Gd-functionalised Au nanoparticles as targeted contrast agents in MRI: Relaxivity enhancement by polyelectrolyte coating

Article abstract of DOI:10.1039/b915223g

Monolayer-protected, Gd³⁺-functionalised gold nanoparticles with enhanced spin-lattice relaxivity (r₁) were prepared; adsorption of polyelectrolytes on these materials further increased r₁ and ligand exchange with a biotin-derivatised disulfide led to a prototype avidin-targeted contrast agent.

Full text of DOI:10.1039/b915223g

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Gd-functionalised Au nanoparticles as targeted contrast agents
in MRI: relaxivity enhancement by polyelectrolyte coatingw
Muhammad Farooq Warsi, Ralph W. Adams, Simon B. Duckett and Victor Chechik*
Received (in Cambridge, UK) 27th July 2009, Accepted 13th November 2009
First published as an Advance Article on the web 2nd December 2009
DOI: 10.1039/b915223g
3
+
Monolayer-protected, Gd -functionalised gold nanoparticles
with enhanced spin–lattice relaxivity (r ) were prepared; adsorption
of polyelectrolytes on these materials further increased r and
they will distribute extravascularly. This could be exploited for
extravascular delivery/imaging; however nonspecific binding
or entrapment could also reduce contrast. Thiol-protected
AuNPs are easy to synthesise and functionalise, thanks to the
1
1
ligand exchange with a biotin-derivatised disulfide led to a
prototype avidin-targeted contrast agent.
7
resistance of Au to oxidation and the affinity of thiols for Au.
Roux et al. pioneered the use of Gd chelate-functionalised
8
AuNPs in MRI. The Gd chelates formed a multilayered
Magnetic resonance imaging (MRI) is an important non-
invasive diagnostic tool that provides information about
biological objects/processes on a molecular level. The inherently
low sensitivity of MRI can be improved by using paramagnetic
compounds (contrast agents) which enhance the relaxivity of
structure on the surface of an Au nanoparticle. Presumably,
the multilayer formation is due to the incorporation of two
thiol groups (for synthetic convenience) into the structure of
the chelating ligand. Nonetheless, a 30% increase in the
1
water. Most contrast agents currently approved for clinical
spin–lattice relaxivity per Gd(III) ion (r
1
) was observed for
of a
use are small molecule Gd(III) chelates. Although these
compounds are efficient proton relaxers, the potential toxicity
of Gd(III) drives the need for further improvements, which will
make it possible to reduce the amount of the contrast agent
Gd-functionalised nanoparticles as compared to r
1
commercially available small molecule analogue, Gd(III) complex
with diethylenetriaminepentaacetic acid (Gd–DTPA). Similar
results were obtained with Au nanoparticles coated with a
2a
9
needed.
DTPA ligand functionalised with two glutathione molecules.
The relaxivity of contrast agents can be enhanced by
reducing their tumbling rate in solution, thus preventing
averaging of electron spin–nuclear spin dipole–dipole inter-
actions, an important relaxation mechanism. Reduction in the
tumbling rate can be achieved by increasing the molecular size
Chung and co-workers used a different approach to prepare
1
0
Gd-functionalised multifunctional Au nanoparticles. The
DTPA ligand was conjugated to an antibody which was
subsequently reacted with G-protein coated Au nanoparticles.
The clear advantages of well-developed bioconjugation
techniques for the preparation of this type of material are
offset by the difficult characterisation and lack of control of
the Gd density on the Au surface. Recently, Gd-functionalised
2
b
of the contrast agent. These considerations led to the
development of contrast agents based on chelated Gd ions
bound to a nanostructure, e.g. dendrimer-attached Gd
3
4a
11
chelates or Gd-loaded mesoporous silica nanospheres.
ligands were introduced into sugar-modified nanoparticles.
Significantly increased relaxivity was observed in several
4
nanostructure-based systems.
These mixed-ligand materials showed high relaxivity which
was strongly dependent on the nature of the sugar ligand;
however, they suffered from relatively low Gd loading. Very
Apart from reduced tumbling rates, nanoparticle-based
contrast agents offer other advantages. Arguably the most
important feature of these materials is multivalency which
makes it possible to attach several functionalities to the same
nanostructure, e.g., for multimodal detection or targeted
delivery. For instance, Mou et al. have developed Gd-loaded
1
2
recently, Moriggi et al. reported the frequency dependence of
relaxivity of Gd-functionalised AuNPs; maximum relaxivity
was observed at 30 MHz.
Here, we report on the preparation of Au nanoparticles
protected by a monolayer of a Gd chelate. The monolayer
protection (as opposed to multilayer) gives rise to nano-
particles which are structurally well defined, and hence
suitable for probing the structure–relaxivity relationship.
Additionally, monolayer coverage is better suited for preparing
multifunctional nanoparticles by co-adsorption of different
ligands on the surface of the same nanoparticle. We demonstrate
the feasibility of such post-functionalisation of nanoparticles
for targeted delivery with a biotin-modified ligand.
multifunctional fluorescent mesoporous silica nanorods
5
suitable for both fluorescence and T or T MRI imaging.
1 2
Baker and co-workers recently reported Gd-loaded dendrimer
nanoparticles targeted for tumor specific magnetic resonance
6
enhancement.
Au nanoparticles (AuNPs) provide an attractive template
for the development of efficient, multifunctional MRI contrast
agents. Although particles below 10 nm in diameter are
expected to have longer vascular half-life than small molecules,
The chelator ligand used in this work was based on a DTPA
ligand commonly employed for making Gd chelates for MRI.
The ligand was synthesised as a disulfide rather than a thiol for
synthetic convenience. The experimental details for the syn-
thesis of new DTPA-based ligand are available in the ESI.w Au
nanoparticles 1 were prepared by a simple reduction of an
Au(III) salt with sodium borohydride in the presence of the
Department of Chemistry, University of York, Heslington, York,
UK YO10 5DD. E-mail: vc4@york.ac.uk
w Electronic supplementary information (ESI) available: Synthesis,
characterisation and relaxivity measurements. See DOI: 10.1039/
b915223g
This journal is ꢀc The Royal Society of Chemistry 2010
Chem. Commun., 2010, 46, 451–453 | 451

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Fig. 1 (a) T
relaxivity (r
1
-weighted images of Gd@AuNPs. (b) Spin–lattice
1
) of Gd–DTPA complex (dotted line), Gd@AuNPs
dashed line), PEI-protected Gd@AuNPs (dotted dashed line), and
(
3
GdCl (solid line).
Gd-loaded AuNPs with different concentrations up to
+
3
0
.25 mM (per Gd
ion). Fig. 1a clearly shows the contrast
enhancement with increased nanoparticles concentration, thus
confirming that these materials can be used for increasing
Scheme
1
Gold nanoparticles (AuNPs) stabilised by a new
DTPA-based ligand (1), and Gd-functionalised AuNPs (2).
spin–lattice relaxation rate (1/T ) of surrounding water protons.
1
In order to obtain quantitative information about the
efficiency of Gd-loaded AuNPs as contrast agents, we determined
chelator ligand (Scheme 1). Unfortunately, the nanoparticles
could not be separated from the excess of unbound ligand
1
by dialysis as evident from H NMR spectra; however,
the molar spin–lattice relaxivity (r ) at 7.0 T using inversion
1
1
recovery method (details for determining r by inversion
purification using gel permeation chromatography afforded
pure nanoparticles free from the unbound ligand.
recovery method and inversion recovery curves for
Gd@AuNPs are available in the ESIw). This increase in
The monolayer-protected AuNPs were soluble in water and
quite stable over pH range from 5.5 to 12. They could be
evaporated to dryness and re-dissolved in water without any
physical or chemical change. The nanoparticles were characterised
1
relaxivity (r ) is most likely due to reduced tumbling of Gd
chelate attached to gold nanoparticles. However the increase is
still quite modest suggesting that the nanoparticle-attached
ligands possess fairly high mobility. This is somewhat
unexpected as dynamic studies of alkanethiol-protected Au
nanoparticles showed significant restriction of rotational
1
by UV-Vis, NMR, TEM, TGA and ICP (ESIw). H NMR spectra
of nanoparticles (Fig. S1, see the ESIw) showed broad peaks at
chemical shifts similar to those of free ligand. The CH S peak was
1
4
diffusion; it is likely that the branched structure of the
chelating ligands prevents good packing on the nanoparticle
surface and thus allows for relatively fast tumbling.
2
not observed which confirmed the absence of the free ligand. TEM
images showed spherical nanoparticles with average diameter
ca. 1.75 Æ 0.42 nm. TGA data showed that nanoparticles lose
ca. 42% of their weight in the 200–400 1C interval.
In order to further decrease the rotational diffusion of
nanoparticle-bound Gd (and hence increase relaxivity), we
have reacted the Gd-loaded AuNPs with poly(ethylene imine)
(PEI). Gd-DTPA complex has a negative charge (À2), and
nanoparticles 1 therefore have significant overall charge.
Polyelectrolytes have previously been reported to self-assemble
Treatment of nanoparticles 1 with GdCl
3
led to the
formation of Gd chelates attached to the nanoparticle surface
3+
Scheme 1). During addition of Gd , the pH of the medium
(
was maintained at 7.5 with 0.01 M NaOH. The amount of
3+
unbound Gd was monitored by UV-Vis spectroscopy using
15
on the surface of charged Au nanoparticles; this process
xylenol orange (a weak lanthanide chelating agent) as an
(driven by electrostatic interactions) is akin to the layer-by-
1
3
indicator. After complete loading, the nanoparticles were
layer assembly used for the preparation of polymer multilayers
16
on charged substrates or colloidal particles. We hoped that
3
+
purified from unbound Gd by dialysis and characterised by
ICP. The Gd-loaded AuNPs 2 showed similar stability and
solubility properties to the Gd-free nanoparticles and did not
adsorption of a single polymer layer on the Gd-loaded AuNPs
will slow down molecular tumbling without adversely affecting
the diffusion of water molecules. To prove the general
applicability of this approach, similar experiments were
carried out using less toxic PAMAM dendrimers as poly-
electrolytes. Polyamines were thus reacted with the nano-
particles using 4 : 1 amine : Gd stoichiometry to enable charge
reversal on the surface. The r1 of PEI-coated-Gd-loaded
3
+
leach any Gd over prolonged (for several months) storage in
solution (as determined by titrations with xylenol orange).
8
This is a significant improvement over previously reported
multilayer-coated Au nanoparticles.
The suitability of Gd-loaded AuNPs for MRI applications
was evaluated by taking T
1
weighted images of solutions of
4
52 | Chem. Commun., 2010, 46, 451–453
This journal is ꢀc The Royal Society of Chemistry 2010

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gel and eluted solutions for BTT-labelled and unlabelled
nanoparticles, respectively. This simple experiment paves the
way for using nanoparticle-bound Gd chelates for functional
magnetic resonance imaging (fMRI).
In summary, we have prepared Au nanoparticles coated
with a monolayer of Gd-complexed, DTPA-based ligand.
The increase in relaxivity (as compared to small molecule
analogues) was moderate, presumably due to the limited
restriction of rotational diffusion of the nanoparticle-attached
Gd ions. The relaxivity can be further increased by self-
assembling a polyelectrolyte on the Gd-studded nanoparticles
and nanoparticles can be readily functionalised, as illustrated
by co-adsorbing a biotin-based recognition unit.
The authors acknowledge Higher Education Commission of
Pakistan for funding this project and Center for Magnetic
Resonance, University of York, for imaging and relaxation
measurements.
Fig. 2 (a) BTT-Gd@AuNPs, (b) BTT-Gd@AuNPs adsorbed on
avidin–agarose column (I), eluent collected after passing BTT-
Gd@AuNPs through avidin–agarose column (II), avidin–agarose
column after elution of biotin-free Gd@AuNPs (III), biotin-free
Gd@AuNPs eluted through avidin–agarose column (IV).
Notes and references
AuNPs increased steadily with increased molecular weight
À1 À1
1 R. B. Lauffer, Chem. Rev., 1987, 87, 901.
(
(
ESIw) and for 60 kDa PEI reached 7.00 mM
Fig. 1b). Remarkably, a similar value (7.10 mM
s
2
(a) P. Caravan, J. J. Ellison, T. J. McMurry and R. B. Lauffer,
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s ) was
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firmed our suggestion that the limited increase in relaxivity
upon
3
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7
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1
9
8
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Chem. Commun., 2010, 46, 451–453 | 453