Luminescent ruthenium(II) polypyridyl functionalized gold nanoparticles; Their DNA binding abilities and application as cellular imaging agents

Article abstract of DOI:10.1021/ja2061159

The synthesis and photophysical and biological investigation of Ru(II)-polypyridyl stabilized water-soluble, luminescent gold nanoparticles (AuNPs) are described. These structures bind to DNA and undergo rapid cellular uptake, being localized within the cell cytoplasm and nucleus within 4 h.

Full text of DOI:10.1021/ja2061159

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Luminescent Ruthenium(II) Polypyridyl Functionalized Gold
Nanoparticles; Their DNA Binding Abilities and Application
As Cellular Imaging Agents
Robert B. P. Elmes,†,§ Kim N. Orange,‡,§ Suzanne M. Cloonan,‡,§ D. Clive Williams,*,‡,§ and
Thorfinnur Gunnlaugsson*,†,§
† School of Chemistry, Centre for Synthesis and Chemical Biology, Trinity College, Dublin, Dublin 2, Ireland
‡School of Biochemistry and Immunology, Trinity College, Dublin 2, Ireland
§Trinity College Biomedical Sciences Institute, Dublin 2, Ireland
S Supporting Information
b
ABSTRACT: The synthesis and photophysical and biolog-
ical investigation of Ru(II)-polypyridyl stabilized water-
soluble, luminescent gold nanoparticles (AuNPs) are de-
scribed. These structures bind to DNA and undergo rapid
cellular uptake, being localized within the cell cytoplasm and
nucleus within 4 h.
he development of functional supramolecular nanostruc-
T
tures for applications in photonics,1 sensing,2 catalysis3 and
medicine,4 etc. is a fast emerging interdisciplinary research field.
The design and synthesis of nanoparticles5 and, in particular,
functionalized gold nanoparticles (AuNPs) has been at the fore-
front of this effort in recent times, with many examples being
developed for use in biological and medical applications,6 due to
their biocompatibility, unique size- and shape-dependence, and
optoelectronic properties. Similarly, Ru(II)-polypyridyl complexes
have been intensively studied due to their photophysical
properties,7 where they have been employed for example in
luminescent recognition and sensing,8 as sensitive and structure-
specific DNA probes.9 Luminescent d6 transition metal ion
complexes have often been proposed as useful fluorophores for
cellular imaging,10,11 but until very recently their use in actual
applications has remained scarce. With our interest in the devel-
opment of luminescent novel cellular targeting (therapeutic/
imaging) agents7a,b,12 and surface modified AuNPs,13 we envisaged
that the combination of Ru(II)-polypyridyl complexes, spatially
separated from the surface of AuNPs, by a covalent spacer, could be
employed as luminescent probes/imaging agents for various bio-
logical applications.15 Herein we describe the synthesis of the
Ru(II) complexes 1À3, Figure 1, all of which possess a terminal
alkyl thiol group which facilitates their adsorption onto AuNPs,
leading to the formation of the three water-soluble systems AuNP-
1, AuNP-2 and AuNP-3 (Figure 1). We demonstrate that these
luminescent AuNPs offer attractive photophysical properties, ideal
for application in cellular imaging, which we demonstrate using
HeLa cells. These are, to the best of our knowledge, the first
examples of such Ru(II)-polypyridyl functionalized AuNPs to be
employed for such cellular applications.
Figure 1. Structure of the complexes 1, 2, and 3 (Ru = Ru(II)), ligand 4,
and cartoon representation of their corresponding AuNPs-1À3 systems.
the synthesis of which was achieved by employing peptidic
(carbodiimide) coupling of 11-mercaptoundecanoic acid14a with
5-amino,-1,10 phenanthroline in CH2Cl2, yielding 4 as an off-
white solid in 78% yield. The microwave irradiation of 4 in the
presence of the Ru(II) bispolypyridyl dichlorides Ru(bpy)2Cl2,
Ru(phen)2Cl2, and Ru(TAP)2Cl2 gave 1, 2, and 3, respectively,
after 40 min.14b These were isolated, by precipitation from water,
using excess NH4PF6, followed by purification using automatic
column chromatography [flash silica; 40:4:1 CH3CN/H2O/
NaNO3 (sat)], yielding 1À3 in 75%, 65%, and 54%, respectively.
These isolated NO3À salts were reconverted to their correspond-
ing PF6À salts and were fully characterized, which included the
use of 1D and 2D NMR. The characteristic MLCT based
absorbance spectra of 1À3, together with the excitation and
emission spectra, are shown in the Supporting Information. All of
the complexes were found to be fully water-soluble as their ClÀ
salts (see Supporting Information), and their photophysical
properties were investigated in 10 mM phosphate-buffered
aqueous solutions at pH 7.4. The absorption spectrum of 1
showed characteristic πÀπ* intraligand transitions centered at
The syntheses of 1, 2, and 3 are shown in Scheme S1 (see also
full details in Supporting Information) and were achieved in a
few steps. The common ligand for all of these complexes is 4,
Received: July 1, 2011
Published: September 16, 2011
r
2011 American Chemical Society
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Figure 2. (A) MLCT absorption band of 1, and AuNP-1 in buffered H2O
at pH 7.4. (B) Transmission electron microscopy image of AuNP-1.
285 nm (ε = 49 500 cmÀ1 MÀ1) assigned to the ancillary ligands,
while the characteristic MLCT transitions of the Ru(II) center
Figure 3. Changes in the emission spectrum of AuNP-3 (λex 450 nm)
with increasing concentration of DNA (0À77 μM). The blue and the
red spectra demonstrated the non-DNA and the DNA bound AuNP-3.
Inset: Plot of the change in integrated MLCT intensity as a function of
P/D ratio.
were observed at 450 nm (ε = 12 400 cmÀ1
M
À1). Similar
transitions were found for 2 and 3. Excitation into the MLCT
bands of 1À3 gave rise to, on all occasions, MLCT based
emission occurring at λmax = 610 nm (ΦF = 0.054), 605 nm
(ΦF = 0.056), and 635 nm (ΦF = 0.028), for 1À3, respectively.
For all of these, the excitation spectra structurally matched those
of the absorption spectra.
the photophysical properties of the surface modified Ru(II)
complexes. In the case of AuNP-1, an initial red shift in the
UVÀvis absorption spectra, followed by a ca. 26% hypochroism,
was observed upon binding to DNA. Similar changes were seen
for AuNP-2 and AuNP-3. Significant changes were also seen in
the emission spectra of AuNP-1À3, as demonstrated in Figure 3,
upon binding of AuNP-3 to DNA, giving rise to 71% quenching
in the MLCT based emission (see Supporting Information for
AuNP-1À2). This is in agreement with the behavior of other
π-deficient Ru(II) complexes of the general formula [Ru
(TAP)2(L)]2+ (L = phen or bpy),16 as those complexes contain-
ing two TAP ligands have been shown to photo-oxidize guanine-
containing nucleotides through a proton coupled photoinduced
electron transfer (PCET) process.17 The affinities of AuNP-1À3
for DNA were further confirmed by carrying out ethidium
bromide (EtBr) displacement assays (Supporting Information),18
which demonstrated that AuNP-1À3 displaced EtBr with an
apparent binding constant (Kapp) of ∼107 MÀ1. From these
results, it is clear that AuNP-1À3 all interact with DNA in a
strong manner. To further confirm this, we investigated the
ability of these nanostructures to bind to DNA using confocal
fluorescence imaging, by treating a solution of DNA (4 mM)
with a solution of AuNP-1À3. This led to immediate forma-
tion of a precipitate and the formation of fibrous networks and
smaller aggregates, which are currently under investigation in
our laboratory.
Having demonstrated high DNA binding affinities of AuNP-
1À3, we next carried out cellular uptake and localization studies
of these systems using confocal fluorescence microscopy. We
anticipated that AuNP-1À3 would be taken up by cells as has
been seen with many AuNP conjugates.19 This may occur
through receptor mediated transport (e.g., endocytosis), or by
plasma membrane driven transport due to their cationic nature.
The results are shown in Figure 4. In Figure 4A, a brighfield
image shows the presence of the AuNP (as aggregates) within
live HeLa (cervical cancer) cells as dense ‘dark spots’, after
incubation with AuNP-1 (∼16 nM) 4 h prior to imaging.
Fluorescence confocal laser scanning microscopy confirmed this
uptake and localization, as demonstrated in Figure 4B, which
shows an image of these cells upon excitation at 450 nm, where
these dense areas of Figure 4A are seen to overlap perfectly with
The syntheses of AuNP-1À3 were achieved using the two-
phase Brust method which gave tetraoctylammoniumbromide
(TOAB) stabilized AuNPs.15a Exchange of TOAB on the surface
of AuNPs with 1À3 was achieved upon stirring TOAB-AuNP
with 1À3, respectively, for 12 h at room temperature, followed
by centrifugation and the removal of any unbound complex by
employing a method developed by Mayer et al.15b involving
double anion exchange. The AuNPs were characterized using
UVÀvisible spectroscopy, where the surface plasmon resonance
(SPR) band was found at 520 nm (Supporting Information)
slightly overlapping with the MLCT absorption bands of 1À3,
Figure 2A. Using UVÀvis spectroscopy, AuNP-1À3 were found
to be stable in aqueous-buffered solution at pH 7.4 at room
temperature for many months. Excitation into the MLCT
absorption bands of AuNP-1À3 gave rise to, on all occasions,
MLCT centered emission with λmax at 610, 605, and 635 nm, for
AuNP-1À3 respectively, with ϕF of ∼0.002, demonstrating that
these were significantly less emissive after adsorption to the
AuNP surface, as has previously by seen for [Ru(bpy)3]2+ in the
presence of AuNPs coated with thiolate monolayers.15c AuNP-
1À3 were further characterized using TEM, which showed
spherical nanoparticles with an average diameter of 4.0 ( 1.3,
4.3 (1.3, and 3.2 (1.1 nm for AuNP-1, AuNP-2, and AuNP-3
respectively, with no evidence of aggregation, Figure 2A, and
using DLS which gave hydrodynamic radii of 3À7 nm
(Supporting Information).
Ru(II)-polypyridyl complexes are known to bind to DNA and
cause photosensitized DNA damage, but the binding constant
and the photocleavage efficiency are highly structurally depen-
dent.7,16 Consequently, the DNA binding affinities of 1À3, and
AuNP-1À3, were evaluated in 10 mM phosphate buffer at
pH 7.4 using salmon testes DNA. In the case of 1À3, DNA
binding gave rise to hypochromic effects in the MLCT band in
the UVÀvis absorption spectra, which in the case of 1 was ca.
30% (Supporting Information). From these, a binding constant
Kb = 5.5 Â 105 MÀ1 was determined, while, for 3, Kb = 6.4 Â 105
M
À1 was calculated. Concomitantly, the MLCT centered emis-
sion was also modulated, being most noticeable for 3, with ca.
75% quenching being observed (see Supporting Information).
Similarly, the binding of AuNP-1À3 also gave rise to changes in
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Figure 4. Live cell confocal laser scanning microscopy images of AuNP-1 (∼16 nM gold concentration20) with HeLa cells: (A) the bright field image;
(B) emission arising from 600 to 700 nm; (C) overlay of the luminescence from AuNP-1 (red), nuclear costain DAPI (blue), and the bright field image;
(D) TEM image of AuNP-1 following incubation with HeLa cells.
Figure 5. Confocal fluorescence Z-section live cell images of AuNP-2 (20 μM) with Hela cells showing luminescence in three dimensions. Shown is
the image obtained with cells costained with nuclear stain DAPI (blue) and AuNP-2 (red). Scanning from left to right.
the red fluorescence emanating from within the cell interior. The
image clearly confirms that AuNP-1 is luminescent within the
cells; with apparent localization in the cytoplasm and the nucleus,
where these can be seen as discrete ‘packets’ or aggregates of
fluorescence. In addition, Z-stack experiments clearly show that
the observed luminescence is spherical in three dimensions;
corresponding to the AuNPs being located inside the cells and
not bound to their exterior, Figure 5, for AuNP-2 (See larger
image in Supporting Information as well as for AuNP-1 and
AuNP-3).
Costaining of the nucleus, using DAPI, further confirmed the
nuclear localization of AuNP-1, as demonstrated in Figure 4C,
where the costained image is overlapped with the bright field
image (Figure 4A) and the luminescent image (Figure 4B).
Similar behavior was observed for AuNP-2, Figure 5, and AuNP-
3 with the luminescence intensity being slightly diminished in the
case of AuNP-3 due to the PCET process discussed above
(Supporting Information). The uptake of AuNP-1 was also
confirmed by TEM imaging within the HeLa cells, confirming
their localization within both the cytosol, Figure 4D, and the
nucleus. We also carried out time dependent fluorescence
confocal imaging studies, where the cellular uptake of AuNP-1
was imaged at 2 and 6 h in addition to the 4 h discussed above.
The results (see Supporting Information) clearly demonstrated
that only minimal uptake had occurred after 2 h. After 6 h, results
similar to those seen at 4 h were observed, though with slightly
enhanced cellular contrast.
To assess the antiproliferative effects of these AuNPs we
attempted to determine the EC50 value of AuNP-1 by carrying
out an Alamar blue cytotoxicity assay with a large range of
concentrations of AuNP-1 in the mesothelioma cell line
CRL5195. In short, and as is evident from Figures 4 and 5 using
HeLa cells, no significant apoptosis was observed for AuNP-1,
and in fact, an IC50 value could not be determined for AuNP-1
from the concentration range used as % viability did not drop
below 60% (see Supporting Information).
In summary, we have developed, to the best of our knowledge,
the first examples of novel supramolecular DNA targeting motifs
based on Ru(II) stabilized AuNPs. We have shown that AuNP-
1À3 all bind to DNA with high affinity, while at the same time
being nontoxic, and that we can monitor the binding of these to
DNA using various spectroscopic techniques. Moreover, by using
fluorescence confocal microscopy and TEM imaging studies, we
have demonstrated their cellular uptake and localization within the
cytoplasm and nucleus. These studies confirm the potential use of
such Ru(II) polypyridyl functionalized AuNPs as highly sensitive
cellular imaging agents. We are currently investigating the mecha-
nism of their uptake, and their fate, in greater detail.
’ ASSOCIATED CONTENT
S
Supporting Information. Synthesis and characterization
b
of 1, 2, and 3; Figures S1ÀS30. This material is available free of
charge via the Internet at http://pubs.acs.org.
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’ AUTHOR INFORMATION
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Corresponding Author
clive.williams@tcd.ie; gunnlaut@tcd.ie
’ ACKNOWLEDGMENT
We thank the Science Foundation Ireland (SFI RFP 2009),
HEA PRTLI Cycle 4, The Irish Research Council for Science,
Engineering and Technology (IRCSET Postgraduate Student-
ship to R.B.P.E.), and TCD for financial support
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