Dissolution of Silver nanoparticles and fate of the released silver ions in hepatocytes

revealed by a synchrotron nanoprobe

Giulia Veronesi1,2, Aurélien Deniaud1, Thomas Gallon1,3, Pierre-Henri Jouneau4, Julie Villanova2, Pascale Delangle3, Marie Carrière3, Isabelle

Kieffer5,6, Peggy Charbonnier1, Elisabeth Mintz1, Isabelle Michaud-Soret1

(1) CNRS/CEA/Univ. Grenoble Alpes LCBM UMR 5249. 17, Av. des Martyrs, F-38054 Grenoble, France(2) ESRF, The European Synchrotron. 71, Avenue des Martyrs, 38043 Grenoble, France.

(3) CEA/Université Grenoble Alpes, INAC-LCIB, F-38054 Grenoble. (4) CEA/Université Grenoble Alpes, INAC-MEM, Grenoble, France.

(5) BM30B/FAME beamline, ESRF and Observatoire des Sciences de l’Univers de Grenoble, UMS 832 CNRS Université Joseph Fourier, F-38041 Grenoble, France.

Nanosafe 2016giulia.veronesi@cea.fr

Nanosafe 2016giulia.veronesi@cea.fr

Silver Nanoparticles (AgNPs)

Gliga et al. P&FT (2014)

Wilson Center (USA)www.nanotechproject.org/cpi

442 products contain AgNPBlueMoonGoods™ Fresh Box Silver Nanoparticle Food Storage Containers

CYTOTOXIC EFFECT:• AgNPs enter cells• Release of toxic Ag(I)• Correlated to in vitro dissolution

BACTERICIDAL EFFECT:Release of toxic Ag(I) in the vicinity of bacteria

Xiu et al. NanoLetters (2012)

Liu et al. ACS Nano (2012)Cell

AgNP

Nucleus

Ag+

Ag+

Ag+

Ag+

Ag+ Ag+

Ag+

Intracellular dissolutionAg(I) trafficking

?

Hepatocytes(liver target organ)

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Synchrotron Nanoprobe

ID16B @ESRFResolution: 50×50 nm2

• X-ray beam focussed to sub-micrometric size

• Multi-analytical platforms

P vs S

Collaboration with Anna Bencsik, ANSES, Lyon

• Hyperspectral imaging (1 XRF spectrum/pixel)

• Provides elemental distributionsID21 @ESRF. Resolution: 500×500 nm2

X-Ray Fluorescence (XRF) imaging

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Visualization and quantification of Ag(I)

500 nm

AgNP-

citrate

24h

18

0

1.2

0

10-3

10-3

10-2

10-1

101

100

10-1

10-2

100

10-1

10-2

𝜇𝑔

𝑐𝑚2

Optical Ag linear Ag log

Control

AgNO3

30 min

AgNP-

PVP 24h

0.009

0

0.14

0

Scale bars = 5 μm

70x70 nm2 resolution

CytotoxicityIntracellular dissolutionin HepG2 hepatocytes

PVP

CitrateAgNO3

Correlation between intracellular dissolution

and cytotoxicity

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TEM observations

Citrate

PVP

5 µm

Ag in HepG2

XRF

Ag+

Vesicles

Cytosol

Ag0

?

NanoXRF + TEM reveal that AgNPs dissolve in endocytic vesicles and release Ag in cytosol

Chemistry of released Ag(I) ?

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X-ray Absorption Spectroscopy (XAS)

XANES: X-ray Absorption Near-Edge Structure

EXAFS: Extended X-ray Absorption Fine Structure

@ FAME-BM30BESRF

XANES analysis: linear combination of spectra of reference compounds

• Measure fraction of dissolved Ag• Retrieve Ag(I) species

EXAFS analysis: Ab initio fitting of spectra

• Characterize Ag(I) coordination sphere (geometry and bond lengths)

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Measuring dissolution rates in cellulo

AgGSH fraction

15 %

21 %

21 %

46 %Linear combination fitting of XANES

• Dissolution is faster for citrate-than for PVP-coated AgNPs

• Ag-S species are formed

• Expose cell culture to AgNP• Wash and centrifuge• Recover cell pellet (~107 cells)• Resuspend, pour a drop on XAS

sample holder• Freeze in liquid N2

• Acquire XAS spectra

Experimental protocol

Disclosing Ag(I) coordination in cellulo

Circumvent the problem of separation

𝜒 𝑘 = 𝑛 ∙ + (1 − 𝑛) ∙

Undissolved NP Dissolved fraction

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Use of a model Measure Ag-S distance in cellulo

Veronesi et al., Nanoscale 2015, 7, 7323.

ExpFit

Ag(I) binding to biological Cu(I) sites

Correlation betweencoordination number and

Ag-S bond length

2.0 2.5 3.02.38

2.40

2.42

2.44

2.46

2.48

2.50

Ag(I)-rMT

Ag(I)-yMT

Ag(I)-Atox1

AgGSH

Ag1.5P2

Ag1P2

Ag2L1

Ag-S

bond length

(Å

)

Ag(I) coordination number

Ag1L1

Veronesi et al. Inorg. Chem. 2015, 54, 11688.

• XAS study of Ag(I) complexation in biological Cu(I)-thiolate sites.

• Solution, pH 7.4

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L1

P2

GSH

Metallothioneins

Atox1

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Disclosing Ag(I) species formed in cellulo

Ag-S = 2.45 Å Mixed AgS2/AgS3

mRNA expression

Overexpression of metallothionein 1 (MT1) and glutamate-cysteine ligase (GCLM)

Ag(I) binds to GSH and Metallothionein

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Conclusions

Veronesi et al, Nanoscale 2016, 8, 17012

5 µm

Ag in HepG2

XRF

Ag+

Vesicles

Cytosol

Ag0

• Visualization and quantification of Ag released from AgNPs in hepatocytes

• Influence of the coating on intracellular dissolution

• Ag(I) binding in Cu(I) sites perturbation of metal homeostasis

Synergistic use of advanced imaging, atomic spectroscopy, and molecular biology

FATE of metallic NPs in cells

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Acknowledgments

Thank you for your attention!

INAC/LCIB CEA-Grenoble:Marie Carrière,Pascale Delangle, Bastien Boff, Christelle Gateau, Colette Lebrun

iBEB/SBTN CEA-Marcoule:Claude Vidaud, Françoise Rollin-Genetet

ESRF:FAME: Isabelle Kieffer, Olivier ProuxID16B: Julie Villanova, Sylvain Bohic, Remi Tucoulou, Vanessa SuárezID21: Marine Cotte, Murielle Salomé,

Hiram Castillo-Michel

LCBM CEA-Grenoble:BioMet: Aurélien Deniaud, Thomas Gallon, Peggy Charbonnier, Elisabeth Mintz, Isabelle Michaud-SoretMireille Chevallet, Isabelle WormsMarianne Marchioni.MCT: Serge Crouzy, Yohann MoreauProMD: Catherine Aude-Garcia, Thierry Rabilloud

LabEx SERENADE

IBS/MEM CEA-Grenoble:Benoit Gallet, Christine Moriscot