INTERACTIONS OF AgNPs WITH BIOLIGANDS: DIRECT QUANTIFICATION OF

DISSOLUTION AND AGGREGATION USING AF4-UVD-MALLS-ICP-MS

I.A.M Worms1,2, W. Liu3, A.Arnould2, F. Rollin-Genetet4, C. Vidaud3, R. Soulas2, J-F. Damlencourt2, S. Motellier2, E. Mintz1, I. Michaud-Soret1, D. Truffier-Boutry2

(1) CEA – Grenoble, LCBM / BioMet UMR5249 CEA/CNRS/UGA, 17, rue des martyrs 38000 Grenoble / France (2) CEA – Grenoble, DRT/LITEN/SEN/LR2N, 17, rue des martyrs 38000 Grenoble / France (3) CEA – Marcoule, DRF/BIAM, 30207 Bagnols-sur-Cèze / France (4) CEA –

Grenoble, DRF/BIG, 17, rue des martyrs 38000 Grenoble

AF4 –UVD-MALLS-ICP-MS (Wyatt/Agilent)From fractionation to detection

Chanel cartoons adapted from E. Alasonati (PhD EPFL, 2009)

size = f(tR)

1/

2/ Direct size determination

3/

AgNP

AgNP

AgNP

Depends on:pH, I, T, Coating agentsAgNPs sizeNature of oxidant (e.g. O2,H2O2…)Light ….

Depends on:pH, I, T, MZ+

Surface chargesCoating agents ….

Oxidative

Dissolution

Behavior of AgNPsin aqueous media

Aggregation

Ag+

AgNP

Size reduction GlobaleSize increase

AF4 –UVD-MALLS-ICP-MS can point-out DISSOLUTION and AGGREGATION

size = f(tR)

1/ Lower tR

2/ Smaller Rg3/ Lower nanoparticulate Ag

1/Higher tR

2/Bigger Rg

3 potential descriptors for dissolution2 potential descriptors for aggregation

1/

2/ Direct size determination

3/

Behavior of AgNPsin the presence of organothiols (L-SH)

AgNP

Promoted

DissolutionPromoted

Aggregation

Depends on [L-SH] Organothiol oxidation status

L-SH (red) vs. L-SS-L (ox)

Size reductionGlobaleSize increase

Coating exchange

Original coatingCitrate

L-Cysteine reduced(Cys)

Glutathione reduced(GSH)

D-Penicillamine reduced(DPen)

Glutathione oxidized(GSSG)

Cit-

AgNPs

20nmBiopure

Nanocomposix

HEPES Buffer 0.01MpH = cte = 7.1T=23°CAbsence of light400 rpm during 24H[Ag]TOT = 0.3mM

AgNPs and organothiols used

[L-SH]TOT=cte= [Ag]TOT

1/ Changes in retention times

ICP-MS107Ag

Fractograms (NaOH pH10= optimized for Cit-AgNPs)

Dissolution vs tR : Change in hydrodynamism (occurs at t=0H) =not reliable

Aggregation vs tR : Peak enlargements to higher tR

2/ Direct sizing by MALLS

ICP-MS107Ag

400nm40nm

MALLSdg (nm)

When coated by GSSG,AgNPs formed aggregatesranging dg = 40 -400nm

Dissolution using MALLS : Interferences due to SPR (backscattering)

3/Quantification of nanoparticulate Ag

Changes in coating (LS-AgNPs/Cit -AgNPs)= changes in AgNPs surface properties

= changes in hydrodynamism

Unreliability of QTAg = f(CPS)

Limiting artefacts

Mass transport efficiency

AgNPs Recovery

Step 3.1/ Recovery of LS-AgNPsRecovery=

QT AgNPs detected/QT AgNPs injected

R (%)=

R (%)

Cit-AgNPs 80

GSH-AgNPs 85

DPen-AgNPs 83

Cys-AgNPs 60*

GSSG-AgNPs 90

Area frato

Area Dir Inj

UVDl=402nm

1/injection Focus flow

Determines if artefacts arise during the injection step

Step 3.2/ Mass transport efficiency of LS-AgNPsICP-MS

107AgAgNPs transport efficiency from the channel to the ICP-MS

When GSH is added the 107Ag CPS detected by ICP-MS is higher than forother AgNPs

Mass transport efficiency

Step 3.3 / AgNPs Quantification

Eluted AgNPs(ng Ag)

Cit-AgNPs 35DPen-AgNPs 34Cys-AgNPs 29GSH-AgNPs 36

GSSG-AgNPs 46

a/ CPS to ppbconversion

Qt AgNPs (ng Ag)Cit-AgNPs 44.4

DPen-AgNPs 41.9Cys-AgNPs 47.0GSH-AgNPs 43.6

GSSG-AgNPs 51.2

c/ R % (from step 3.1)

b/ Peaksintegration

Raw fractograms

Quantification of AgNPs Dissolutionin the presence of organothiols

Without any correction()of ICP-MS fractograms

Leads mainly to overestimationOf dissolution process

Assessment of AgNPs and aggregates sizes using Cryo-TEM

Cit-AgNPsGSH-AgNPs

DPen-AgNPs

GSSG-AgNPs

Assessment of AgNPs and aggregates sizes using Cryo-TEM

Cit-AgNPsGSH-AgNPs

DPen-AgNPs

GSSG-AgNPs

suspension is heterogeneous in size with well dispersed aggregates (40-200nm) and some isolated AgNPs

suspensions are well dispersed and formed of isolated AgNPs

Assessment of AgNPs and aggregates sizes using Cryo-TEM

Cit-AgNPsGSH-AgNPs

DPen-AgNPs

GSSG-AgNPs

No clear changes in AgNPs primary size distributions(+/-) 5nm

suspension is heterogeneous in size with well dispersed aggregates (40-200nm) and some isolated AgNPs

suspensions are well dispersed and formed of isolated AgNPs

Quantification of AgNPs Dissolutionin the presence of organothiols

1/ No clear promoted dissolution/ Cit-AgNPsUnder our conditions

2/ 20% max dissolution(AF4-ICP-MS) 2-3 nm shift in average sizeNo change in size distribution is expected!

3/ GSSG aggregates stabilized AgNPs

DPen-AgNPs GSH-AgNPsCit-AgNPs

GSSG-AgNPs

Interactions of AgNPs with Proteins

Promoted

Dissolution

Promoted

Aggregation

Depends on Protein surface charge, size = Stability of the coronaand biochemical properties

GlobaleSize increase

Corona formation

AgNP

Size reduction

AgNP

AgNP

AgNP

AgNP

Changesin metal loading

Interactions of AgNPs with 2 metalloproteinsPublication coming soon!

Promoted

Dissolution

Promoted

Aggregation

Corona formation

AgNP

AgNP

AgNP

AgNP

AgNP

Changesin metal loading

Can be quantified by AF4- UVD-MALLS-ICP-MS

ICP-MS107Ag

ICP-MSAg, Cu, Zn, Cd

UVDl=280nm MALLS

dg (nm)

Interactions of AgNPs with 2 metalloproteinsPublication coming soon!

Promoted

Dissolution

Promoted

Aggregation

Corona formation

AgNP

AgNP

AgNP

AgNP

AgNP

Changesin metal loading

Can be quantified by AF4- UVD-MALLS-ICP-MSAnd measured by (Cryo)-TEM

ICP-MS107Ag

ICP-MSAg, Cu, Zn, Cd

UVDl=280nm MALLS

dg (nm)

Prot-AgNPs (2H)

Prot-AgNPs (24H)

CONCLUSIONS

AF4-UVD-MALLS-ICP-MS

- Information obtained from raw fractograms must not be over interpreted!- Take the time to make global corrections (recovery and ICP-MS calibrations)

- BOTH aggregation and/or dissolution (even lower than 20%) can be quantified in the presence of bioligands

1/ tR 2/ MALLS 3/ ICP-MS

Dissolution NO(hydrodyn)

NO (SPR) YES*

Aggregation YES YES -

Both - - YES*

CORONAformation

YES - YES (recovery)

*Discrimination betweenstabilizing (GSSG)

ordissolving conditions (all other coatings)

CONCLUSIONS

Cryo-TEM analysis

1/ AgNPs size 2/ AgNPsaggregates sizes

Dissolution NO**

Aggregation - YES

Both YES

Need expertises but « less » time-consuming at the end.

**Need dissolution > 20% to reach significant changes in size distribution(start 23nm D2nm) Increase number of AgNPs analyzed to decrease the PDI of size distribution

CONCLUSIONS

Cryo-TEM analysis

Need expertises but « less » time-consuming at the end. The structure of aggregates can be followed within time…

1/ AgNPs size 2/ AgNPsaggregates sizes

Dissolution NO**

Aggregation _ YES

Both YES

**Need dissolution > 20% to reach significant changes in size distribution(start 23nm D2nm) Increase number of AgNPs analyzed to decrease the PDI of size distribution= increase the quantity of material to be analyzed

THANKS FOR YOUR ATTENTION

Postdoctoral fellowship

DRF/BIG/CBM/BioMetMarianne MarchioniThomas GallonAurélien DeniaudMireille Chevallet……BioMet team

DRT/LITEN/SEN/LR2NChristophe BrouardArnaud GuiotDominique Locatelli

Assessment of AgNPs and aggregates sizes using Cryo-TEM

Directobservation

AgNPs aggregated

(S)TEM OSIRIS (FEI)

1/Sample 3µL direct deposition on grid

3/Cryo-transferin N2(liq)

VITROBOT (FEI)

Cryo N2(liq)observation

AgNPs dispersedand isolated

« Snap-shot »

2/Fast vitrification processin Ethane(liq)

1/Sample 3µL direct deposition on grid

2/Dry at room T°C

Assessment of AgNPs and aggregates sizes using Cryo-TEM

AgNPs dispersedand isolated

1/ AgNPs diameters= dissolution assessmentby shift in the size distribution

Dispersedaggregates

1/AgNPs diameters= intra-aggregatesdissolution assessmentSmall to large aggregates

1/Direct sizing of aggregates