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Probing the Ligand Shell of Nanoparticles:
Opportunities and Limitations
Martin Volk
University of Liverpool
NPL Focus User Meeting: DCS
29 November 2016
Outlook
Gold Nanoparticles (5-40 nm)
ligand shell determination
dependence of shell thickness on
ligand length
capping density
protein (BSA) corona
Ligand Shells on Gold Nanoparticles
gold NPs: 10 – 40 nm diameter @ ligands: ~ 1 - 4 nm
a-helical
[Krpetic et al, ACS Nano 2013]
Ligand Shells – Differential Centrifugal Sedimentation
13.5nm Au-NP @ ligands: ~ 1 - 4 nm
DCS can distinguish
between different shells
larger ligands
smaller apparent NP size
(citrate)
(citrate)
Ligand Shells – Differential Centrifugal Sedimentation
sedimentation time:larger ligands
smaller apparent NP size
average density (dcore, s, rshell)
… because software assumes
shell (s) to have density of gold!
BUT:
software assumes reffective = rcore = 19.3 g/cm3
2)2)(( sd
Ct
e
corefluidffective rr
3
333
)2(
))2((
sd
dsdd
core
shellcorecorecorecoreeffective
rrr
ligand shell thickness
correct value MUCH smaller: rshell ~ 1.4 g/cm3 (small organic molecules)
ligand shell adds “drag”, but not much weight
slower sedimentation reported as smaller NP
Ligand Shells – Differential Centrifugal Sedimentation
simulated DCS results for dcore = 10 nm:
measured
“apparent”
NP size
shell
thicknessLimitations:
value of rshell ? (only for thicker shells)
measured
“apparent”
NP size
thicker shells cannot be measured
BUT:
requires knowledge
of precise core size
dcore!
TWO unknowns,
but only one
experimental value
DCS Calibration – CALNN as “internal ruler”
Peptide P1: CALNN
C A L N N
capping density: 2.4 peptides/nm2
density of protein interior < 1.4 g/cm3
s > 1.5 nm
extended backbone: s < 1.7 nm
DCS Calibration – CALNN as “internal ruler”
Peptide P1: CALNN
C A L N N
capping density: 2.4 peptides/nm2
density of protein interior < 1.4 g/cm3
s > 1.5 nm
extended backbone: s < 1.7 nm
s (P1) ~ 1.6 nm
gold NP core diameter
shell thickness for other layers
(also supported by
IR and MD data)
Ligand Shells – PEG
DCS-Results
P1 citrate PEG1 PEG2 PEG3 PEG4 PEG5
s/nm 1.6 1.00 1.15 1.30 1.45 2.00 2.15
(independent of core size)generic calibration !
Ligand Shells – Capping Density
CALNN ligand exchange: [CALNN] = 0.2 mM 2.4 peptides/nm2
0.01 mM 1.7 peptides/nm2
Duchesne et al, Langmuir 2008, 24, 13572
FTIR:
high capping density forces
peptide into extended conformation
1643 cm-1 extended
~1650 cm-1: random coil
Ligand Shells – Capping Density
Peptide P1: CALNN
DCS:
Layer thickness
1.7 peptides/nm2: 1.47 nm
2.4 peptides/nm2: 1.6 nm
layer thickness NOT
is proportional to
capping density /
amount of peptide on NP
Ligand Shells – Capping Density
Peptide P1: CALNN - Molecular Dynamics Simulations
(10 nm NP in explicit solvent)
DCS correctly reports reduced ligand shell
(effective) size for lower capping density
2.4 peptides/nm21.7 peptides/nm2
low capping
density layer is
less compact !
BSA-Corona on Au-NPs
Protein Corona
Walczyk et al, JACS 2010, 132, 5761
physisorbed
(“soft” corona)
chemisorbed
(“hard” corona)
BSA-Corona on Au-NPs
Incubation of Au-NPs (11 nm) in Bovine Serum Albumin (over night)
corona formation on
citrate-NPs
corona formation on
PEG-COOH ligand layer
PEG: HS-(CH2)11-(EG)6-
BSA-Corona on Au-NPs
Density of Protein Corona
must be < 1.25 g/cm3
assume 1.1-1.2 g/cm3
(density of protein crystals)
BSA
BSA-Corona on Citrate Au-NPs
Bovine Serum Albumin on citrate-stabilized NPs (11 nm)
no BSA in DCS-gradient
protein detaches as
it enters gradient ?
BSA in DCS-gradient
corona in protein solution
DCS allows study of chemi- and physisorbed corona !
BSA-Corona on Citrate Au-NPs
Chemisorbed Bovine Serum Albumin on citrate-stabilized NPs (11 nm)
BSA-Corona on Citrate Au-NPs
Chemisorbed Bovine Serum Albumin on citrate-stabilized NPs (11 nm)
very rapid formation of chemisorbed corona !
BSA-Corona on Citrate Au-NPs
Chemisorbed Bovine Serum Albumin on citrate-stabilized NPs (11 nm)
smax = 5.9 nm
Dissociation constant
KD = 6.0 mM
Hill coefficient
n = 0.55
anti-cooperative binding
nn
n
KN
N
D[BSA
BSA
]
][
max
Hill-model for adsorption
as more proteins bind, chemisorption gets hindered
BSA-Corona on Citrate Au-NPs
Chemisorbed Bovine Serum Albumin on citrate-stabilized NPs (11 nm)
maximum corona thickness: smax = 5.9 nm
3.15 nm
7.3 nm
single cystein
Au-S bond?
[Röcker et al, Nat Nanotechn. 2009]
BSA-Corona on Citrate Au-NPs
Physisorbed Bovine Serum Albumin on citrate-stabilized NPs (11 nm)
physisorbed corona
shows steeper isotherm
more cooperativity?
BSA-Corona on Au-NP@PEG-COOH
Bovine Serum Albumin on 11nm Au-NPs@PEG-COOH
rPEG = 1.4 g/cm3
rBSA = 1.15 g/cm3
citrate-NPs
BSA-Corona on Au-NP@PEG-COOH
Bovine Serum Albumin on 11nm Au-NPs@PEG-COOH
vs. citrate NPs
BSA corona on
NP@PEG-COOH
close to the situation
where DCS not useful!
NP@PEG-COOH
Summary
DCS for measurement of ligand shell thickness
requires “internal ruler”
Capping layers formed by small molecules
PEG ligands of different length
peptide capping density
Protein Corona
DCS allows investigation of chemisorbed AND
physisorbed corona
BSA corona on citrate-NPs forms rapidly, with anti-
cooperative binding via the only free cysteine
BSA corona on NP@PEG-COOH less well definded
no corona (physi- or chemisorbed) on NP@PEG-OH
Acknowledgements
Ligand Shell Thickness Determination
Zeljka Krpetic
A. Michael Davidson
Mathias Brust, Raphael Levy, David L. Cooper
MD-Simulations
Elena Colangelo
Raphael Levy
David Paramelle, Qiubo Chen, Michael Sullivan (Singapore)
Protein Corona
A. Michael Davidson
Mathias Brust
European Research Council, EPSRC, University of Liverpool
