Functionalisation of gold nanoparticles with ruthenium(ii) polypyridyl complexes for their application in cellular imaging

Article abstract of DOI:10.1039/d0dt02754e

Two new dinuclear Ru(ii) polypyridyl complexes containing an alkyl disulphide functionalised bipyridine-based ligand and either 1,10-phenanthroline (phen) or 1,4,5,8-tetraazaphenanthrene (TAP) as ancillary ligands have been synthesised and characterised. Their attachment onto the surface of gold nanoparticles (AuNPs, average diameter of ca. 2.5 nm) resulted in the formation of two new water-soluble Ru(ii)-AuNP conjugates that combine the advantageous properties of both moieties. Both free complexes show the attractive photophysical properties of Ru(ii) polypyridyl complexes and a rapid cellular uptake in HeLa cervical cancer cells. However, their corresponding gold conjugates displayed lower quantum yields than those determined for the free complexes presumed to be due to an energy transfer quenching of the Ru(ii) luminescence by interaction with the gold surface. Despite their diminished luminescence, confocal fluorescence microscopy studies revealed that the Ru(ii)-AuNP conjugates are successfully internalised into HeLa cells and better tolerated than their free complex counterparts after 24 h incubation, which makes them potential luminescent nanomaterials for bioimaging applications.

Full text of DOI:10.1039/d0dt02754e

Dalton
Transactions
View Article Online
View Journal
PAPER
Functionalisation of gold nanoparticles with
ruthenium(II) polypyridyl complexes for their
Cite this: DOI: 10.1039/d0dt02754e
application in cellular imaging†
Sandra Estalayo-Adrián,
Aramballi J. Savyasachi,a John M. Kelly a and Thorfinnur Gunnlaugsson
*
a,b Gavin J. McManus,c Hannah L. Dalton,a
a
*
Two new dinuclear Ru(II) polypyridyl complexes containing an alkyl disulphide functionalised bipyridine-
based ligand and either 1,10-phenanthroline (phen) or 1,4,5,8-tetraazaphenanthrene (TAP) as ancillary
ligands have been synthesised and characterised. Their attachment onto the surface of gold nanoparticles
(AuNPs, average diameter of ca. 2.5 nm) resulted in the formation of two new water-soluble Ru(II)-AuNP
conjugates that combine the advantageous properties of both moieties. Both free complexes show the
attractive photophysical properties of Ru(II) polypyridyl complexes and a rapid cellular uptake in HeLa cer-
vical cancer cells. However, their corresponding gold conjugates displayed lower quantum yields than
those determined for the free complexes presumed to be due to an energy transfer quenching of the Ru
(II) luminescence by interaction with the gold surface. Despite their diminished luminescence, confocal
fluorescence microscopy studies revealed that the Ru(II)-AuNP conjugates are successfully internalised
into HeLa cells and better tolerated than their free complex counterparts after 24 h incubation, which
makes them potential luminescent nanomaterials for bioimaging applications.
Received 6th August 2020,
Accepted 23rd September 2020
DOI: 10.1039/d0dt02754e
rsc.li/dalton
tration of most gold nanoconjugates has also been confirmed
by several research groups. Differences in their surface chem-
Introduction
In recent years, gold nanoparticle (AuNP) conjugates have istry, particle size, shape, charge and lipophilicity have been
been extensively explored for use in biology and medicine due shown to play
to their attractive properties.1–4 Defined as submicroscopic mechanism.5,6
a crucial role in the cellular uptake
particles with sizes between 1 and 100 nm, they display unique
AuNPs have a large surface area to volume ratio which pro-
and tuneable optoelectronic properties. For instance, AuNPs vides them with the ability to achieve high loading of ligands,
absorb light strongly at a particular wavelength due to their and thus, high concentrations of cargo can be delivered into
surface plasmon resonance (SPR) properties, an effect that can cells while maintaining a lower overall concentration of
be tuned via synthesis.3 The AuNP surface also offers a stable AuNPs.7 In this context, luminophores have been attached to
and easily functionalised platform for conjugation with AuNPs and used as probes for non-invasive biomolecular
ligands containing functional groups that exhibit affinity for imaging.8 Furthermore, the conjugation of photosensitisers to
gold such as thiols, phosphines and amines.1,3 Furthermore, AuNPs has emerged as an alternative to classic photodynamic
AuNPs have shown to be biocompatible, with any toxicity therapy (PDT) agents.9 The resulting photosensitiser-AuNP
being normally associated with the chemical composition of conjugates offer some advantages over classic PDT agents,
the ligands attached to the gold surface.2 Intracellular pene- such as increase of the local photosensitiser concentration,
prevention of nonspecific accumulation in normal tissues, or
tumour-targeted delivery of the photosensitiser due to an
enhanced permeability and retention (EPR) effect of the par-
ticle scaffold.9
aSchool of Chemistry and Trinity Biomedical Sciences Institute (TBSI), Trinity
College Dublin, The University of Dublin, Dublin 2, Ireland. E-mail: estalays@tcd.ie,
gunnlaut@tcd.ie
The attractive photophysical properties and selective photo-
chemistry of Ru(II) polypyridyl complexes have been intensively
explored over the years.10 In addition, their ability to be used
in a biological context as DNA photoprobes, cellular imaging
agents and light-activated anticancer drugs has played a major
role in the development of many of the Ru(II)-based systems
bAdvanced Materials and BioEngineering Research (AMBER) Centre, Trinity College
Dublin, The University of Dublin, Dublin 2, Ireland
cSchool of Biochemistry and Immunology, Trinity Biomedical Sciences Institute
(TBSI), Trinity College Dublin, The University of Dublin, Dublin 2, Ireland
†Electronic supplementary information (ESI) available: Characterisation and cel-
lular studies. See DOI: 10.1039/d0dt02754e
This journal is © The Royal Society of Chemistry 2020
Dalton Trans.

View Article Online
Paper
Dalton Transactions
described in the literature in recent times.11–13 Therefore, the
combination of the advantageous properties of both AuNPs
and Ru(II) polypyridyl complexes should result in attractive
Results and discussion
Synthetic procedures
systems for application in cellular imaging or photothermal The Ru(II) polypyridyl complexes 1 and 2 and their gold conju-
therapy (PTT) in the treatment of cancer although, despite this gates 1·AuNP and 2·AuNP were prepared in a few steps syn-
biological potential, only a few examples of Ru(II) polypyridyl thetic procedure (Scheme 1).15,17 First, the synthesis of the
complex-functionalised AuNPs have been reported in the ligand 11,11′-disulfanediylbis(N-(3-([2,2′-bipyridin]-4-yl)phenyl)
literature.14–17 Herein, we describe the synthesis of two new undecanamide) (3) was achieved by an amide coupling reac-
dinuclear Ru(II) polypyridyl complexes, 1 and 2 (Fig. 1), con- tion between 3-([2,2′-bipyridin]-4-yl)aniline (4) and 11,11′-disul-
taining an alkyl disulphide moiety and either 1,10-phenanthro- fanediyldiundecanoic acid (5) using N-ethyl-N′-(3-dimethyl-
line (phen) or 1,4,5,8-tetraazaphenanthrene (TAP) as ancillary aminopropyl)carbodiimide hydrochloride (EDC) and 4-di-
ligands, and the subsequent conjugation of these complexes methylaminopyridine (DMAP) in dry CH2Cl2, yielding 3 as a
to the surface of AuNPs (via the sulphur atoms) which resulted beige solid in 72% yield.18 Microwave-assisted synthesis was
in the formation of the water-soluble gold conjugates 1·AuNP then carried out for the complexation of 3 with the appropriate
and 2·AuNP. We report the photophysical properties of both Ru(II) precursors cis-[Ru(phen)2Cl2] and cis-[Ru(TAP)2Cl2] in a
the free complexes and their gold conjugates, as well as their H2O/EtOH mixture (1 : 1) at 140 °C for 40 min (1) or 30 min
biological profiling in HeLa cervical cancer cells, demonstrat- (2). Purification by column chromatography on neutral
ing the potential use of 1·AuNP and 2·AuNP as real-time lumi- alumina using MeCN/H2O (10 : 0 to 9 : 1) as eluent afforded
nescent cellular imaging agents. We demonstrate that the con- the chloride salts of 1 and 2 in 80% and 26% yield, respect-
jugation of such ‘free’ complex to the surface of the AuNPs can ively. Both 1 and 2 and ligand 3 were fully characterised using
have significant effect on their cytotoxicity properties, and as conventional techniques (Fig. S1–S11, ESI†). Successful for-
such facilitating the ‘switching’ between therapeutic and mation of 1 and 2 was confirmed by their 1H NMR spectra
imaging applications between structurally related systems.
(Fig. 2), while MALDI+-HRMS showed their expected isotopic
patterns (Fig. S7 and S11, ESI†) with peaks at m/z 1815.5494
and 1823.5002 which corresponded to the [M − H]+ ions of 1
and 2, respectively.
The synthesis of 1·AuNP and 2·AuNP was performed using
AuNPs, produced by a modified version of the two-phase
Brust-Schiffrin method used previously in our group.15,19,20
This method results in the formation of thermally- and air-
stable tetraoctylammonium bromide (TOAB)-stabilised AuNPs
of controlled size (with a Au(0) metallic core diameter in the
range of 1.5 to 5.2 nm).21 Functionalisation of the surface of
these AuNPs with the Ru(II) complexes was next carried out by
exchanging the TOAB stabiliser coat with 1 and 2. For this
purpose, an aqueous solution of the appropriate Ru(II)
complex was added to a toluene solution of the TOAB-stabil-
ised AuNPs and the mixture stirred vigorously for 16 h.
Complete transfer of the AuNPs from the toluene layer to the
aqueous layer was confirmed by the initial orange aqueous
layer turning dark brown, indicating functionalisation of the
AuNPs with the water-soluble Ru(II) complexes.
After separation of the aqueous layer containing the Ru(II)
polypyridyl functionalised-AuNPs by centrifugation, any
unbound complex was removed through a series of anion
exchange steps based on a method developed by Mayer et al.
and previously used in our group.15,17,22 This involves the
addition of a saturated aqueous solution of NH4PF6 to the
aqueous solution containing the Ru(II) polypyridyl functiona-
lised-AuNPs and isolation of the resulting flocculate by cen-
trifugation. The obtained solid was dissolved in MeCN and
repeatedly precipitated by adding Et2O until the supernatant
became colourless. Once the resultant solid was redispersed in
MeCN, the next anion exchange step consisted of addition of a
concentrated acetone solution of tetrabutylammonium chlor-
ide (TBACl), followed by stirring for 1 h. After centrifugation,
Fig. 1 Chemical structures of the dinuclear Ru(II) polypyridyl com-
plexes, 1 and 2, and their corresponding gold conjugates, 1·AuNP and
2·AuNP.
Dalton Trans.
This journal is © The Royal Society of Chemistry 2020

View Article Online
Dalton Transactions
Paper
Scheme 1 Synthesis of Ru(II) polypyridyl complexes 1 and 2: (i) sodium pyruvate, EtOH, 2 M NaOH, 0 °C, 3 h; (ii) 1-(2-pyridacyl)pyridinium iodide,
NH4OAc, H2O, reflux, 5 h; (iii) Δ; (iv) N2H4, 10% Pd/C, EtOH, reflux, 5 h; (v) Br2, DMF (anh.), 0 °C, 3 h; (vi) EDC, DMAP, CH2Cl2, r.t., 2 days; (vii) cis-[Ru
(L)2Cl2] (L = phen or TAP), EtOH/H2O (1 : 1), microwave reaction, 140 °C, 40 min (1) or 30 min (2).
nalised-AuNPs. This was achieved by drop casting aqueous
solutions of 1·AuNP and 2·AuNP onto a 200-mesh formvar film
stabilised with carbon copper grid, followed by TEM imaging
of airdried samples. The TEM images obtained for both
1·AuNP and 2·AuNP revealed spherical nanoparticles (Fig. 3a
and b) with diameters in the range of 1.5–7.0 nm for 1·AuNP
and 1.0–6.5 nm for 2·AuNP, which are consistent with the syn-
thetic procedure employed.21 Analysis of the particle size of ca.
300 nanoparticles from different regions of TEM images
Fig. 2 1H NMR (400 MHz, DMSO-d6) spectra of (a) 1 and (b) 2 showing
the aromatic region. Signals corresponding to phen and TAP ligands are
in green and signals assigned to ligand 3 are in red.
the dark brown solid obtained was washed several times with
acetone and MeCN, dried under vacuum and finally sus-
pended in H2O to give dark brown coloured colloidal solutions
of 1·AuNP or 2·AuNP.
Transmission electron microscopy studies
Fig. 3 TEM images of (a) 1·AuNP and (b) 2·AuNP (250 µM) after depo-
sition onto formvar stabilised carbon coated copper grids and the
corresponding particle size distribution profile calculated from analysing
the TEM images, and particle size distribution determined by DLS ana-
Transmission electron microscopy (TEM) studies were per-
formed to gain further insight into the size, size distribution
and morphology of the synthesised Ru(II) polypyridyl functio- lysis for (c) 1·AuNP and (d) 2·AuNP in aqueous solutions at 298 K.
This journal is © The Royal Society of Chemistry 2020
Dalton Trans.

View Article Online
Paper
Dalton Transactions
showed that the majority of the AuNPs functionalised with 1
displayed an average gold core diameter between 2.5 and
3.0 nm (Fig. 3a). However, a slightly smaller average gold core
diameter was observed for the majority of the AuNPs functio-
nalised with the TAP analogue complex 2 with diameters
values between 2.0 and 2.5 nm (Fig. 3b). Interestingly, the re-
placement of the N in positions 1 and 8 in the TAP ligand by
CH in the phen ligand was shown to have a clear effect on the
aggregation properties of the resulting gold conjugate. Thus,
aggregation was found to be more prominent in the case of
1·AuNP, with no major evidence of aggregates formation by the
TAP conjugate 2·AuNP. Such behaviour has been previously
observed in our group for similar systems and was attributed
to the ability of the TAP ligand to participate in hydrogen
bonding with water which reduces the aggregation of the
resulting Ru(II) TAP complexes-based systems.17
Dynamic light scattering studies
Dynamic light scattering (DLS) measurements were also con-
ducted on aqueous solutions containing 1·AuNP or 2·AuNP
(Fig. 3c and d). Average hydrodynamic diameters of 12 nm
(94% volume) and 13 nm (98% volume) were determined for
both surface-modified gold particles 1·AuNP and 2·AuNP,
respectively.15,20,23 Similarity in the hydrodynamic diameters
of both systems suggests that the presence of phen or TAP as
ancillary ligands in the Ru(II) complexes bound to the surfaces
of the AuNPs does not have a significant influence in the size
of the resulting Ru(II) polypyridyl functionalised-AuNPs. It
should be noticed that, while the particle size determined by
TEM analysis only depends on the size of the particle gold
core, DLS measurements include the alkyl disulphide functio-
nalised Ru(II) complex bound to the gold surface. Thus, larger
diameter values are expected when employing DLS, which is
consistent with the results showed here.
Fig. 4 UV-vis absorption and emission spectra of (a)
1 (red),
TOAB·AuNP (blue) and 1·AuNP (green), and (b) 2 (red), TOAB·AuNP
(blue) and 2·AuNP (green) in 10 mM sodium phosphate-buffered
aqueous solution at pH 7.4 and 298 K (λexc = 453 (1), 460 (1·AuNP), 416
(2) or 416 (2·AuNP) nm).
Furthermore, while the AuNPs functionalised with the Ru characteristic of the MLCT transition of the Ru(II) centre.
(II) TAP-based complex 2·AuNP showed a narrow size distri- Likewise, 2 showed intense absorption bands at 232 nm (ε =
bution in the range of 6.5–24.4 nm (Fig. 3c), a broader size dis- 122 000 M−1 cm−1) and 275 nm (ε = 148 000 M−1 cm−1) that
tribution in the range of 5.6–78.8 nm (Fig. 3d) was observed correspond to π–π* intra-ligand transitions in the TAP ligand,
for the phen analogue system 1·AuNP indicating the presence and a broad band centred at 416 nm (ε = 39 000 M−1 cm−1
)
of much larger species in solution. These results point to a assigned to the MLCT transition of the Ru(II) centre.
greater tendency of 1·AuNP to form aggregates when compared
Functionalisation of AuNPs with 1 and 2 resulted in the
to the TAP analogue gold conjugate 2·AuNP, which is in agree- appearance of a broad shoulder in their MLCT absorption
ment with the TEM studies discussed above. Having character- bands extending to ca. 700 nm (Fig. 4) and attributed to the
ised the size of both 1·AuNP and 2·AuNP, we next investigated SPR band characteristic of metallic nanoparticles for 1·AuNP
their photophysical properties in buffered-aqueous solution.
and 2·AuNP. The presence of the SPR band confirms the suc-
cessful attachment of the Ru(II) complexes on the AuNP
surface. Interestingly, no major changes were observed in the
Photophysical characterisation
The photophysical properties of the free complexes 1 and 2 π–π* intra-ligand and MLCT bands position when compared to
and their gold conjugates 1·AuNP and 2·AuNP were evaluated those shown by the free complexes 1 and 2 (Fig. 4). Generally,
in 10 mM sodium phosphate-buffered aqueous solution at pH the overlap of both MLCT and SPR bands makes it difficult to
7.4 and 298 K (Fig. 4). As expected, the UV-vis absorption spec- identify any shifts in the position of the SPR band after incor-
trum of 1 showed three main absorption maxima at 225, 263 poration of the Ru(II) complexes onto the gold surface, as it
and 453 nm. Bands at 225 nm (ε = 130 000 M−1 cm−1) and has been shown that the absorption maximum and bandwidth
263 nm (ε = 181 000 M−1 cm−1) were attributed to π–π* intra- of the SPR band is sensitive to the size, shape and composition
ligand transitions within the ancillary phen ligands while the of the nanoparticle.24 However in our case these effects appear
broad band centred at 453 nm (ε = 36 000 M−1 cm−1) was to be small. Comparing the relative absorbances (at 453 nm) of
Dalton Trans.
This journal is © The Royal Society of Chemistry 2020

View Article Online
Dalton Transactions
Paper
1 the unfunctionalised AuNP nanoparticle and 1·AuNP, an respectively, upon excitation into their MLCT absorption
approximate estimate of the ratio of Ru : Au of 1 : 7.9 can be bands (Fig. 4), as was shown for 1 and 2. Likewise, excitation
made Similar calculations for 2·AuNP gives Ru : Au of 1 : 9.8 spectra of 1·AuNP and 2·AuNP were shown to be identical to
(see Experimental section for further details).
those of the corresponding free complexes indicating no con-
Excitation into the MLCT bands of the free complexes tribution to the observed luminescence from the AuNP SPR
resulted in broad luminescence bands centred at 616 nm for 1 band (Fig. S12c and S12d, ESI†). Luminescence quantum
and 648 nm for 2 (Fig. 4). The excitation spectra recorded for 1 yields of both systems were found to be significantly lower in
and 2 were found to be identical to their absorption spectra comparison to those determined for the free complexes, with
(Fig. S12a and S12b, ESI†). Luminescence quantum yields values of 0.002 and 0.001 determined for 1·AuNP and 2·AuNP,
values of 0.048 and 0.007 were determined for 1 and 2, respect- respectively (Table 1). This behaviour was not unexpected as
ively in aerated solution (Table 1). In addition, luminescence quenching of the Ru(II) complex luminescence by an energy-
lifetimes of 804 and 254 ns were measured, respectively. In the transfer mechanism resulting from the interaction with the
case of 1, this value is longer to that reported in the literature AuNP surface has already been reported in the literature.16,34
for [Ru(phen)2bpy]2+,25 whereas 2 exhibits a much lower lumine- In addition, lifetime values of 827 and 303 ns were measured
scence lifetime than its analogue [Ru(TAP)2bpy]2+.26,27 As has in aerated 10 mM sodium phosphate-buffered aqueous solu-
been previously discussed in the literature, the presence of the tion at pH 7.4 for the gold conjugates 1·AuNP and 2·AuNP,
electron-withdrawing TAP ligands confers an oxidising character respectively. These values are similar to those observed for
to the excited state of the resulting Ru(II) complex.28 Therefore, their free complex counterparts in the same conditions and
the 3MLCT excited state of TAP containing Ru(II) complexes were attributed to the unquenched complexes bound to the
would be sufficiently photo-oxidizing to extract an electron from gold surface. Moreover, 1·AuNP and 2·AuNP showed lifetimes
reductant molecules resulting in a quenching of their lumine- values of 999 and 421 ns when the solution was deaerated,
scence. Sulphur containing molecules such as H2S or cysteine which are only slightly larger values than those measured in
have shown to be efficient reductants.29 Thus, the low quantum aerated solution. This behaviour is in contrast to that showed
yield and lifetime values observed for 2 can be explained by an by the free complex 1 for which a more significant increase of
intramolecular photoinduced electron transfer (PET) from the the lifetime was observed when the solution was deaerated. It
alkyl disulphide moiety to the oxidising Ru(TAP)2 core. can be presumed that the attachment of a large number of
Furthermore, when the solution was deaerated, significant complex molecules around the gold surface would keep them
increase of the lifetime of 1 was observed, indicating a quench- close to each other resulting in the protection of their excited
ing of the excited state by dissolved oxygen in solution. In con- state from oxygen quenching. Additionally, the aforemen-
trast, no significant change in the lifetime was observed for 2 in tioned aggregation would possibly also reduce collision
deaerated solution. This could be due both to the known lower quenching with solvated oxygen. Thus, deaeration of the solu-
sensitivity of the excited state of TAP complexes to dissolved tion would not have a major effect as molecules of the
oxygen and also to the proposed quenching by the disulphide complex would be already protected in their new arrangement
group.13,30,31
around the gold core. In the case of the TAP containing gold
In the case of the AuNP conjugates 1·AuNP and 2·AuNP, conjugate 2·AuNP, results are consistent with those observed
broad emission bands were observed at 624 and 649 nm, for the free complex and thus similarity in lifetime values
measured in both aerated and deaerated conditions would be
due to the negligible contribution of dissolved oxygen to the
quenching of the excited state of TAP containing Ru(II)
complexes.13,30,31
Table 1 Emission properties of 1, 2, 1·AuNP and 2·AuNP in 10 mM
sodium phosphate-buffered aqueous solution at pH 7.4 and 298 K.
Complexes [Ru(phen)2bpy]2+ and [Ru(TAP)2bpy]2+ are included for
comparison
Cellular uptake and viability studies
em
Cellular uptake studies in HeLa cervical cancer cells were con-
ducted on both the free complexes 1 and 2 and their gold con-
jugates 1·AuNP and 2·AuNP with the aim of investigating the
applicability of the new systems in biological imaging. HeLa
cells were incubated either with 1, 2, 1·AuNP or 2·AuNP (ca.
20 µM Ru(II) complex concentration)35 at 37 °C for 2, 4 and
24 h. Cells were then treated with the nuclear stain Hoechst
33258 and imaged using confocal fluorescence microscopy.
Confocal fluorescence images showed that both 1 and 2 loca-
lised in the cytoplasm of HeLa cells with a time-dependent cel-
lular uptake (Fig. 5). An increase of the emission intensity of 1
and 2 was detected over time, from almost negligible emission
after 2 h incubation, and then more significant emission
λ
(nm) Φem a buffer τem (ns)b
τem (ns)
buffer (air) buffer (N2)
max
Complex
buffer
(air)
[Ru(phen)2bpy]2+ c 608
—
555
605
804
254e
827
303
—
[Ru(TAP)2bpy]2+ d
1
2
1·AuNP
2·AuNP
649
616
648
624
649
0.040
0.048
0.007
0.002
0.001
778
1237
253e
999
421
a Air-saturated aqueous solution of [Ru(bpy)3]Cl2 as reference (Φem
=
0.028).32 Estimated errors 2%. b If not otherwise indicated, the
luminescence decays are monoexponential. Estimated errors 5%.
c Pyle et al.25 The reported values are in 5 mM Tris, 50 mM NaCl buffer
at pH 7.5. d Masschelein et al.26 Karlsson et al.27 The reported values
are in aqueous solution. e The luminescence decays are bi-exponen-
tial.33 Estimated errors 5%.
This journal is © The Royal Society of Chemistry 2020
Dalton Trans.

View Article Online
Paper
Dalton Transactions
cell morphology of the HeLa cells treated with 1 for 24 h
suggests that this complex is more toxic than its TAP analogue
and as such nuclear localisation of 1 could be a sign of cell
damage after long exposure to this complex.
Confocal fluorescence images of the gold conjugates
1·AuNP and 2·AuNP in the same conditions as those employed
for the corresponding free complexes showed an increase in
the cellular uptake over time for both systems (Fig. 5). After 2
and 4 h incubation, both gold conjugates mainly localised in
the cell membrane. However, brighter luminescent emission
from 1·AuNP and 2·AuNP appeared in the cytoplasm of the
cells after 24 h incubation. Moreover, cellular internalisation
of the gold systems was also observed in the bright field
images with the appearance of the AuNPs as dense dark areas
(Fig. S14, ESI†). The effective cellular internalisation of the
AuNPs conjugates was further confirmed by Z-stack studies
(Fig. 6). Both systems 1·AuNP and 2·AuNP were clearly loca-
lised in the cytoplasm of the cells around the nuclei as
observed from the red emission from the ruthenium-based
nanomaterials. Thus, these results gave further evidence of the
successful internalisation of the gold conjugates 1·AuNP and
2·AuNP in HeLa cells. The cellular uptake effectiveness and
mechanism of AuNPs have been shown to be dependent on
the size, shape and surface charge of the nanomaterial.3,5,36
This may occur through receptor-mediated endocytosis as has
been described for many other AuNP conjugates.3,5,17,36 Such
an uptake mechanism can be supported by the punctate
appearance of the gold systems 1·AuNP and 2·AuNP inside the
cells. The exact mechanism involved in the cellular uptake of
the free complexes 1 and 2 and their AuNP conjugates 1·AuNP
and 2·AuNP is currently under investigation.
Fig. 5 Confocal fluorescence microscopy images of HeLa cells
showing the uptake of 1, 1·AuNP, 2 and 2·AuNP (red) at ca. 20 µM Ru(II)
complex concentration after 2, 4 and 24 h incubation. The nucleus is
stained blue with Hoechst 33258.
The cytotoxicity of the free complexes 1 and 2 against HeLa
cells was next evaluated using the Alamar Blue viability assay.
intensity after 4 and 24 h incubation. However, 2 was found to Their phototoxicity was also investigated based on the poten-
be less emissive inside the cells when compared with 1. This is tial of Ru(II) polypyridyl complexes to be used as light-activated
in agreement with the photophysical properties of these com- therapeutic agents previously reported.12 HeLa cells were
plexes discussed above where 1 was found to be much more treated with 1 and 2 and incubated for 24 h at 37 °C before
emissive than 2 as a result of an intramolecular PET from the being exposed to 18 J cm−2 of light for 1 h using a UV-filtered
alkyl disulphide moiety to the oxidising Ru(TAP)2 core. Hg–Xe arc lamp or keeping in the dark. After a further 24 h
Furthermore, previous studies have shown that complexes con- incubation, HeLa cells were treated with the Alamar Blue
taining phen as ancillary ligands exhibited a greater cellular reagent and the toxicity assessed under both light and dark
uptake than their TAP analogues.13,31 Thus, a lower intracellu- conditions (Fig. S15, ESI†). Both complexes displayed modest
lar concentration can be expected for 2 than for 1 resulting in toxicity against HeLa cells in dark conditions with 1 being
a lower emission intensity inside the cells when treated with twice as toxic as 2 (IC50 (1) = 19 2 µM, IC50 (2) = 38 2 µM)
the TAP complex. In addition, luminescence intensity of TAP probably due to the lower lipophilicity of TAP complexes when
complexes might be quenched by reductant biomolecules compared to their phen analogues resulting in a larger intra-
such as the nucleobase guanine and the amino acid trypto- cellular concentration of the latter.13,31 Furthermore, upon
phan through a PET process, as has been fully discussed in activation with light, 1 was shown to be significantly more
the literature.28
phototoxic than 2 (IC50 (1) = 1.77 0.05 µM, IC50 (2) = 17.1
Furthermore, partial nuclear localisation of 1 was observed 0.8 µM) with phototoxic index values of 11 and 2, respectively.
after 24 h incubation (Fig. S13, ESI†). Overlap of the fluo- This can be explained by the fact that phen complexes have
rescence from the nuclear stain Hoechst 33258 (blue) with the been shown to produce more singlet oxygen upon illumination
luminescence emanating from 1 (red) confirmed its nuclear with light than their TAP analogues.13,31 It was also noted that
uptake in some cells. Nuclear localisation was also seen in the the morphology of HeLa cells observed by confocal microscopy
bright field image where cells exhibited a darker appearance after 24 h incubation with 1 and 1·AuNP showed that the gold
as a result of the high overload of 1 inside the cells. However, conjugate was better tolerated by the cells than its free
Dalton Trans.
This journal is © The Royal Society of Chemistry 2020

View Article Online
Dalton Transactions
Paper
Fig. 6 3D confocal fluorescence microscopy images generated from Z-stack studies showing the cytoplasm effective internalisation of (a) 1·AuNP
and (b) 2·AuNP (red) at ca. 20 µM Ru(II) complex concentration in HeLa cells after 24 h incubation. The nucleus is stained blue with Hoechst 33258.
The fluorescence of the extracellular medium was saturated with fluorescein sodium salt (5 µM) to maximise the contrast between the inside and
the outside of the cells and obtain a better definition of their outline.
complex counterpart, suggesting a decrease in the cytotoxicity 1·AuNP and 2·AuNP has not yet been assessed, the morphology
of 1 when bound to the gold surface.
of the cells treated with 1·AuNP for 24 h pointed to a lower tox-
icity of this system when compared to its free complex ana-
logue 1. In conclusion, these results represent a contribution
to the potential bioimaging applications of the nanomaterials
resulting from the combination of Ru(II) polypyridyl complexes
and AuNPs. Furthermore, we foresee that this work can be a
stepping-stone in paving the way for development of alterna-
tively drug-delivery systems in the future, where Ru(II)-polypyri-
dyl complexes can be selectively delivered/removed and the
release monitored in real time, from the AuNPs surface, such
as upon enzymatic cleavage.
Conclusions
In this study, we have reported the synthesis, photophysical
characterisation and biological evaluation of two new dinuc-
lear Ru(II) polypyridyl complexes containing either phen (1) or
TAP (2) as ancillary ligands and their corresponding gold con-
jugates (1·AuNP and 2·AuNP) resulted from their attachment
onto the surface of AuNPs with an average diameter of ca.
2.5 nm. The free complexes 1 and 2 have shown the distinctive
absorption and emission properties associated with Ru(II) poly-
pyridyl complexes that has made them attractive candidates to
be explored as conjugates for AuNPs. Thus, absorption pro-
perties characteristic of both the AuNP SPR and the Ru(II)
complex MLCT absorption bands have been observed for the
Experimental
Materials and methods
Ru(II)-AuNPs conjugates 1·AuNP and 2·AuNP, while their emis- All chemicals and solvents were obtained commercially and,
sive behaviour is due to the Ru(II) MLCT excited state. unless specified, used without further purification. Solvents
However, these nanosystems have shown a lower quantum for synthetic purposes were used at general purpose reagent
yield when compared to the free complexes 1 and 2 presumed (GPR) grade unless otherwise stated. Dry solvents were
to be due to an energy-transfer quenching of the Ru(II) lumine- obtained from a solvent purification system (SPS) purchased
scence by interaction with the gold surface.
from Innovative Technology Inc.
The ability of 1 and 2 to be rapidly taken up by HeLa cells
All NMR spectra were recorded using a Bruker Avance III
1
has been confirmed by confocal microscopy studies. Despite 400 NMR spectrometer operating at 400 MHz for H NMR and
their diminished luminescence, 1·AuNP and 2·AuNP have also 101 MHz for 13C NMR. Chemical shifts (δ) were referenced
been found to be successfully internalised into HeLa cells with relative to the internal solvent signals. Matrix-assisted laser de-
localisation in their cytoplasm over time. Additional cyto- sorption/ionization (MALDI) mass spectra were recorded on a
toxicity studies have demonstrated that while both 1 and 2 MALDI QToF Premier (Waters Corporation, Micromass MS
were only moderately toxic in dark conditions, their illumina- Technologies, Manchester, UK) and high resolution (HR) mass
tion with visible light resulted in an increase in toxicity, in par- spectra were determined by a peak matching method using
ticular for the phen derivative 1. Although the origin of such Glu-Fib as an internal reference (m/z = 1570.677). Infrared
photoactivation has not yet been investigated, singlet oxygen spectra were recorded on a PerkinElmer Spectrum One FT-IR
production is probably one of the major pathways by which spectrometer fitted with a Universal ATR Sampling Accessory
these complexes become toxic after light activation. for solid samples. Melting points were determined using an
Furthermore, despite the cytotoxicity of the gold conjugates IA9000 digital melting point apparatus. Elemental analyses
This journal is © The Royal Society of Chemistry 2020
Dalton Trans.

View Article Online
Paper
Dalton Transactions
were conducted at the Department of Chemistry, Maynooth was then treated with 20 µL of Alamar Blue (BioSource) and
University. left incubating at 37 °C in the dark for 4 h. Fluorescence was
UV-vis absorption spectra were recorded on a Varian CARY read using a fluorescence microplate reader (SpectraMax
50 spectrophotometer with a wavelength range of 200–900 nm Gemini XS, Molecular Devices) at 590 nm (excitation at
and a scan rate of 600 nm min−1. Baseline correction measure- 544 nm). The data were analysed using the SoftMax® Pro
ments were used for all spectra. Emission and excitation Software. The background fluorescence of the media without
spectra were recorded on a Varian Carey Eclipse Fluorimeter. cells plus Alamar Blue was taken away from each group, and
Luminescence quantum yields were calculated using [Ru the control untreated cells represented 100% cell viability.
(bpy)3]Cl2 as reference (Φem = 0.028, in air-saturated aqueous Data points represent the mean
S.E.M. of triplicate treat-
solution).32 Luminescence lifetimes were measured by single- ments performed on three independent days with activity
photon timing (SPT) on a Fluorolog FL 3–22 equipped with a expressed as percentage cell viability compared to vehicle
FluoroHub v2.0 single-photon timing module using a 458 nm treated controls. For photoactivation studies, cells were sub-
pulsed nanosecond light-emitting diode (Horiba N-460) as jected to 18 J cm−2 using a Hamamatsu L2570 200 W Hg–Xe
excitation source.
arc lamp equipped with a NaNO2 filter.
Dynamic Light Scattering (DLS) measurements were per-
formed at 298 K with a Malvern Instruments Zetasizer Nano
Synthesis and characterisation
ZS provided with a 633 nm He–Ne laser (4 mW) as the light 1,4,5,8-Tetraazaphenanthrene (TAP),37 4-(3-aminophenyl)-2,2′-
source. Prior to each measurement, the appropriate Ru(II) poly- bipyridine,38 and the precursors complexes cis-[Ru(phen)2Cl2]
pyridyl functionalised-AuNP sample (ASPR ≈ 0.03) was centri- and cis-[Ru(TAP)2Cl2],39 were synthesised according to pro-
fuged for 60 min at 3900 rpm to break up any possible aggre- cedures previously reported in the literature.
gates. Transmission Electron Microscopy (TEM) analysis were
11,11′-Disulfanediylbis(N-(3-([2,2′-bipyridin]-4-yl)phenyl)-undec-
conducted in the CRANN – Advanced Microscopy Laboratory anamide) (3). 4-(3-Aminophenyl)-2,2′-bipyridine (88.2 mg,
(AML) at Trinity College Dublin using a Jeol 2100 transmission 357 µmol, 1 eq.) was added to dry CH2Cl2 (10 mL) and the
electron microscope operating at 200 kV. Samples were pre- solution was cooled to 0 °C before 11,11′-disulfanediyldiunde-
pared by adsorbing a drop of the appropriate Ru(II) polypyridyl canoic acid (77.6 mg, 178 µmol, 0.5 eq.), N-ethyl-N′-(3-di-
functionalised-AuNP solution (250 µM) onto a 200-mesh methylaminopropyl)carbodiimide hydrochloride (181 mg,
formvar film stabilised with carbon copper grid, with the 942 µmol, 2.6 eq.) and 4-dimethylaminopyridine (53.2 mg,
excess solution being wicked off using filter paper after 10 min 436 µmol, 1.2 eq.) were added to the solution. The resulting
of adsorption time.
mixture was stirred under inert atmosphere and at 0 °C for 1 h
and then for a further 2 days at room temperature. After remov-
ing the solvent under reduced pressure an orange oil was
General biological procedures
HeLa cells were grown in a cell culture flask using Dulbecco’s obtained which was dried in vacuo. The addition of H2O
Modified Eagle Medium supplemented with 10% fetal bovine caused precipitation of a beige solid which was isolated by cen-
serum, 1% penicillin/streptomycin and 0.2% plasmocin at trifugation and washed several times with more H2O. The
37 °C in a humidified atmosphere of 5% CO2.
resulting solid was redispersed in MeCN, collected by centrifu-
Confocal microscopy experiments were carried out using gation and dried in vacuo, yielding the product as a beige solid
HeLa cells seeded at a density of 5 × 104 cells per mL and (115 mg, 129 µmol, 72%). M.p. 85–87 °C. δH (400 MHz, DMSO-
3
treated as indicated. Cells were then washed twice with fresh d6): 10.08 (1H, s, NH), 8.75 (1H, d, H13, J = 5.2 Hz), 8.73 (1H,
medium and stained blue with Hoechst 33258 (10 µg mL−1). ddd, H2, 3J = 4.8 Hz, 4J = 1.8 Hz, 4J = 0.8 Hz), 8.65 (1H, d, H6, 4J
3
For the Z-stack studies, an aqueous solution of fluorescein = 1.8 Hz), 8.44 (1H, d, H5, J = 7.9 Hz), 8.12 (1H, s, H11), 7.97
3
4
3
sodium salt (5 µM) was also added to the cells in order to (1H, td, H4, J = 7.9 Hz, J = 1.8 Hz), 7.74 (1H, d, H9, J = 8.3
3
4
make the extracellular medium fluorescent, as it is a water- Hz), 7.72 (1H, dd, H12, J = 5.2 Hz, J = 1.8 Hz), 7.49 (3H, m,
soluble fluorescent dye which is not taken up by live cells. By H3, H7 and H8), 2.63 (2H, t, H11′, 3J = 7.2 Hz), 2.32 (2H, t, H2′, 3J
fluorescent saturation of the extracellular medium, the con- = 7.3 Hz), 1.57 (4H, m, H3′ and H10′), 1.25 (12H, m, H4′–H9′).40
trast between the inside and the outside of the cells is maxi- δC (101 MHz, DMSO-d6): 171.59 (CvO), 155.95 (q), 155.04 (q),
mised resulting in better definition of the outline of the cells. 150.10, 149.29, 148.12 (q), 140.28 (q), 137.68 (q), 137.41,
Cells were then imaged by live microscopy using a Leica 129.74, 124.40, 121.48, 121.34, 120.65, 119.81, 117.38, 117.03,
SP8 gated STED confocal microscope with a 40× oil immersion 37.88, 36.48, 28.83, 28.81, 28.74, 28.64, 28.51, 28.48, 27.68,
lens (NA 1.30). A 405 nm diode laser was used to excite both 25.05. νmax (ATR)/cm−1: 3283 (amide N–H stretch), 3061 (aro-
Hoechst and the appropriate ruthenium system and the emis- matic C–H stretch), 2918 and 2848 (alkane C–H stretch), 1656
sion was measured at 415–505 nm and 610–715 nm, respect- (CvO stretch), 1543 (amide N–H bend), 1464 (aromatic CvC
ively. The software used to collect images was Leica stretch), 1425 (C–N stretch). MALDI+-HRMS: m/zcalc = 893.4610
Application Suite X (LAS X).
for C54H65N6O2S2; m/zfound = 893.4639 [M + H]+.
Tetra(1,10-phenanthroline)(11,11′-disulfanediylbis(N-(3-([2,2′-
Viability assays were undertaken by seeding of 2.5 × 103
cells per mL in a 96-well plate and treated with different con- bipyridin]-4-yl)phenyl) undecanamide))diruthenium(II) chloride
centration of the appropriate Ru(II) complex for 48 h. Each well (1). The precursor complex cis-[Ru(phen)2Cl2] (50.5 mg,
Dalton Trans.
This journal is © The Royal Society of Chemistry 2020

View Article Online
Dalton Transactions
Paper
94.8 µmol, 2.2 eq.) and ligand 3 (38.6 mg, 43.2 µmol, 1 eq.) 3052 (aromatic C–H stretch), 2922 and 2851 (alkane C–H
were suspended in EtOH/H2O (1 : 1, 8 mL). The mixture was stretch), 1665 (CvO stretch), 1536 (amide N–H bend), 1485
deoxygenated by sparging with argon for 15 min and heated at (aromatic CvC stretch), 1406 (C–N stretch). MALDI+-HRMS:
140 °C for 40 min using microwave irradiation. Solvent was m/zcalc
=
1823.4911 for C94H87N22O2S2Ru2; m/zfound
=
removed at reduced pressure and the resulting solid was puri- 1823.5002 [M − H]+.
fied by alumina chromatography using MeCN/H2O (10 : 0 to
Synthesis of Ru(II) stabilised AuNPs (1·AuNP and
9 : 1) as eluent, yielding the product as a red solid which was 2·AuNP).15,21,22 HAuCl4·3H2O (0.10 g, 0.25 mmol, 1 eq.) was
dried in vacuo (65.6 mg, 34.8 µmol, 80%). Calculated for dissolved in H2O (10 mL) before a solution of TOAB (0.36 g,
C102H96N14Cl4O2S2Ru2 + 16H2O: C, 54.54; H, 5.74; N, 8.73. 0.64 mmol, 2.6 eq.) in toluene (25 mL) was added. The result-
Found: C, 54.04; H, 5.21; N, 9.07. δH (400 MHz, DMSO-d6): ing mixture was stirred vigorously at room temperature for
10.43 (1H, s, NH), 9.16 (1H, d, H5L3, J = 8.2 Hz), 9.10 (1H, d, 10 min and NaBH4 (0.12 g, 3.17 mmol, 12.7 eq.) dissolved in
3
4
3
H6L3, J = 1.4 Hz), 8.85 (2H, d, Hphen, J = 8.2 Hz), 8.75 (2H, dt, H2O (10 mL) was added dropwise. The reaction mixture was
Hphen, 3J = 8.3 Hz, 4J = 1.1 Hz), 8.38 (5H, m, H5phen and H6
,
stirred for a further 2 h. Following successful transfer of the Au
phen
Hphen), 8.33 (1H, s, H11L3), 8.26 (1H, dd, Hphen, 3J = 5.2 Hz, 4J = (0) into the toluene layer, the organic and aqueous layers were
3
4
1.1 Hz), 8.17 (1H, td, H4L3, J = 8.2 Hz, J = 1.3 Hz), 7.96 (4H, separated, and the toluene layer was washed with H2O (2 ×
m, Hphen, H2 and H13L3), 7.73 (4H, m, Hphen), 7.67 (2H, dd, 20 mL), 0.1 M HCl (2 × 20 mL) and 0.1 NaOH (2 × 20 mL). The
L3
H8L3 and H9L3, 3J = 8.0 Hz, 4J = 1.7 Hz), 7.60 (1H, dd, H12L3, 3J = TOAB-stabilised AuNPs (5 mL) were added to an aqueous solu-
6.0 Hz, 4J = 1.4 Hz), 7.48 (1H, t, H7L3, 3J = 8.0 Hz), 7.43 (1H, m, tion of 1 or 2 (0.5 eq., 5 mL) and the resulting mixture was
H3L3), 2.65 (2H, t, H11′L3, 3J = 7.2 Hz), 2.34 (2H, t, H2′L3, 3J = 7.4 stirred at room temperature for 16 h. Following successful
Hz), 1.57 (4H, m, H3′ and H10′L3), 1.23 (12H, m, H4′–H9′L3).40 transfer of the AuNPs into the aqueous layer, the organic and
L3
δC (101 MHz, DMSO-d6): 171.68 (CvO), 157.40 (q), 157.00 (q), the aqueous layer were separated, and the aqueous layer was
152.72, 152.58, 152.44, 152.40, 151.86, 151.70, 152.44, 152.40, subsequently filtered through a PDVF 0.45 µm microsyringe to
148.56 (q), 147.17 (q), 147.15 (q), 146.91 (q), 146.89 (q), 140.36 give a dark brown solution. Any unbound Ru(II) complex was
(q), 137.78, 136.93, 136.91, 136.80, 135.73 (q), 130.53 (q), then removed by addition of a saturated aqueous solution of
130.49 (q), 130.43 (q), 129.61, 128.07, 127.79, 126.46, 126.35, NH4PF6 (3 mL) affording flocculation of a dark solid which
124.78, 124.61, 121.97, 121.54, 120.88, 117.82, 37.79, 36.32, 28.86, was collected by centrifugation and washed with a mixture of
28.85, 28.79, 28.65, 28.54, 28.48, 27.69, 25.04. νmax (ATR)/cm−1
:
H2O/MeOH (3 : 1, 3 × 5 mL). The solid was redissolved in
3247 (amide N–H stretch), 3053 (aromatic C–H stretch), 2923 and MeCN (1 mL) and repeatedly precipitated by adding Et2O. The
2851 (alkane C–H stretch), 1665 (CvO stretch), 1550 (amide N–H resulting solid was redispersed in MeCN (1 mL) before
bend), 1469 (aromatic CvC stretch), 1426 (C–N stretch). MALDI+- addition of an acetone concentrated solution of TBACl (3 mL)
HRMS: m/zcalc = 1815.5331 for C102H95N14O2S2Ru; m/zfound
=
resulting in the formation of a dark brown flocculate which
was collected by centrifugation, washed with acetone (3 ×
1815.5494 [M − H]+.
Tetra(1,4,5,8-tetraazaphenantherene)(11,11′-disulfanedi-ylbis 5 mL) and dried under high vacuum before being resuspended
(N-(3-([2,2′-bipyridin]-4-yl)phenyl)undecanamide))diru-thenium in H2O to give a dark brown colloidal solution.
(II) chloride (2). Complex 2 was synthesised according to the
For 1·AuNP the Ru : Au ratio was estimated from the absor-
same procedure described for 1 but using cis-[Ru(TAP)2Cl2] bance at 453 nm assuming extinction coefficients ε453 for the
(104 mg, 193 µmol, 2.1 eq.) as precursor complex and ligand 3 bound ruthenium complex of 18 000 (i.e. half of that for the
(84.1 mg, 94.1 µmol, 1 eq.), yielding the product as a red dinuclear complex 1) and of 1.41 × 106 for the functionalised
solid (48.1 mg, 24.5 µmol, 26%). Calculated for nanoparticle at the SPR maximum. This latter value is calcu-
C94H88N22Cl4O2S2Ru2 + 16.5H2O: C, 49.88; H, 5.39; N, 13.62. lated for a nanoparticle of average diameter 2.75 nm and 640
Found: C, 49.22; H, 4.62; N, 13.37. δH (400 MHz, DMSO-d6): Au atoms using the equations in the paper of Liu et al.41
L3
10.58 (1H, s, NH), 9.22 (3H, m, H5 and HTAP), 9.13 (1H, d, Similarly for 2·AuNP measured at 416 nm and using ε416
=
4
3
H6L3, J = 1.7 Hz), 9.02 (2H, d, HTAP, J = 2.8 Hz), 8.66 (4H, m, 19 500 for bound 2 and ε530 = 7.2 × 105 for an average 2.25 nm
H9TAP and H10TAP), 8.52 (1H, d, HTAP, J = 2.9 Hz), 8.44 (2H, m, particle. These calculations give Ru : Au of 1 : 7.9 for 1·AuNP
3
HTAP), 8.38 (2H, m, H11L3 and HTAP), 8.26 (1H, td, H4L3, 3J = 7.9 and 1 : 9.8 for 2·AuNP.
Hz, 4J = 1.2 Hz), 7.91 (2H, m, H2 and H13L3), 7.71 (2H, m,
L3
L3
3
4
H7 and H9L3), 7.60 (1H, dd, H12L3, J = 6.1 Hz, J = 1.7 Hz),
7.49 (2H, m, H3 and H8L3), 2.66 (2H, t, H11′L3, J = 7.2 Hz),
L3
3
Conflicts of interest
3
L3
2.34 (2H, t, H2′L3, J = 7.5 Hz), 1.58 (4H, m, H3′ and H10′L3),
1.26 (12H, m, H4′–H9′L3).40 δC (101 MHz, DMSO-d6): 171.74 There are no conflicts to declare.
(CvO), 156.76 (q), 156.37 (q), 152.94, 152.85, 149.78, 149.76,
149.29, 149.25, 148.62, 148.52, 144.55 (q), 144.50 (q), 141.88
(q), 141.59 (q), 140.46 (q), 139.00, 135.22 (q), 132.49, 132.44,
132.32, 129.64, 128.05, 124.96, 124.69, 122.02, 121.71, 121.04
Acknowledgements
(q), 121.03, 117.88, 37.81, 36.33, 28.87, 28.81, 28.68, 28.55, We thank Science Foundation Ireland (SFI PI Awards 10/45
28.49, 27.70, 25.07. νmax (ATR)/cm−1: 3244 (amide N–H stretch), IN.1/B2999 and 13/IA/1865) and Trinity College Dublin (TCD)
This journal is © The Royal Society of Chemistry 2020
Dalton Trans.

View Article Online
Paper
Dalton Transactions
for financial support. We also thank Drs Feeney, Hessman,
O’Brien and Ruether for the help with MS and NMR studies. TEM
analysis was performed at the AMBER Centre and the CRANN
Advanced Microscopy Laboratory at Trinity College Dublin. Finally,
we would like to thank Prof. Susan Quinn (University College
Soc. Rev., 2017, 46, 7706; M. Jakubaszek, B. Goud, S. Ferrari
and G. Gasser, Chem. Commun., 2018, 54, 13040; S. Monro,
K. L. Colón, H. Yin, J. Roque, P. Konda, S. Gujar,
R. P. Thummel, L. Lilge, C. G. Cameron and
S. A. McFarland, Chem. Rev., 2019, 119, 797.
Dublin) and Robert Elmes (Maynooth University) for their helpful 13 S. Estalayo-Adrián, S. Blasco, S. A. Bright, G. J. McManus,
discussion and suggestions during the preparation of this manu-
script and for their long-standing collaborations.
G. Orellana, D. C. Williams, J. M. Kelly and
T. Gunnlaugsson, Chem. Commun., 2020, 56, 9332–9335.
14 P. Pramod, P. K. Sudeep, K. G. Thomas and P. V. Kamat,
J. Phys. Chem. B, 2006, 110, 20737; N. J. Rogers, S. Claire,
R. M. Harris, S. Farabi, G. Zikeli, I. B. Styles, N. J. Hodges
and Z. Pikramenou, Chem. Commun., 2014, 50, 617;
J. Moreau, F. Lux, M. Four, J. Olesiak-Banska,
K. Matczyszyn, P. Perriat, C. Frochot, P. Arnoux,
O. Tillement, M. Samoc, G. Ponterini, S. Roux and
G. Lemercier, Phys. Chem. Chem. Phys., 2014, 16, 14826;
P. Zhang, J. Wang, H. Huang, B. Yu, K. Qiu, J. Huang,
S. Wang, L. Jiang, G. Gasser, L. Ji and H. Chao,
Biomaterials, 2015, 63, 102; S. A. M. Osborne and
Z. Pikramenou, Faraday Discuss., 2015, 185, 219.
Notes and references
1 C. Quintana, M.P. Cifuentes and M. G. Humphrey, Chem.
Soc. Rev., 2020, 49, 2316; D. A. Giljohann, D. S. Seferos,
W. L. Daniel, M. D. Massich, P. C. Patel and C. A. Mirkin,
Angew. Chem., Int. Ed., 2010, 49, 3280; K. Saha, S. S. Agasti,
C. Kim, X. Li and V. M. Rotello, Chem. Rev., 2012, 112,
2739; C. M. Cobley, J. Chen, E. Chul Cho, L. V. Wang and
Y. Xia, Chem Soc. Rev., 2011, 40, 44; A. J. Mieszawska,
W. J. M. Mulder, Z. A. Fayad and D. P. Cormode, Mol.
Pharmaceutics, 2013, 10, 831; H. Jans, and Q. Huo, Chem. 15 R. B. P. Elmes, K. N. Orange, S. M. Cloonan, D. C. Williams
Soc. Rev., 2012, 41, 2849. and T. Gunnlaugsson, J. Am. Chem. Soc., 2011, 133, 15862.
2 E. C. Dreaden, M. A. Mackey, X. Huang, B. Kang and 16 F. C.-M. Leung, A. Y.-Y. Tam, V. K.-M. Au, M.-J. Li and V.
M. A. El-Sayed, Chem. Soc. Rev., 2011, 40, 3391. W.-W. Yam, ACS Appl. Mater. Interfaces, 2014, 6, 6644.
3 E. C. Dreaden, A. M. Alkilany, X. Huang, C. J. Murphy and 17 M. Martínez-Calvo, K. N. Orange, R. B. P. Elmes, B. la Cour
M. A. El-Sayed, Chem. Soc. Rev., 2012, 41, 2740.
4 N. Elahi, M. Kamali and M. H. Baghersad, Talanta, 2018,
184, 537.
5 B. D. Chithrani, A. A. Ghazani and W. C. W. Chan, Nano
Lett., 2006, 6, 662.
6 W. Jiang, B. Y. S. Kim, J. T. Rutka and W. C. W. Chan, Nat.
Nanotechnol., 2008, 3, 145; X. Xie, J. Liao, X. Shao, Q. Li
and Y. Lin, Sci. Rep., 2017, 7, 3827; L. Ding, C. Yao, X. Yin,
C. Li, Y. Huang, M. Wu, B. Wang, X. Guo, Y. Wang and
M. Wu, Small, 2018, 14, 1801451; P. Foroozandeh and
A. A. Aziz, Nanoscale Res. Lett., 2018, 13, 339.
Poulsen, D. C. Williams and T. Gunnlaugsson, Nanoscale,
2016, 8, 563.
18 11,11′-Disulfanediyldiundecanoic acid was used instead of
11-mercaptoundecanoic acid as formation of the disul-
phide provides protection of the thiol group which can
otherwise undergo nucleophilic attack with the carbonyl
group under the conditions required for the next synthetic
step. Previous experiments have demonstrated no for-
mation, or formation in a low yield, of the desired product
when 11-mercaptoundecanoic acid was used instead of
11,11′-disulfanediyldiundecanoic acid.
7 M. E. Davis, Z. Chen and D. M. Shin, Nat. Rev. Drug 19 J. Massue, S. J. Quinn and T. Gunnlaugsson, J. Am. Chem.
Discovery, 2008, 7, 771.
8 P. M. Tiwari, K. Vig, V. A. Dennis and S. R. Singh,
Nanomaterials, 2011, 1, 31; N. J. Rogers, H. C. Jeffery,
Soc., 2008, 130, 6900; S. Comby, E. M. Surender, O. Kotova,
L. K. Truman, J. K. Molloy and T. Gunnlaugsson, Inorg.
Chem., 2014, 53, 1867.
S. Claire, D. J. Lewis, G. Zikeli, N. J. Hodges, S. Egginton, 20 S. Comby and T. Gunnlaugsson, ACS Nano, 2011, 5, 7184;
G. B. Nash and Z. Pikramenou, Nanomedicine, 2017, 12,
2725; E. M. Surender, S. Comby, B. Cavanagh, O. Brennan,
T. C. Lee and T. Gunnlaugsson, Chem, 2016, 1, 438.
9 S. S. Lucky, K. C. Soo and Y. Zhang, Chem. Rev., 2015, 115,
1990.
L. K. Truman, S. Comby and T. Gunnlaugsson, Angew.
Chem., Int. Ed., 2012, 51, 9624; L. K. Truman,
S. J. Bradberry, S. Comby, O. Kotova and T. Gunnlaugsson,
ChemPhysChem, 2017, 18, 1746; J. K. Molloy, A. M. Nonat,
J. E. O’Brien, D. F. Brougham and T. Gunnlaugsson,
Supramol. Chem., 2020, 32, 373–382.
10 A. Juris, V. Balzani, F. Barigelletti, S. Campagna, P. Belser
and A. von Zelewsky, Coord. Chem. Rev., 1988, 84, 85; 21 M. Brust, M. Walker, D. Bethell, D. J. Schiffrin and
V. Balzani, A. Juris, M. Venturi, S. Campagna and R. Whyman, J. Chem. Soc., Chem. Commun., 1994, 801.
S. Serroni, Chem. Rev., 1996, 96, 759; J. G. Vos and 22 C. R. Mayer, E. Dumas and F. Sécheresse, J. Colloid
J. M. Kelly, Dalton Trans., 2006, 4869. Interface Sci., 2008, 328, 452.
11 M. R. Gill and J. A. Thomas, Chem. Soc. Rev., 2012, 41, 23 B. J. Berne and R. Pecora, Dynamic Light Scattering: With
3179.
Applications to Chemistry, Biology, and Physics, Dover
Publications, 2013; L. Tong, E. Lu, J. Pichaandi, P. Cao,
M. Nitz and M. A. Winnik, Chem. Mater., 2015, 27, 4899.
12 C. Mari, V. Pierroz, S. Ferrari and G. Gasser, Chem. Sci.,
2015, 6, 2660; F. E. Poynton, S. A. Bright, S. Blasco,
D. C. Williams, J. M. Kelly and T. Gunnlaugsson, Chem. 24 M. C. Daniel and D. Astruc, Chem. Rev., 2004, 104, 293.
Dalton Trans.
This journal is © The Royal Society of Chemistry 2020

View Article Online
Dalton Transactions
Paper
25 A. M. Pyle, J. P. Rehmann, R. Meshoyrer, C. V. Kumar, 32 A. M. Brouwer, Pure Appl. Chem., 2011, 83, 2213.
N. J. Turro and J. K. Barton, J. Am. Chem. Soc., 1989, 111, 3051.
26 A. Masschelein, L. Jacquet, A. Kirsch-De Mesmaeker and
J. Nasielski, Inorg. Chem., 1990, 29, 855.
27 K. Karlsson and A. Kirsch-De Mesmaeker, J. Phys. Chem.,
1991, 95, 10681.
33 Reported data correspond to the pre-exponential weighted
mean lifetimes (τM) and were calculated according to
τM = Σ(aiτi)/Σai (air: τ1 = 229 ns (83%), τ2 = 572 ns (17%);
N2: τ1 = 232 ns (86%), τ2 = 592 ns (14%)).
34 T. Huang and R. W. Murray, Langmuir, 2002, 18, 7077.
28 L. Marcélis, J. Ghesquière, K. Garnir, A. Kirsch-De 35 T. A. Taton, Curr. Protoc. Nucleic Acid Chem., 2002, 9,
Mesmaeker and C. Moucheron, Coord. Chem. Rev., 2012, 12.2.1.
256, 1569; S. Estalayo-Adrián, K. Garnir and C. Moucheron, 36 B. Yameen, W. I. Choi, C. Vilos, A. Swami, J. Shi and
Chem. Commun., 2018, 54, 322. O. C. Farokhzad, J. Controlled Release, 2014, 190, 485.
29 I. Urriza-Arsuaga, M. Bedoya and G. Orellana, Anal. Chem., 37 R. Nasielski-Hinkens, M. Benedek-Vamos, Y. Hautain and
2019, 91, 2231. J. Nasielski, Bull. Soc. Chim. Belg., 1976, 85, 781.
30 J.-P. Lecomte, A. Kirsch-De Mesmaeker, J. M. Kelly, 38 R. B. P. Elmes, M. Erby, S. A. Bright, D. C. Williams and
A. B. Tossi and H. Görner, Photochem. Photobiol., 1992, 55, T. Gunnlaugsson, Chem. Commun., 2012, 48, 2588.
681; J.-P. Lecomte, A. Kirsch-De Mesmaeker, M. M. Feeney 39 T. Doi, H. Nagamiya, M. Kokubo, K. Hirabayashi and
and J. M. Kelly, Inorg. Chem., 1995, 34, 6481; S. Kajouj, T. Takahashi, Tetrahedron, 2002, 58, 2957.
L. Marcélis, V. Lemaur, D. Beljonne and C. Moucheron, 40 Ligand 3 and complexes 1 and 2 are dimeric in nature and
Dalton Trans., 2017, 46, 6623.
31 L. Marcélis, S. Kajouj, J. Ghesquière, G. Fettweis,
I. Coupienne, R. Lartia, M. Surin, E. Defrancq, J. Piette,
only half of the molecule was considered for the signal
integration since protons from both halves of the molecule
are equivalent.
C. Moucheron and A. Kirsch-De Mesmaeker, Eur. J. Inorg. 41 X. Liu, M. Atwater, J. Wang and Q. Huo, Colloids Surf., B,
Chem., 2016, 2016, 2902.
2007, 58, 3.
This journal is © The Royal Society of Chemistry 2020
Dalton Trans.