings that synthetic surfactant enhances IL-8 release in response to

multi-walled carbon nanotubes (Gasser et al., 2012). Further experimental work is

warranted to establish the mechanism of this pro-inflammatory effect.

There has been much debate in the literature regarding the mechanism linking DENP

inhalation with adverse events in the vasculature. Our observation that a BALF-

derived corona is pro-inflammatory suggests events occurring at the point of

deposition in the lung may trigger inflammation that has downstream implications for

adverse cardiovascular events, many of which have an inflammatory component

(Mangge et al., 2014). Central to the debate surrounding the link between DENP

inhalation and cardiovascular disease has been the key issue of whether it is possible

for nanoparticles deposited in the lung to translocate to secondary organs including

the systemic circulation (Kermanizadeh et al., 2015, Kreyling et al., 2014b), or if,

alternatively, particle deposition in the lung causes inflammation and a pulmonary

acute phase response that constitutes the mechanistic link between DENP exposure

and cardiovascular disease (Saber et al., 2014). Whilst some studies appear to show

particle translocation does occur (Nemmar et al., 2002), others have refuted this

(Wiebert et al., 2006, Mills et al., 2006) or found it to be a rare phenomenon

24

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

applicable only to low numbers of nanoparticles (Kreyling et al., 2002). Our results

suggest that if the protein corona formed on particles is stable, particles that are able

to translocate from the lung into the circulation may evoke a larger inflammatory

response after passing through the lung where they will acquire a lung lining fluid

derived corona. However, the protein corona is dynamic (Monopoli et al., 2011)

therefore it’s composition may change as any translocated nanoparticles pass from the

lung to the circulation. Interestingly, our preliminary observations suggested plasma

proteins and fibrinogen may be anti-inflammatory when present in the corona, yet

fibrinogen β- and γ-chain are present in the BALF-derived corona on DENP which

was pro-inflammatory. Therefore, it may that the biological reactivity of a

nanoparticle in any given organ is determined by the net balance of pro- and anti-

inflammatory proteins present in the corona. Further experimental work is warranted

to determine if a nanoparticle that has acquired a pro-inflammatory corona in the lung

retains this biological identity in the circulation, or if contact with blood proteins will

modify the corona in favor of anti-inflammatory effects.

Conclusion

In conclusion, we have shown that coronae formed on nanoparticles play a key role in

determining nanoparticle uptake and inflammatory response. We have demonstrated

that both the nanoparticle itself and the composition of the biological fluid in which

the corona is formed are critical to determining the response to nanoparticles. Future

studies must take into account the route of exposure and likely composition of the

biological milieu encountered when considering the effects, both detrimental and

desirable, of nanoparticles.

25

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

1

ACKNOWLEDGEMENTS

The authors gratefully acknowledge Professor Anders Blomberg and Dr Jamshed

Pourazar of the University of Umea, Sweden, for their kind gift of human BALF

samples.

SOURCES OF FUNDING

This work was supported by a British Heart Foundation Programme Grant

(PG/10/009).

DECLARATION OF INTEREST STATEMENT

The authors declare no conflicts of interest regarding this work.

27

1

2

3

4

5

6

7

8

910111213

14

Table 1

H2O RPMI Plasma BALFRat Human

DENP 132 ± 35 125 ± 29 138 ± 32 151 ± 44 162 ± 46nanoCB 154 ± 9.0 114 ± 3.6 128 ± 4.3 187 ± 3.3 228 ± 43

Table 1: Hydrodynamic Particle Sizes. Particle hydrodynamic diameters are shown

for particles prepared in aqueous suspension, RPMI tissue culture medium or PBS

plus plasma (human), or BALF (human and rat) as used in subsequent experiments.

Figures quoted in nm and represent mean ± SEM (n=3).

Table 2

2D Gel Spot

ID Number

Protein Identification

1 Alpha-2-HS-Glycoprotein

2 Fibrinogen-γ-chain

3 Fibrinogen-β-chain

4 Compliment c3

5 IG-γ-chain C

6 IG-κ-chain C

7 Apo-lioprotein A

Table 2: Plasma Proteins Bound by DENP. Two dimensional gel spot

identification numbers correspond to the protein spots encircled on Figure 1B.

Proteins listed in the table are those identified by liquid chromatography tandem mass

spectrophotometry and the ProteinPilot database following excision of protein spots 1

– 7 shown on figure 1B.

28

12

34

5

6

7

89

1011

1213

14

15

16

17

1819

Table 3

Band 163.4 kDa

Band 244.2 kDa

Band 332.6 kDa

Band 425.1 kDa

Serum albumin 68.7 Actin cytoplasmic 1 41.7 Annexin A5 35.7 Peroxiredoxin-6 24.8

Liver carboxylesterase

60.2 Pulmonary surfactant-associated protein D

37.6 Annexin A8 36.7 Glutathione S-transferase alpha 4 25.5

Serine protease inhibitor A3L 46.3 Tropomyosin alpha-3 chain OS

29 Glutathione S-transferase P 23.4

SEC 14-like protein 46.0 Purine nucleoside phosphorylase

32.3 Ras-related protein Rab-1A 22.6

Alpha enolase OS 47.1 14-3-3 protein zeta/delta 27.7 Pulmonary surfactant associated protein A

26.3

Keratin type II cytoskeleton 54 F-actin-capping protein subunit beta

30.6 Tropomyosin alpha-3 chain 29.0

Keratin type I cytoskeleton 47.8 Annexin A3 36.4

Alpha-2-HS-glycoprotein 37.9

Alpha-1-antiproteinase 46.1

Table 3: BALF Proteins Bound by DENP. Bands 1-4 correspond to bands indicated by arrowheads on Figure 2A. Molecular weights in the

header are those calculated for each band on the one-dimensional gel. Proteins listed in the table are those identified by liquid chromatography

tandem mass spectrophotometry and the ProteinPilot database following excision of bands 1-4 from Figure 3. Molecular weights quoted in the

29

12

34

5

6

table for each protein are those supplied by the UniProt Protein Knowledgebase for the relevant protein accession number identified by

ProteinPilot.

30

1

2

34

Figure Legends

Figure 1: Binding of plasma proteins by DENP. A – Representative one-

dimensional SDS-PAGE of plasma proteins bound by diesel exhaust nanoparticles

(DENP; 0.5mg/ml) following 0–3 washes with PBS as indicated above gel. Whole

human plasma is shown for comparison. Particles incubated in H2O served as a

negative control. B–Two-dimensional gel of DENP (0.5mg/ml) bound plasma

proteins. Numbered circles indicate areas excised for further analysis by liquid

chromatography tandem mass spectrophotometry. C–Binding of fibrinogen with

increasing concentrations of DENP indicated approximately 12µg DENP bind 1.15 µg

fibrinogen (n = 3).

Figure 2: Inflammatory cytokine release by THP-1 cells in response to DENP.

The concentration of IL-8 (A), IL-1β (B) and TNF-α (C) present in the supernatant of

control (untreated) THP-1 cells was below the limit of detection of the assay (<20

pg/mL). Treatment with DENP (100 µg/mL) increased the concentration of IL-8 (A),

IL-1β (B) and TNF-α (C) present in THP-1 supernatants; an effect which was

abolished by human plasma (10%) and fibrinogen (10 µg/mL) where the result was

below the limit of detection of the assay (<20pg/mL). LPS (1µg/mL) served as a

positive control of THP-1 activation and caused a large increase in the concentration

of all cytokines (A-C). Results are mean±SEM (n=3), statistical analyses were not

performed on these data as the control values were below the limit of detection of the

assay.

31

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

Figure 3 – Binding of BALF proteins by nanoparticles. Representative one-

dimensional SDS-PAGE of BALF proteins bound by a range of nanoparticles as

indicated above the gel (all 0.5mg/ml). Whole BALF is shown for comparison.

Particles incubated in H2O served as a negative control. Arrow heads 1-4 indicate

bands excised for further analysis by liquid chromatography tandem mass

spectrophotometry.

Figure 4 – Uptake of DENP and NanoCB into dTHP-1 cells. A–Representative

photomicrographs of cytospin preparations (x40 magnification) showing the presence

of black DENP and NanoCB nanoparticles (both 10µg/ml) in the cytoplasm of dTHP-

1 cells following 24-hour incubation. The number of intracellular black nanoparticles

was increased by pre-incubation of nanoparticles with BALF as indicted. B–The

number of black cytosolic pixels was enumerated and used a surrogate marker of

nanoparticle uptake into dTHP-1 cells. No black pixels were detected in cells

exposed to control media without particles. The presence of a protein corona formed

in BALF significantly increased the internalisation of DENP and NanoCB. Results

are the mean±SEM (n = 4).

Figure 5 – Inflammatory cytokine release from human monocyte-derived

macrophages in response to DENP pre-incubated with human BALF. The

presence of a protein corona formed in human BALF tended to increase the

concentration of IL-8 in the supernatant of human macrophages in response to DENP,

reaching statistical significance at 100µg/ml DENP-BALF. Results are mean ±SEM

(n = 4); results were compared by two-tailed t-test.

32

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

REFERENCES

ASHBY, J., PAN, S. & ZHONG, W. 2014. Size and surface functionalization of iron oxide nanoparticles influence the composition and dynamic nature of their protein corona. ACS Appl Mater Interfaces.

BAKAND, S., HAYES, A. & DECHSAKULTHORN, F. 2012. Nanoparticles: a review of particle toxicology following inhalation exposure. Inhal Toxicol, 24, 125-35.

BOURDON, J. A., SABER, A. T., JACOBSEN, N. R., JENSEN, K. A., MADSEN, A. M., LAMSON, J. S., WALLIN, H., MOLLER, P., LOFT, S., YAUK, C. L. & VOGEL, U. B. 2012. Carbon black nanoparticle instillation induces sustained inflammation and genotoxicity in mouse lung and liver. Part Fibre Toxicol, 9, 5.

BROWN, D. M., DONALDSON, K., BORM, P. J., SCHINS, R. P., DEHNHARDT, M., GILMOUR, P., JIMENEZ, L. A. & STONE, V. 2004. Calcium and ROS-mediated activation of transcription factors and TNF-alpha cytokine gene expression in macrophages exposed to ultrafine particles. Am J Physiol Lung Cell Mol Physiol, 286, L344-53.

DAIGNEAULT, M., PRESTON, J. A., MARRIOTT, H. M., WHYTE, M. K. & DOCKRELL, D. H. 2010. The identification of markers of macrophage differentiation in PMA-stimulated THP-1 cells and monocyte-derived macrophages. PLoS One, 5, e8668.

DENG, Z. J., LIANG, M., MONTEIRO, M., TOTH, I. & MINCHIN, R. F. 2011. Nanoparticle-induced unfolding of fibrinogen promotes Mac-1 receptor activation and inflammation. Nat Nanotechnol, 6, 39-44.

DENG, Z. J., MORTIMER, G., SCHILLER, T., MUSUMECI, A., MARTIN, D. & MINCHIN, R. F. 2009. Differential plasma protein binding to metal oxide nanoparticles. Nanotechnology, 20, 455101.

DONALDSON, K., DUFFIN, R., LANGRISH, J. P., MILLER, M. R., MILLS, N. L., POLAND, C. A., RAFTIS, J., SHAH, A., SHAW, C. A. & NEWBY, D. E. 2013. Nanoparticles and the cardiovascular system: a critical review. Nanomedicine (Lond), 8, 403-23.

FOUCAUD, L., WILSON, M. R., BROWN, D. M. & STONE, V. 2007. Measurement of reactive species production by nanoparticles prepared in biologically relevant media. Toxicol Lett, 174, 1-9.

GASSER, M., WICK, P., CLIFT, M. J., BLANK, F., DIENER, L., YAN, B., GEHR, P., KRUG, H. F. & ROTHEN-RUTISHAUSER, B. 2012. Pulmonary surfactant coating of multi-walled carbon nanotubes (MWCNTs) influences their oxidative and pro-inflammatory potential in vitro. Part Fibre Toxicol, 9, 17.

GEISER, M. 2010. Update on macrophage clearance of inhaled micro- and nanoparticles. J Aerosol Med Pulm Drug Deliv, 23, 207-17.

GESSNER, A., WAICZ, R., LIESKE, A., PAULKE, B., MADER, K. & MULLER, R. H. 2000. Nanoparticles with decreasing surface hydrophobicities: influence on plasma protein adsorption. Int J Pharm, 196, 245-9.

HERZOG, E., BYRNE, H. J., DAVOREN, M., CASEY, A., DUSCHL, A. & OOSTINGH, G. J. 2009. Dispersion medium modulates oxidative stress response of human lung epithelial cells upon exposure to carbon nanomaterial samples. Toxicol Appl Pharmacol, 236, 276-81.

33

123456789

101112131415161718192021222324252627282930313233343536373839404142434445464748

JAMES, S. A., FELTIS, B. N., DE JONGE, M. D., SRIDHAR, M., KIMPTON, J. A., ALTISSIMO, M., MAYO, S., ZHENG, C., HASTINGS, A., HOWARD, D. L., PATERSON, D. J., WRIGHT, P. F., MOORHEAD, G. F., TURNEY, T. W. & FU, J. 2013. Quantification of ZnO nanoparticle uptake, distribution, and dissolution within individual human macrophages. ACS Nano, 7, 10621-35.

KENDALL, M., DING, P. & KENDALL, K. 2011. Particle and nanoparticle interactions with fibrinogen: the importance of aggregation in nanotoxicology. Nanotoxicology, 5, 55-65.

KENDALL, M., GUNTERN, J., LOCKYER, N. P., JONES, F. H., HUTTON, B. M., LIPPMANN, M. & TETLEY, T. D. 2004. Urban PM2.5 surface chemistry and interactions with bronchoalveolar lavage fluid. Inhal Toxicol, 16 Suppl 1, 115-29.

KERMANIZADEH, A., BALHARRY, D., WALLIN, H., LOFT, S. & MOLLER, P. 2015. Nanomaterial translocation-the biokinetics, tissue accumulation, toxicity and fate of materials in secondary organs-a review. Crit Rev Toxicol, 45, 837-72.

KONDEJ, D. & SOSNOWSKI, T. R. 2013. Alteration of biophysical activity of pulmonary surfactant by aluminosilicate nanoparticles. Inhal Toxicol, 25, 77-83.

KREYLING, W. G., FERTSCH-GAPP, S., SCHAFFLER, M., JOHNSTON, B. D., HABERL, N., PFEIFFER, C., DIENDORF, J., SCHLEH, C., HIRN, S., SEMMLER-BEHNKE, M., EPPLE, M. & PARAK, W. J. 2014a. In vitro and in vivo interactions of selected nanoparticles with rodent serum proteins and their consequences in biokinetics. Beilstein J Nanotechnol, 5, 1699-711.

KREYLING, W. G., HIRN, S., MOLLER, W., SCHLEH, C., WENK, A., CELIK, G., LIPKA, J., SCHAFFLER, M., HABERL, N., JOHNSTON, B. D., SPERLING, R., SCHMID, G., SIMON, U., PARAK, W. J. & SEMMLER-BEHNKE, M. 2014b. Air-blood barrier translocation of tracheally instilled gold nanoparticles inversely depends on particle size. ACS Nano, 8, 222-33.

KREYLING, W. G., SEMMLER, M., ERBE, F., MAYER, P., TAKENAKA, S., SCHULZ, H., OBERDORSTER, G. & ZIESENIS, A. 2002. Translocation of ultrafine insoluble iridium particles from lung epithelium to extrapulmonary organs is size dependent but very low. J Toxicol Environ Health A, 65, 1513-30.

LADEN, F., NEAS, L. M., DOCKERY, D. W. & SCHWARTZ, J. 2000. Association of fine particulate matter from different sources with daily mortality in six U.S. cities. Environ Health Perspect, 108, 941-7.

LESNIAK, A., FENAROLI, F., MONOPOLI, M. P., ABERG, C., DAWSON, K. A. & SALVATI, A. 2012. Effects of the presence or absence of a protein corona on silica nanoparticle uptake and impact on cells. ACS Nano, 6, 5845-57.

LEWIS, J. A., RAO, K. M., CASTRANOVA, V., VALLYATHAN, V., DENNIS, W. E. & KNECHTGES, P. L. 2007. Proteomic analysis of bronchoalveolar lavage fluid: effect of acute exposure to diesel exhaust particles in rats. Environ Health Perspect, 115, 756-63.

LUCKING, A. J., LUNDBACK, M., MILLS, N. L., FARATIAN, D., BARATH, S. L., POURAZAR, J., CASSEE, F. R., DONALDSON, K., BOON, N. A., BADIMON, J. J., SANDSTROM, T., BLOMBERG, A. & NEWBY, D. E. 2008. Diesel exhaust inhalation increases thrombus formation in man. Eur Heart J, 29, 3043-51.

34

123456789

10111213141516171819202122232425262728293031323334353637383940414243444546474849

LUNDQVIST, M., STIGLER, J., ELIA, G., LYNCH, I., CEDERVALL, T. & DAWSON, K. A. 2008. Nanoparticle size and surface properties determine the protein corona with possible implications for biological impacts. Proc Natl Acad Sci U S A, 105, 14265-70.

MA, J., MERCER, R. R., BARGER, M., SCHWEGLER-BERRY, D., COHEN, J. M., DEMOKRITOU, P. & CASTRANOVA, V. 2015. Effects of amorphous silica coating on cerium oxide nanoparticles induced pulmonary responses. Toxicol Appl Pharmacol, 288, 63-73.

MANGGE, H., BECKER, K., FUCHS, D. & GOSTNER, J. M. 2014. Antioxidants, inflammation and cardiovascular disease. World J Cardiol, 6, 462-77.

MILLER, M. R. 2014. The role of oxidative stress in the cardiovascular actions of particulate air pollution. Biochem Soc Trans, 42, 1006-11.

MILLER, M. R., MCLEAN, S. G., DUFFIN, R., LAWAL, A. O., ARAUJO, J. A., SHAW, C. A., MILLS, N. L., DONALDSON, K., NEWBY, D. E. & HADOKE, P. W. 2013. Diesel exhaust particulate increases the size and complexity of lesions in atherosclerotic mice. Part Fibre Toxicol, 10, 61.

MILLER, M. R., SHAW, C. A. & LANGRISH, J. P. 2012. From particles to patients: oxidative stress and the cardiovascular effects of air pollution. Future Cardiol, 8, 577-602.

MILLS, N. L., AMIN, N., ROBINSON, S. D., ANAND, A., DAVIES, J., PATEL, D., DE LA FUENTE, J. M., CASSEE, F. R., BOON, N. A., MACNEE, W., MILLAR, A. M., DONALDSON, K. & NEWBY, D. E. 2006. Do inhaled carbon nanoparticles translocate directly into the circulation in humans? Am J Respir Crit Care Med, 173, 426-31.

MILLS, N. L., DONALDSON, K., HADOKE, P. W., BOON, N. A., MACNEE, W., CASSEE, F. R., SANDSTROM, T., BLOMBERG, A. & NEWBY, D. E. 2009. Adverse cardiovascular effects of air pollution. Nat Clin Pract Cardiovasc Med, 6, 36-44.

MILLS, N. L., TORNQVIST, H., GONZALEZ, M. C., VINK, E., ROBINSON, S. D., SODERBERG, S., BOON, N. A., DONALDSON, K., SANDSTROM, T., BLOMBERG, A. & NEWBY, D. E. 2007. Ischemic and thrombotic effects of dilute diesel-exhaust inhalation in men with coronary heart disease. N Engl J Med, 357, 1075-82.

MINCHIN, R. F. & MARTIN, D. J. 2010. Nanoparticles for molecular imaging--an overview. Endocrinology, 151, 474-81.

MONOPOLI, M. P., ABERG, C., SALVATI, A. & DAWSON, K. A. 2012. Biomolecular coronas provide the biological identity of nanosized materials. Nat Nanotechnol, 7, 779-86.

MONOPOLI, M. P., WALCZYK, D., CAMPBELL, A., ELIA, G., LYNCH, I., BOMBELLI, F. B. & DAWSON, K. A. 2011. Physical-chemical aspects of protein corona: relevance to in vitro and in vivo biological impacts of nanoparticles. J Am Chem Soc, 133, 2525-34.

MONTEILLER, C., TRAN, L., MACNEE, W., FAUX, S., JONES, A., MILLER, B. & DONALDSON, K. 2007. The pro-inflammatory effects of low-toxicity low-solubility particles, nanoparticles and fine particles, on epithelial cells in vitro: the role of surface area. Occup Environ Med, 64, 609-15.

MORTIMER, G. M., BUTCHER, N. J., MUSUMECI, A. W., DENG, Z. J., MARTIN, D. J. & MINCHIN, R. F. 2014. Cryptic epitopes of albumin determine mononuclear phagocyte system clearance of nanomaterials. ACS Nano, 8, 3357-66.

35

123456789

10111213141516171819202122232425262728293031323334353637383940414243444546474849

MUSTAFIC, H., JABRE, P., CAUSSIN, C., MURAD, M. H., ESCOLANO, S., TAFFLET, M., PERIER, M. C., MARIJON, E., VERNEREY, D., EMPANA, J. P. & JOUVEN, X. 2012. Main air pollutants and myocardial infarction: a systematic review and meta-analysis. JAMA, 307, 713-21.

NEMMAR, A., HOET, P. H., VANQUICKENBORNE, B., DINSDALE, D., THOMEER, M., HOYLAERTS, M. F., VANBILLOEN, H., MORTELMANS, L. & NEMERY, B. 2002. Passage of inhaled particles into the blood circulation in humans. Circulation, 105, 411-4.

PARK, M. V., NEIGH, A. M., VERMEULEN, J. P., DE LA FONTEYNE, L. J., VERHAREN, H. W., BRIEDE, J. J., VAN LOVEREN, H. & DE JONG, W. H. 2011. The effect of particle size on the cytotoxicity, inflammation, developmental toxicity and genotoxicity of silver nanoparticles. Biomaterials, 32, 9810-7.

PEREZ-GIL, J. 2008. Structure of pulmonary surfactant membranes and films: the role of proteins and lipid-protein interactions. Biochim Biophys Acta, 1778, 1676-95.

POULSEN, S. S., SABER, A. T., WILLIAMS, A., ANDERSEN, O., KOBLER, C., ATLURI, R., POZZEBON, M. E., MUCELLI, S. P., SIMION, M., RICKERBY, D., MORTENSEN, A., JACKSON, P., KYJOVSKA, Z. O., MOLHAVE, K., JACOBSEN, N. R., JENSEN, K. A., YAUK, C. L., WALLIN, H., HALAPPANAVAR, S. & VOGEL, U. 2015. MWCNTs of different physicochemical properties cause similar inflammatory responses, but differences in transcriptional and histological markers of fibrosis in mouse lungs. Toxicol Appl Pharmacol, 284, 16-32.

ROBERTSON, S., GRAY, G. A., DUFFIN, R., MCLEAN, S. G., SHAW, C. A., HADOKE, P. W., NEWBY, D. E. & MILLER, M. R. 2012. Diesel exhaust particulate induces pulmonary and systemic inflammation in rats without impairing endothelial function ex vivo or in vivo. Part Fibre Toxicol, 9, 9.

ROOS-ENGSTRAND, E., POURAZAR, J., BEHNDIG, A. F., BUCHT, A. & BLOMBERG, A. 2011. Expansion of CD4+CD25+ helper T cells without regulatory function in smoking and COPD. Respir Res, 12, 74.

RUGE, C. A., SCHAEFER, U. F., HERRMANN, J., KIRCH, J., CANADAS, O., ECHAIDE, M., PEREZ-GIL, J., CASALS, C., MULLER, R. & LEHR, C. M. 2012. The interplay of lung surfactant proteins and lipids assimilates the macrophage clearance of nanoparticles. PLoS One, 7, e40775.

SABER, A. T., JACOBSEN, N. R., JACKSON, P., POULSEN, S. S., KYJOVSKA, Z. O., HALAPPANAVAR, S., YAUK, C. L., WALLIN, H. & VOGEL, U. 2014. Particle-induced pulmonary acute phase response may be the causal link between particle inhalation and cardiovascular disease. Wiley Interdiscip Rev Nanomed Nanobiotechnol, 6, 517-31.

SAPTARSHI, S. R., DUSCHL, A. & LOPATA, A. L. 2013. Interaction of nanoparticles with proteins: relation to bio-reactivity of the nanoparticle. J Nanobiotechnology, 11, 26.

SCHULZE, C., SCHAEFER, U. F., RUGE, C. A., WOHLLEBEN, W. & LEHR, C. M. 2011. Interaction of metal oxide nanoparticles with lung surfactant protein A. Eur J Pharm Biopharm, 77, 376-83.

SCHWARZE, P. E., TOTLANDSDAL, A. I., LAG, M., REFSNES, M., HOLME, J. A. & OVREVIK, J. 2013. Inflammation-related effects of diesel engine exhaust particles: studies on lung cells in vitro. Biomed Res Int, 2013, 685142.

36

123456789

101112131415161718192021222324252627282930313233343536373839404142434445464748

SEATON, B. A., CROUCH, E. C., MCCORMACK, F. X., HEAD, J. F., HARTSHORN, K. L. & MENDELSOHN, R. 2010. Review: Structural determinants of pattern recognition by lung collectins. Innate Immun, 16, 143-50.

SHAW, C. A., ROBERTSON, S., MILLER, M. R., DUFFIN, R., TABOR, C. M., DONALDSON, K., NEWBY, D. E. & HADOKE, P. W. 2011. Diesel exhaust particulate--exposed macrophages cause marked endothelial cell activation. Am J Respir Cell Mol Biol, 44, 840-51.

THORLEY, A. J. & TETLEY, T. D. 2013. New perspectives in nanomedicine. Pharmacol Ther, 140, 176-85.

VRANIC, S., GARCIA-VERDUGO, I., DARNIS, C., SALLENAVE, J. M., BOGGETTO, N., MARANO, F., BOLAND, S. & BAEZA-SQUIBAN, A. 2013. Internalization of SiO(2) nanoparticles by alveolar macrophages and lung epithelial cells and its modulation by the lung surfactant substitute Curosurf. Environ Sci Pollut Res Int, 20, 2761-70.

WIEBERT, P., SANCHEZ-CRESPO, A., FALK, R., PHILIPSON, K., LUNDIN, A., LARSSON, S., MOLLER, W., KREYLING, W. G. & SVARTENGREN, M. 2006. No significant translocation of inhaled 35-nm carbon particles to the circulation in humans. Inhal Toxicol, 18, 741-7.

ZHANG, W., ZHANG, Q., WANG, F., YUAN, L., XU, Z., JIANG, F. & LIU, Y. 2014. Comparison of interactions between human serum albumin and silver nanoparticles of different sizes using spectroscopic methods. Luminescence.

ZHENG, M., CASS, G. R., KE, L., WANG, F., SCHAUER, J. J., EDGERTON, E. S. & RUSSELL, A. G. 2007. Source apportionment of daily fine particulate matter at Jefferson Street, Atlanta, GA, during summer and winter. J Air Waste Manag Assoc, 57, 228-42.

37

123456789

101112131415161718192021222324252627