Post on 06-Oct-2021
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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