“Laboratory-generated soot with well-defined organic coating for in-vitro cytotoxicity assessment”

Interdisciplinary project funded by the Federal Office for Environment (FOEN, N° 17.0094.PJ)

ZairaLeniKonstan'naVasilatou,MichaelaEss,AlejandroKeller,MarianneGeiser

ETHConference19June2019

# 6 Air pollution is the 6th highest-ranking risk

factor for death globally

Health Effects Institute. 2018. State of Global Air 2018. Boston MA

Global map of modelling annual median concentration of PM2.5 in µg/m3 http://maps.who.int/airpollution/

Source: WHO 2016

92% More than 9 out of 10 of the world’s population lives in areas

exceeding WHO guidelines for safe limits.

<1% In China and India, less than 1% of the population lives in areas

meeting the WHO guidelines

Adapted from “Recent advances in understanding SOA: implications for global climate forcing” M. Shrivastava et al., 2017.

Genera'onofwellcharacterizedagedsootpar'clescoatedwithoxidizedorganicma?er–  Anthropogenicorigin(1,3,5-trimethylbenzene,urbansite)–  Biogenicorigin(α-pinene,suburban)

Projectgoalsandexperimentalsetup(I)

miniCAST5201TypeBCM.EssandK.Vasilatou,AerosolScienceandTechnology(2019)

Dilu'on Photo-oxida'onreactor

Coated-sootpar'cles

Aethalometer,PAX(870nm)

TEOM

DiffusionSizeClassifier(minidisc)

EC/OCANALYSIS,Chromatography

Par*clesize&numberconcentra*on

Es*ma*onofcoa*ngthickness

Op*calproper*es

Massconcentra*on

Lung-depositedsurfacearea

Chemistry

1:10

Projectgoalsandexperimentalsetup(II)

54 × 49 × 48 cm (WDH), 30.5 kg

(Nano-)Aerosol Chamber for In-Vitro Toxicology, NACIVT (www.nacivt.ch)

Jeannet N. et al., Nanotoxicology , 2015; Geiser M. et al., Nanomaterials, 2017

Ø  “All-in-one”,mobilesystemfordirectuseatanypar'clesource

Ø  Mimicspar'cledeposi'oninlungs(T,RH,gascomposi'on,airflow,NP,Ndep)

Ø  Simultaneousexposureof24cellcultures

Ø  Controlled&stablecondi'onsallowlong-termexposures

Aerosol(externalproduc'on/source)

Aerosolcondi'oning(T;RH)

Aerosoldistribu'ontodeliverytubes

Par'cledeposi'onon24cellcultures

Customelectronics(aerosolcharger,integratedpump,

flowcontrol)

Objec'veandexperimentalsetup(III)The three experimental campaigns took place at the Federal Institute of Metrology (METAS)

1st: 13-16/08/2018 (TMB) 2nd: 03-17/12/2018 (TMB) 3rd: 13-24/05/2019 (α-pinene)

The equipment of University of Bern transferred to METAS:

•  NACIVT chamber •  Cell culture lab (e.g. sterile bench, cell incubator) •  Light microscope

Exposureofrealis*ccellmodelstowell-controlled,chemicallydefinedsynthe*creferenceaerosols,toiden*fytheaerosolcomponents/proper*esresponsibleforadverserespiratoryeffects.

TEOM

EC/OC

PAX(870nm)AE33

IC

MSC

Mini-CAST

CellcultureequipmentNACIVTchamber

Par'cledeliverystrategy

EC/TC(%)99543126Size(nm)9188103122

Cytotoxicitydependsonorganiccoa?ng(experimentsrepeatedwithaddi%onalcoa%ngthickness)

EC(sootcore)ànotcytotoxic

Firstteststoevaluatecoa*ngeffectsoncytotoxicity

4h 24h

Exposure time (1h)

Par'clenumberconcentra'onremainsthesamebetween30nmand90nminallthreecampaigns:30nm∼4x105NP/cm3

90nm∼1.3x105NP/cm3

1stand2ndcampaign:TMBasSOA-precursor(I)

Ø  Nosignificantdifferenceintermsofcytotoxicitybetweenincubatorcontrol(I.C),p-freeairandsynthe?cairexposedcells

Ø  Sootpar%clesconsis%ngonlyofEC(90nmUnCoatedsoot)causenocytotoxiceffects

Ø  Cytotoxicityincreaseswithincreasingcoa%ngthickness.Max-Coat90nminducesthehighestcytotoxicity,i.e.releaseofLDH(mean23.35%)

0 30 60 90 120 1500

10

20

30

40

CorrelationSize/Cytotoxicity

R2= 0.6061p-value= 0.0134

Size (nm)

Cyt

otox

icity

(%) L

DH

rele

ase

I.C

Synth.ai

r

p-free

air

UnCoat

Min-Coat

Med-C

oat

Max-C

oat

UnCoat

Min-Coat

Med-C

oat

Max-C

oat0

10

20

30

40

Ctrl Soot 90 nm Soot 30 nm

****

**

LDH

rele

ase

(%)

Mini-CASTSoot90nm

Mini-CASTSoot30nm

Ctrl

3rdcampaign:α-pineneasSOA-precursor

Ø  Cytotoxicityincreaseswithcoa%ngthicknessasinthecasewithTMBasSOA-precursor

Ø  General,slightlylowercytotoxiceffectwithα-pineneasprecursorcomparedtoTMB

0 30 60 90 120 1500

10

20

30

40

50

CorrelationSize/Cytotoxicity

R2= 0.5116p-value= 0.0303

Size (nm)

Cyt

otox

icity

(%) L

DH

rele

ase

I.C

Synth.ai

r

p-free

air

UnCoat

Min-Coat

Med-C

oat

Max-C

oat

UnCoat

Min-Coat

Med-C

oat

Max-C

oat0

10

20

30

40

Ctrl Soot 90 nm Soot 30 nm

**** *

* **

LDH

rele

ase

(%)

Mini-CASTSoot90nm

Mini-CASTSoot30nm

Ctrl

Ongoinganalysis:Secretome102cytokinesanalyzed

Outof102cytokinesandchemokinesanalyzed,35aredown-orup-regulatedincomparisontoincubatorcontrol

Adiponectin

Angiogenin

CD40 lig

and

Cystat

in CDkk

-1EGF

EMMPRIN

ENA-78

G-CSF

GDF-15

GM-CSF

GRO-alpha

ICAM-1

IGFBP-2

IGFBP-3

IL-1raIL-4IL-6IL-8

IL-11

IL-17AIL-23IL-24

Lipocalin

-2

MCP-1MIF

MIP-3alpha

MMP-9

Myelopero

xidas

e

Osteopontin

PDGF-AA

Serpin E1

SHBGuPAR

VEGF

p-free air

UnCoat

Min-Coat

Med-Coat

Max-Coat

Fold

cha

nge

rela

tive

to In

c. C

trl

5

10

15

20

25

30Soot90nm

Adiponectin

Angiogenin

CD40 lig

and

Cystat

in CDkk

-1EGF

EMMPRIN

ENA-78

G-CSF

GDF-15

GM-CSF

GRO-alpha

ICAM-1

IGFBP-2

IGFBP-3

IL-1raIL-4IL-6IL-8

IL-11

IL-17AIL-23IL-24

Lipocalin

-2

MCP-1MIF

MIP-3alpha

MMP-9

Myelopero

xidas

e

Osteopontin

PDGF-AA

Serpin E1

SHBGuPAR

VEGF

p-free air

UnCoat

Min-Coat

Med-Coat

Max-Coat

Fold

cha

nge

rela

tive

to In

c. C

trl

0

5

10

15

20

25

30Soot30nm

1stand2ndcampaign:TMBasSOA-precursor

ConclusionandoutlookPilotstudyfundedbyFOEN

Ø  Sootpar'clesconsis'ngonlyofEC(90nmUnCoatedsoot)causenocytotoxiceffects.Ø  Cytotoxicityincreaseswithcoa*ngthicknesswithTMBandα-pineneasSOA-precursors.

Max-Coat90nminducesthehighestcytotoxicity.Ø  α-pineneasSOA-precursorcauseslightlylowercytotoxiceffectcomparedtoTMB.

Ongoingandfutureac'vi'es:Ø  Transmissionelectronmicroscopy(TEM)ofcellculturesec'onsandofpar'clessampledonTEMgrids.Ø  Secretomeanalysisforthe3rdcampaign

ThepilotstudyhasledtotheEUproject:EMPIRAeroTox(h?p://empir.npl.co.uk/aerotox/)Ø  Confocalmicroscopyofsamplesfrom2ndand3rdcampaignattheNa'onalPhysicalLaboratory(NPL,UK).Ø  Analysisofthedegreeofoxida'onoftheorganicma?erbyAerosolMassSpectrometry(NPL,UK).

Acknowledgments

Institute of Anatomy

Group Geiser

Ø  Prof. M. Geiser Ø  B. Kupferschmid

Former members: Ø  M. Delaval Ø  H. Jonsdottir

METAS & FHNW

Laboratory Particles and Aerosols

Ø  K. Vasilatou Ø  M. Ess

Laboratory Aerosol Measurement

Technology

Ø  A. Keller

BAFU

Air quality section Ø  Richard Ballaman

Traffic section

Ø  Giovanni D’Urbano

Projectfunding

AeroToxPartners

Ongoinganalysis:TEMinserts2ndEXPERIMENTALCAMPAIGN:TMBasSOA-precursor

Inc.Ctrl

p-free

air

Synth. a

ir0

5

10

15

20 ***ns

**

LDH

rele

ase

(%)

KS normality test

Passed normality test (alpha=0.05)? Yes Yes Yes P value summary ns ns ns D'Agostino & Pearson normality test Passed normality test (alpha=0.05)? Yes Yes Yes P value summary ns ns ns Shapiro-Wilk normality test Passed normality test (alpha=0.05)? Yes Yes Yes P value summary ns ns ns

3rdcampaign:α-pinene as SOA-precursor

0

10

20

30

40

Ctrl

I.C

Synt

. Air

Soot 90 nm

UnCoated

Exp

p-fr

ee Exp

p-fr

ee Exp

p-fr

ee Exp

p-fr

ee

Min-Coat Med-Coat Coated

Soot 30 nmEx

p

p-fr

ee Exp

p-fr

ee Exp

p-fr

ee Exp

p-fr

ee

UnCoated Min-Coat Med-Coat Coated

** * *** **** ns *** *

LDH

rele

ase

(%)

Cytotoxicityuponexposuretocoatedsootmuchhigherthanthatofincubatorcontrolandpar?cle-freeairtests->Theincreaseincytotoxicityisindeedcausedbycoatedsootpar%cles

1stand2ndcampaign:TMBasSOA-precursor(II)

Goodreproducibilitybetweenthe1stand2ndcampaign

0

10

20

30

40

Ctrl

I.C

Synt

h. A

ir

Soot 90 nm

UnCoated

Exp

p-fr

ee Exp

p-fr

ee Exp

p-fr

ee Exp

p-fr

ee

Min-Coat Med-Coat Max-Coated

Soot 30 nmEx

p

p-fr

ee Exp

p-fr

ee Exp

p-fr

ee Exp

p-fr

ee

1st Campaign

UnCoated Min-Coat Med-Coat Max-Coated

**ns ns *** ****** * ns ***

2nd Campaign

LDH

rele

ase

(%)

Current project EMPIR project

Model aerosols: internal mixture of soot core particles with coating from secondary organic matter Properties tested: uncoated vs. coated particles, role of soot core size, coating thickness and coating chemistry

Further tests to clarify the role of: - primary vs. secondary organic coating - secondary inorganic coating - internal metal admixtures -  externally mixed dust particles (e.g. from

brake abrasion) Biomedical analyses will be complemented with toxicogenomics and particle uptake studies

metals different coatings

Spring 2018 - Spring 2019 June 2019 - May 2022

Genera'onofagedsootaerosol

Oxida'onflowreactor

ozone,OH

UVClamp

UVClamp

0.0E+00

5.0E+04

1.0E+05

1.5E+05

2.0E+05

2.5E+05

3.0E+05

3.5E+05

4.0E+05

4.5E+05

10 100 1000

Num

bercon

centra*o

n[#/cm3]

Mobilitydiameter[nm]

0.00.10.20.30.40.50.60.70.80.9

90 110 130 150 170 190

SSA

(870

nm) [

-]

Mobilitydiameter[nm]

SSA= 𝑙𝑖𝑔ℎ𝑡 𝑠𝑐𝑎𝑡𝑡𝑒𝑟𝑖𝑛𝑔/𝑙𝑖𝑔ℎ𝑡 𝑒𝑥𝑡𝑖𝑛𝑐𝑡𝑖𝑜𝑛 

PrototypeminiCASTsootgeneratorModel5201BC

(JingLtd.,Bern,Switzerland)

0102030405060708090100

Par*clecompo

si*o

n[%

]

EC/TC[%]

OC/TC[%]

organicvaporconcentra*on(ppm)

Elementalcarbon(EC)

Organiccarbon(OC)