Upconverting Nanoparticles Prompt Remote Near-Infrared Photoactivation of Ru(II)-Arene Complexes

Article abstract of DOI:10.1002/chem.201503991

The synthesis and full characterisation (including X-ray diffraction studies and DFT calculations) of two new piano-stool Ru^II-arene complexes, namely [(η⁶-p-cym)Ru(bpy)(m-CCH-Py)][(PF)₆]₂ (1) and [(η⁶-p-cym)Ru(bpm)(m-CCH-Py)][(PF)₆]₂ (2; p-cym=p-cymene, bpy=2,2′-bipyridine, bpm=2,2′-bipyrimidine, and m-CCH-Py=3-ethynylpyridine), is described and discussed. The reaction of the m-CCH-Py ligand of 1 and 2 with diethyl-3-azidopropyl phosphonate by Cu-catalysed click chemistry affords [(η⁶-p-cym)Ru(bpy)(P-Trz-Py)][(PF)₆]₂ (3) and [(η⁶-p-cym)Ru(bpm)(P-Trz-Py)][(PF)₆]₂ (4; P-Trz-Py=[3-(1-pyridin-3-yl-[1,2,3]triazol-4-yl)-propyl]phosphonic acid diethyl ester). Upon light excitation at λ=395 nm, complexes 1-4 photodissociate the monodentate pyridyl ligand and form the aqua adduct ions [(η⁶-p-cym)Ru(bpy)(H₂O)]²⁺ and [(η⁶-p-cym)Ru(bpm)(H₂O)]²⁺. Thulium -doped upconverting nanoparticles (UCNPs) are functionalised with 4, thus exploiting their surface affinity for the phosphonate group in the complex. The so-obtained nanosystem UCNP@4 undergoes near-infrared (NIR) photoactivation at λ=980 nm, thus producing the corresponding reactive aqua species that binds the DNA-model base guanosine 5′-monophosphate.

Full text of DOI:10.1002/chem.201503991

DOI: 10.1002/chem.201503991
Full Paper
&
Photochemotherapeutics
Upconverting Nanoparticles Prompt Remote Near-Infrared
Photoactivation of Ru(II)–Arene Complexes
Emmanuel Ruggiero,[a] Claudio Garino,[b] Juan C. Mareque-Rivas,[a, c]
Abraha Habtemariam,*[a, c, d] and Luca Salassa*[a, e]
In memory of Jon Mikel Azpiroz
Abstract: The synthesis and full characterisation (including
X-ray diffraction studies and DFT calculations) of two new
piano-stool RuII–arene complexes, namely [(h6-p-cym)Ru(b-
py)(m-CCH-Py)][(PF)6]2 (1) and [(h6-p-cym)Ru(bpm)(m-CCH-
Py)][(PF)6]2 (2; p-cym=p-cymene, bpy=2,2’-bipyridine,
bpm=2,2’-bipyrimidine, and m-CCH-Py=3-ethynylpyridine),
is described and discussed. The reaction of the m-CCH-Py
ligand of 1 and 2 with diethyl-3-azidopropyl phosphonate
by Cu-catalysed click chemistry affords [(h6-p-cym)Ru(bpy)(P-
Trz-Py)][(PF)6]2 (3) and [(h6-p-cym)Ru(bpm)(P-Trz-Py)][(PF)6]2
(4; P-Trz-Py=[3-(1-pyridin-3-yl-[1,2,3]triazol-4-yl)-propyl]phos-
phonic acid diethyl ester). Upon light excitation at l=
395 nm, complexes 1–4 photodissociate the monodentate
pyridyl ligand and form the aqua adduct ions [(h6-p-cym)-
Ru(bpy)(H2O)]2+ and [(h6-p-cym)Ru(bpm)(H2O)]2+. Thulium
-doped upconverting nanoparticles (UCNPs) are functional-
ised with 4, thus exploiting their surface affinity for the
phosphonate group in the complex. The so-obtained nano-
system UCNP@4 undergoes near-infrared (NIR) photoactiva-
tion at l=980 nm, thus producing the corresponding reac-
tive aqua species that binds the DNA-model base guanosine
5’-monophosphate.
systems have been designed as neuroscience tools,[2] drug de-
livery platforms,[3,4] and anticancer prodrugs.[5,6]
Introduction
Encouraged by the clinical success of photodynamic therapy
(PDT),[1] light-activatable molecules and nanomaterials are
being increasingly investigated for their capacity to generate
biologically active species in situ with high spatiotemporal
control. In principle, such an attractive strategy allows the bio-
logical effects of drugs to be localised, thus potentially reduc-
ing their therapeutic drawbacks. For this reason, light activa-
tion has found application in a number of fields and diverse
Promising metal-based photo-chemotherapeutics have been
developed that exploit the unique photochemical and antitu-
mor properties of transition-metal complexes. Several research
groups worldwide have demonstrated that light-activatable
complexes of various transition metals (e.g., Pt, Ru, Rh, and Ir)
show encouraging antineoplastic profiles and unconventional
mechanisms of action relative to their ground-state ana-
logues.[4–7]
Nevertheless, the poor absorption properties of metal com-
plexes in the therapeutic window of the red and near-infrared
(NIR) spectrum (ca. l=600–1000 nm) pose a serious limitation
to advancing their use toward preclinical and clinical studies
because maximal light penetration into tissues is achieved in
this wavelength range and damage to biological components
is minimised.[8,9] Relative to PDT photosensitisers, photoactivat-
able anticancer metal complexes generally display low-energy
absorption bands that rarely extend over l=550 nm and have
modest extinction coefficients. Although a few exceptions
have been reported,[10–12] it is extremely challenging to improve
absorption features without altering the ground-state stability
and photoreactivity of the metal complexes.
[a] E. Ruggiero, Prof. J. C. Mareque-Rivas, Dr. A. Habtemariam, Dr. L. Salassa
CIC biomaGUNE, Paseo de Miramón182
20009, Donostia-San Sebastiµn Euskadi (Spain)
E-mail: a.habtemariam@warwick.ac.uk
lsalassa@cicbiomagune.es
[b] Dr. C. Garino
Department of Chemistry and NIS Centre of Excellence
University of Turin, via Pietro Giuria 7, 10125 Turin (Italy)
[c] Prof. J. C. Mareque-Rivas, Dr. A. Habtemariam
Ikerbasque, Basque Foundation for Science
48011 Bilbao (Spain)
[d] Dr. A. Habtemariam
Department of Chemistry, University of Warwick
Coventry CV4 7AL (UK)
For this reason, we and other groups have recently started
to explore the use of upconversion nanoparticles (UCNPs) as
phototriggers for the NIR photoactivation of (anticancer) metal
complexes.[9,13–20] UCNPs are typically NaYF4 or NaGdF4 nano-
crystals doped with lanthanide ions, such as Yb3+:Tm3+ or
Yb3+:Er3+, which convert NIR light (l=980 nm) into UV/Vis
[e] Dr. L. Salassa
Kimika Fakultatea, Euskal Herriko Unibertsitatea and
Donostia International Physics Center (DIPC) P.K.
1072 Donostia-San Sebastiµn, Euskadi (Spain)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201503991.
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light through multiphotonic processes.[21,22] Conveniently, UV/
Vis photons emitted by UCNPs upon NIR-light excitation can
promote photochemical reactions in metal complexes and po-
tentially switch on their biological effects. Ford and co-workers
showed that the NIR irradiation of UCNPs loaded with either
the Roussin black salt anion or the photoactivated CO-releas-
ing moiety (photoCORM) trans-[Mn(2,2’-bipyridine)(PPh3)2(CO)2]
results in the release of NO and CO, respectively.[13,14] Similarly,
we have demonstrated that related upconversion nanosystems
promoted the release of pyridine from a Ru–polypyridyl com-
plex[16] and the generation of a PtII species from a PtIV prodrug
candidate.[17] Alternatively, He et al. designed an UCNP system
capable of releasing doxorubicin in a controlled fashion by
taking advantage of a photoactivatable Ru complex that acts
as a valve.[20] Conversely, Bonnet and co-workers also reported
a promising system for activation at l=630 nm of a model
Ru–polypyridyl complex by using triplet–triplet annihilation
upconversion.[23]
For this purpose, we prepared two new Ru–pyridinato deriv-
atives, namely [(h6-p-cym)Ru(bpy)(m-CCH-Py)][(PF)6]2 (1) and
[(h6-p-cym)Ru(bpm)(m-CCH-Py)][(PF)6]2 (2; p-cym=p-cymene,
bpy=2,2’-bipyridine, bpm=2,2’-bipyrimidine, and m-CCH-Py=
3-ethynylpyridine) and exploited a click-chemistry strategy to
derivatise the monodentate 3-ethynylpyridine pendant arm
with a phosphonate group, which has high affinity for the
UCNP surface.[38] The functionalised complexes 3 and 4 and
their precursors were characterised and their photochemistry
studied in detail by using different methods, including DFT cal-
culations.
On the basis of its photoreactivity, complex
4 was
selected to anchor onto the core@shell NaYF4:Yb(30%)/
Tm(0.5%)@NaYF4 nanoparticles and perform NIR photochemis-
try experiments. Results proved the obtained UCNP@4 nano-
construct is activated at l=980 nm, thus generating the reac-
tive aqua photoproduct [(h6-p-cym)Ru(bpm)(H2O)]2+, which
binds the DNA-model base guanosine 5’-monophosphate
(GMP).
The unique optical (upconversion emission) and chemical
(e.g., Gd3+ and 18FÀ ions on the surface) features of UCNPs are
also exploited for multimodal imaging (i.e., single-photon
emission computed tomography/positron emission tomogra-
phy (SPECT/PET),[24,25] computed tomography (CT),[26] magnetic
resonance imaging (MRI),[27] optical,[28] and photoacustic),[29] as
demonstrated by numerous studies in vivo, which have ap-
The synthetic strategy and NIR photoactivation reported
herein may offer new intriguing opportunities for the design
of innovative prodrug nanosystems that rely on the rich anti-
cancer properties of metallo–arene complexes.
peared in the last few years. These properties and the low tox- Results and Discussion
icity of UCNPs[30] make hybrid nanomaterials based on UCNPs
Synthesis of complexes 1–4 and UCNP@4
and metal complexes suitable for application in theranostics.[31]
Herein, we devise a new approach to demonstrate the use-
fulness of UCNPs in the photoactivation of RuII–arene com-
plexes, an attractive class of metal complex with remarkable
anticancer activity in vitro[32,33] and in vivo.[34] Pyridinato RuII–
arene derivatives display dark-stability and selectively release
pyridinato ligands upon visible-light excitation, thus generat-
ing highly reactive aqua species.[35–37] Controlling such a reac-
tion is key for activating the biological activity of RuII–arene de-
rivatives.
Complexes [(h6-p-cym)Ru(bpy)(m-CCH-Py)][(PF)6]2 (1) and [(h6-p-
cym)Ru(bpm)(m-CCH-Py)][(PF)6]2 (2) were synthesised in mod-
erate yields (ca. 35%) by treating the precursors [(h6-p-cym)-
Ru(bpy)Cl][PF6] and [(h6-p-cym)Ru(bpm)Cl][PF6] with AgNO3 and
subsequently performing ligand-exchange reactions with 3-
ethynylpyridine in MeOH/H2O (Scheme 1A).[39–42] A range of
techniques was used to characterise both complexes, including
X-ray, multinuclear NMR, mass-spectrometry, and elemental
analysis.
Scheme 1. Schematic representation of the approaches employed for the synthesis of A) 1–4 and B) UCNP@4. NaAsc=sodium ascorbate.
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We employed 3-ethynylpyridine because pyridyl ligands are
known to render Ru–arene complexes hydrolytically stable in
the dark.[35–37] Moreover, the presence of an alkyne group on
the ligand allowed us to adopt click chemistry for the function-
alisation of 1 and 2 and anchoring onto UCNPs. Click chemistry
is a convenient strategy for ligand design in inorganic chemis-
try and has attracted significant attention in recent years.[43]
Such an approach affords (bio)orthogonal reactions that are
high yielding, selective, and robust under mild conditions.[44]
Inspired by the work of Branda and co-workers,[45] we em-
ployed diethyl-3-azidopropyl phosphonate and its azido func-
tion to incorporate the phosphonate group into 1 and 2 by
using click chemistry. Phosphonates have good affinity for the
NaYF4 surface of UCNPs and improve their biocompatibility, as
demonstrated by various groups.[38,46]
Diethyl-3-azidopropyl phosphonate was obtained reacting
(3-bromopropyl)phosphonic acid with sodium azide in ace-
tone.[47] Clicked complexes [(h6-p-cym)Ru(bpy)(P-Trz-Py)][(PF)6]2
(3) and [(h6-p-cym)Ru(bpm)(P-Trz-Py)][(PF)6]2 (4; P-Trz-Py=[3-(1-
pyridin-3-yl-[1,2,3]triazol-4-yl)-propyl]phosphonic acid diethyl
ester) were prepared by treating 1 and 2 with diethyl-3-azido-
propyl phosphonate in THF/water at 608C for 72 hours in the
presence of CuSO4 and sodium ascorbate (Scheme 1A). Com-
plexes 3 and 4 were obtained in good yields and purity and
1
were characterised by H, 13C, and 31P NMR spectroscopic and
mass-spectrometric analysis.
Although the photochemical behaviour of the complexes is
rather similar (see below), complex 4 was selected for loading
onto the core@shell NaYF4:Yb3+/Tm3+@NaYF4 UCNPs on the
basis of the higher reactivity of its bpm analogue 2 relative to
1 (i.e., with the bpy ligand). For this reason, we first activated
the phosphonate group of 4 into the acid form by de-esterifi-
cation with tribromo(methyl)silane (TMSBr) in CH2Cl2. The so-
obtained complex was stirred overnight with oleate-free
core@shell UCNPs in H2O, and an orange pellet corresponding
to UCNP@4 was collected after several washing and centrifu-
gation steps (Scheme 1B). Characterisation of the hybrid mate-
rial was achieved by using a combination of different tech-
niques (see below).
Figure 1. Crystal structures of A) 1 and B) 2. Thermal ellipsoids are depicted
at the 50% probability level. Counterions (PF6À) and hydrogen atoms have
been omitted for clarity.
Table 1. Selected X-ray and DFT-calculated bond lengths [Š] for 1 and 2
in the ground-state (S0) and in two triplet-state (T0 and T1) geometries.
Compd. RuÀN(m-CCH-Py) RuÀN(N,N’) RuÀN(N,N’) RuÀp-cym(centroid)
X-ray
1
2
2.125(2)
2.1241(17)
2.085(2)
2.084(2)
1.701
2.0995(16) 2.0873(16) 1.704
Ground state (S0)
1
2
2.159
2.158
2.103
2.111
2.095
2.106
1.848
1.850
Characterisation and photochemical properties of 1 and 2
Lowest-lying triplet state (T0)
1
2
2.164
2.143
2.404
2.457
2.113
2.131
2.079
2.082
X-ray and DFT structures
Triplet state (T1)
The crystal structure of the hexafluorophosphate salts of 1 and
2 were determined by means of X-ray diffraction studies
(Figure 1). These structures are fairly similar, and the first-coor-
dination spheres of both Ru complexes display comparable
bond lengths and angles (Table 1). Complexes 1 and 2 present
the typical piano-stool geometry of related RuII–arene com-
plexes with N,N’ ancillary ligands.[41] Crystal packing and experi-
mental details, including the X-ray diffraction data, are report-
ed in the Supporting Information (see Table S1 and Figures S1
and S2 in the Supporting Information). DFT-optimised ground-
state geometries (Table 1, B3LYP/LanL2DZ/6-31G**) for 1 and 2
describe the structure of the complexes satisfactorily. The cal-
culated and experimental RuÀN bond lengths differ only by
less than 0.02 Š. However, the RuÀp-cym(centroid) distances
1
2
2.556
2.511
2.087
2.093
2.082
2.113
2.157
2.140
are approximately 0.15 Š longer in the DFT calculations relative
to the X-ray determinations.
Two triplet excited-state structures were also DFT-minimised
for 1 and 2 (Table 1) because triplet states are likely to be in-
volved in the photochemistry of the complexes. The lowest-
lying triplet state (T0) for both compounds has a distorted
structure with one RuÀN(N,N’) bond strongly elongated (>
2.40 Š). However, the higher-energy triplet geometries (T1) for
both compounds display elongated RuÀN(m-CCH-Py) distances
(>2.51 Š).
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Photophysical and photochemical properties of 1 and 2
have a significant contribution from s-antibonding orbitals,
which confer a dissociative nature (see Figure S4 in the Sup-
porting Information). Through generation of these dissociative
singlet states and subsequent intersystem crossing, the low-
energy triplet states T0 and T1 (Figure 2B) can be populated.
T1 is dissociative toward the 3-ethynylpyridine ligand, whereas
T0 might promote the partial dissociation of a pyridyl ring of
the bpy/bmp ligand (see Figures S7–S9 in the Supporting In-
formation). Nevertheless, release of the chelating ligands is
prevented by the strong coordination of the second ring. For
this reason, light excitation of 1 and 2 results in selective pho-
todissociation of the monodentate 3-ethynylpyridine ligand.
Figure 2B reports the energy level of selected singlet and trip-
let states for 2 and their corresponding EDDMs (singlet states)
and spin densities (triplet states; see Figure S10 for 1 in the
Supporting Information).
The UV/Vis spectrum (see Figure S3 in the Supporting Informa-
tion) of 1 exhibits five distinct bands at l=245, 269, 305, 317,
and 370 nm (e=7600, 6400, 5400, 5700, and 1300mÀ1 cmÀ1, re-
spectively), whereas 2 (Figure 2A) displays two major bands
and one shoulder, respectively at l=246, 370, and 291 nm
(e=3700, 1200, and 3200mÀ1 cmÀ1). Time dependent (TD)-DFT
is a valuable tool to assign the character of the absorption
bands. Calculation of singlet–singlet transitions and analysis of
their orbital composition (see Tables S2 and S3 and Figure S4
in the Supporting Information) provide information on the
nature of the singlet-excited states, which can be conveniently
visualised by using electron-density difference maps (EDDMs;
see Figures S5 and S6 in the Supporting Information).
In agreement with related complexes, the high-energy
bands of 1 and 2 have mixed metal-to-ligand charge transfer
(MLCT) and intraligand character (<325 nm). Conversely the
lowest-energy bands have mainly MLCT character. Furthermore,
transitions of weak intensity with mixed MLCT/MC (metal-cen-
tred) character are present in their tail. These latter transitions
The low energy of the T0 state (1.33 and 1.29 eV for 1 and
2, respectively) calculated by DFT calculations and the small
energy difference between this state and T1 are consistent
with the lack of emission from the two complexes, which tend
to relax to the ground state by nonradiative pathways, and
Figure 2. A) UV/Vis absorption spectrum of 2 in aqueous solution (95:5=H2O/DMSO); vertical bars represent singlet–singlet TD-DFT transitions (see Table S2
in the Supporting Information). B) Singlet and triplet energy-level diagram with EDDMs (singlets) and spin-density surfaces (triplet states) for 2. In the EDDMs,
black indicates a decrease in electron density, whereas gray indicates an increase. Time-course photolysis reactions for 2 in aqueous solution (95:5=D2O/
1
[D6]DMSO) upon l=395 nm excitation (15 mWcmÀ2) followed by C) UV/Vis and D) 1H NMR spectroscopic analysis. H NMR: 2 (gray), [(h6-p-cym)Ru(bpm)(-
H2O)]2+ (black), and free 3-ethynylpyridine (white); =bpm, =p-cym, =3-ethynylpyridine.
~
&
*
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with their modest photodissociation quantum yields of less
than 1%, as determined by using actinometry methods[48]
(F1 =0.003 and F2 =0.008; see Table S4 and Figures S11–S14
in the Supporting Information).
Figure 2C,D shows the photolysis of 2 (D2O/[D6]DMSO,
95:5%) upon light irradiation at l=395 nm (15 mWcmÀ2) fol-
1
lowed by UV/Vis and H NMR spectroscopic analysis. Light irra-
diation induces variations in the UV/Vis absorption profile of 2,
with a decrease at l=370 nm and an increase at l=260–
320 nm of the bands. The presence of isosbestic points at l=
230 and 315 nm reveals the formation of a single photoprod-
uct.
1
Furthermore, diagnostic changes in the H NMR spectra of 2
and ultraperformance (UP) LC-MS analysis (see Figure S15 in
the Supporting Information) clearly confirm the release of 3-
ethynylpyridine and the formation of the aqua [(h6-p-cym)-
Ru(bpm)(H2O)]2+ ionic species.[35–37] A control experiment indi-
cates 2 is stable in the dark up to at least 24 hours (see Fig-
ure S16 in the Supporting Information). Analogue results were
observed for 1 and are summarised in Figures S17–S19 (see
the Supporting Information).
Figure 3. Photolysis time course for 4 in aqueous solution (95:5=H2O/
1
[D6]DMSO) upon l=395 nm excitation (15 mWcmÀ2) followed by H NMR
1
spectroscopic analysis. H NMR: 4 (gray), [(h6-p-cym)Ru(bpm)(H2O)]2+ (black),
~
&
*
and free P-Trz-Py (white); =bpm, =p-cym, =P-Trz-Py.
NIR photochemistry with UCNPs
lowing the procedure of Branda and co-workers.[51] By assum-
ing a particle density of 4.2 gcmÀ3 and a diameter of 36.9 nm,
the 4/UCNP ratio was calculated to be approximately 3000:1
(3.5% wt). The UCNPs display a typical upconversion emission
in THF of Tm3+ ions (Figure 4) with peaks at l=345 and
Photochemistry of 3, 4, and UCNP@4 under visible- and NIR-
light excitation
Initially, the behaviour of clicked complexes 3 and 4 was veri-
fied and compared with their analogues 1 and 2 in the dark
and under visible-light conditions. Analysis by NMR spectrosco-
py and UPLC-MS (see Figure 3 and Figures S20–S23 in the Sup-
porting Information) shows that 3 and 4 indeed react as the
precursors 1 and 2; namely, 3 and 4 undergo photodissocia-
tion of the P-Trz-Py ligand upon excitation at l=395 nm. The
addition of excess GMP to irradiated solutions of 3 and 4 re-
sults in the formation of the adducts [(h6-p-cym)Ru(b-
py)(GMP)]2+ and [(h6-p-cym)Ru(bpm)(GMP)]2+ after 12 hours of
incubation at ambient temperature (see Figures S24–S27 in the
Supporting Information). Next, we selected 4 to perform deco-
ration of UCNPs because of the slightly higher photodissocia-
tion yield of bpm complexes with respect to their bpy ana-
logues.
3
1
3
3
360 nm (3P0! F4 and D2! H6); l=450 and 475 nm (1D2! F4
1
3
3
3
and G4! H6); l=645, 690, and 720 nm (1G4! F4 and F3!
3H6), and at l=800 nm (3H4! H6). Photoluminescence studies
3
confirm that core@shell NPs present more intense emissions
relative to their core counterparts, especially in the blue region
of the UV spectrum (see Figure S32 in the Supporting Informa-
tion). The undoped NaYF4 shell is essential to improve the effi-
ciency of the upconversion process as it decreases nonradia-
tive decay due to the solvent and capping ligands.[49]
As we demonstrated previously,[17] such an improvement is
key to take full advantage of the overlap between the emis-
sion spectrum of UCNPs and the absorption spectrum of metal
complexes (Figure 4B). No significant changes in UCNP lumi-
nescence was detected once the particles were treated with
hydrochloric acid to remove the oleic acid, thus favouring the
interaction with the phosphonic acid group of (activated) 4.
Surface modification was confirmed first by the colour of the
UCNPs, which became pale orange, and then by UV/Vis spec-
troscopic analysis of UCNP@4 suspended in aqueous solution
and by XPS. The UV/Vis spectrum of UCNP@4 (Figure 4B) ex-
hibits the absorption profile of the complex with bands at l=
261 and 370 nm. Notably, the spectrum shows the characteris-
tic band of the dopant Yb3+ ion at l=980 nm.
For anchoring of 4 to UCNPs, we first synthesised core@shell
NaYF4:Yb3+/Tm3+(30/0.5%)@NaYF4 nanoparticles by thermal
decomposition as previously described by several groups (see
the Experimental Section and the Supporting Informa-
tion).[17,49,50] UCNPs were thoroughly characterised by TEM, X-
ray photospectroscopy (XPS), and FTIR and emission spectros-
copy (see Figures S28–S32 in the Supporting Information). TEM
images of the core@shell UCNPs show uniform size and shape
(diameter=37 nm; Figure 4A).
Functionalisation with 4 was achieved as described in
Scheme 1B and did not cause any observable change to the
nanoparticles, rather TEM images of aqueous solutions of
UCNP@4 showed improved dispersion and lower aggregation
relative to the core-only and core@shell UCNPs capped with
oleic acid. We obtained a rough estimate of the complex-graft-
ing density onto UCNPs by UV/Vis spectroscopic analysis fol-
Aqueous solutions of 4 and UCNP@4 were deposited onto
titanium supports for XPS measurements (see Figure 4C,D and
Figures S33 and S34 in the Supporting Information). The ex-
pected Ru and P peaks for the Ru2+ ion and the phosphonic
group of the ligand, respectively, were observed in both sam-
ples; however, a dramatic difference appeared in the P 2p3/2
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Figure 4. A) TEM image of UCNP@4. B) Overlap between the normalised absorption (gray) and emission (dark gray) spectrum of UCNP@4 of core@shell
UCNPs (THF) upon excitation at l=980 nm (15 WcmÀ2). Inset: enlargement of the region l=900–1000 nm for the absorption of UCNP@4 in aqueous solu-
tion. P 2p3/2, 2p1/2 XPS spectra of C) 4 and D) UCNP@4.
and 2p1/2 region. In particular, the protonation state of the
phosphonic group was clearly different in the case of UCNP@4
relative to 4. The former showed a much greater proportion of
2À
RÀPO3
versus RÀPO(OH)2 groups (90:10 and 33:67 for
UCNP@4 and 4, respectively) in agreement with the formation
of electrostatic interactions between the phosphonic group of
the complex and the surface of the UCNPs.
Therefore, we investigated the effects of NIR light on
UCNP@4 in aqueous solution by irradiating at l=980 nm and
monitoring the course of photoreaction by 1H NMR spectro-
scopic analysis, which was possible despite the decreased reso-
1
lution of the H NMR spectrum of UCNP@4 due to the intrinsic
paramagnetism of UCNPs.
Remarkably, the NMR spectra of the NIR-irradiated UCNP@4
display changes in the signal pattern (Figure 5), thus resem-
bling the spectra observed for 4 under excitation at l=
395 nm and ultimately confirming the formation of the [(h6-p-
cym)Ru(bpm)(H2O)]2+ ion. Moreover, UPLC-MS experiments
consistently show an optimal agreement in retention time and
m/z values for irradiated solutions of 4 and UCNP@4 (at l=
395 and 980 nm; see Figures S21 and S35 in the Supporting In-
formation).
Figure 5. Photolysis time course for UCNP@4 in aqueous solution upon exci-
1
tation at l=980 nm (8.1 WcmÀ2) followed by H NMR spectroscopic analysis.
1H NMR: UCNP@4 (gray), P-Trz-Py (black), and [(h6-p-cym)Ru(bpm)(H2O)]2+
~
&
*
(white); =bpm, =p-cym, =P-Trz-Py.
The aqua photoproduct generated by NIR light at the UCNP
surface reacts with GMP to afford the adduct [(h6-p-cym)-
Ru(bpm)(GMP)]2+ (as confirmed by using NMR spectroscopy
and UPLC-MS; see Figures S36 and S37 in the Supporting Infor-
mation), hence demonstrating that the reactivity of RuII com-
plexes toward biological targets can be triggered by low-
energy photons.
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hydroxide (ꢀ97%), ammonium fluoride (98%), iron(III) chloride
(97%), potassium oxalate monohydrate (99%), sodium azide (ꢀ
95%), guanosine 5’-monophosphate disodium salt hydrate (ꢀ
99%), and all the solvents were obtained from Sigma–Aldrich and
were used as received.
Control experiments indicate that UCNP@4 is stable and
does not undergo ligand dissociation in the dark, whereas 4
(and 2) does not photoreact when irradiated by NIR light for
several hours (see Figures S38 and S39 in the Supporting Infor-
mation).
The photochemical efficiency of these hybrid systems still
needs to be improved for more advanced applications; howev-
er, the combination of UCNP optical features with their multi-
modal-imaging capability can open new intriguing opportuni-
ties for innovative application in theranostics.
Synthesis of ruthenium complexes
The dimer [{(h6-p-cym)RuCl2}2] and the RuII complexes [(h6-p-cym)-
Ru(bpy)Cl][PF6] and [(h6-p-cym)Ru(bpm)Cl][PF6] were prepared
based on previous reports.[39–41] The RuII–pyridinato complexes 1–4
were prepared based on a previously reported methodology with
some modifications.[42]
Conclusion
[(h6-p-cym)Ru(bpy)(m-CCH-Py)][(PF)6]2
(1):
AgNO3
(0.2 g,
1.175 mmol) was added to an aluminium-foil-covered round-
bottom flask charged with [(h6-p-cym)Ru(bpy)Cl][PF6] (500 mg,
1.2 mmol) in a 1:1 mixture of MeOH/H2O (50 mL). The reaction mix-
ture was stirred at ambient temperature for 18 h. The solution
turned light yellow, and the off-white AgCl precipitate was filtered
off. 3-Ethynylpyridine (620 mg, 6 mmol) was added to the clear
yellow solution of [(h6-p-cym)Ru(bpy)(H2O)]2+ to give a dark-red so-
lution. The reaction mixture was heated at 408C for 18 h, and KPF6
(1.3 g, 7.2 mmol) was added after cooling. The precipitate that
formed was dissolved by the addition of acetone to give a clear so-
lution, which was filtered. The solution was reduced in volume
until the onset of precipitation on the rotary evaporator and left to
evaporate slowly at ambient temperature to afford yellowish
flakes, which were collected by filtration, washed with methanol
and diethyl ether, and dried in air (330 mg, 35%). 1H NMR
Herein, we have reported the synthesis of two new photoacti-
vatable Ru–arene complexes 1 and 2 and the thorough charac-
terisation of their structural and photochemical proprieties. In
addition, we have demonstrated that click chemistry is a valua-
ble strategy to introduce a phosphonate group directly on
complexes 1 and 2 by exploiting the alkyne function on the 3-
ethynylpyridyl ligand coordinated to their Ru centres. As in the
case of their precursors, the so-obtained derivatives 3 and 4
selectively release the pyridyl ligand under direct light excita-
tion of their lowest-energy absorption band. Moreover, phos-
phonate groups on 3 and 4 confer these complexes high affin-
ity for the surface of core@shell NaYF4:Yb/Tm@NaYF4 upcon-
verting nanoparticles.
3
Notably, ligand photodissociation and GMP coordination is
triggered under excitation at l=980 nm when 4 is anchored
on UCNPs (UCNP@4), thus providing, to the best our knowl-
edge, the first example of remote near-infrared photoactiva-
tion of a RuII–arene model prodrug.
(500 MHz, D2O): d=0.82 (d, J(H,H)=6.9 Hz, 6H), 1.77 (s, 3H), 2.41
(spt, 3J(H,H)=7.0 Hz, 1H), 5.99 (d, 3J(H,H)=6.4 Hz, 2H), 6.40 (d,
3J(H,H)=6.5 Hz, 2H), 7.30 (dd, 3J(H,H)=8.0, 5.9 Hz, 1H), 7.86 (td,
3J(H,H)=7.3, 5.7, 2H), 7.93 (dt, 3J(H,H)=8.1, 1.5 Hz, 1H), 8.22 (td,
3J(H,H)=7.9, 1.4 Hz, 2H), 8.30 (m, 3H), 8.51 (s, 1H), 9.64 ppm (d,
3J(H,H)=5.0 Hz, 2H); the proton resonance of the alkyne group
overlaps with residual water signal in D2O, whereas this proton res-
onates at d=4.73 ppm (s, 1H) in [D6]DMSO; 13C NMR (125 MHz,
[D6]DMSO): d=17.9, 22.2, 30.6, 78.5, 84.9, 87.5, 92.5, 103.7, 108.8,
122.0, 125.1, 127.1, 129.5, 141.8, 143.3, 152.8, 155.1, 155.4,
Although UCNPs have been intensively investigated for
medical applications, research into their combination with co-
ordination compounds is still very limited. The approach that
we proposed herein is of general applicability in terms of syn-
thetic and photoactivation strategies. The 3-ethynylpyridyl
ligand is indeed a convenient candidate on which to perform
click chemistry reactions in the proximity of transition-metal
centres. Furthermore, the aliphatic arm of the azido-phospho-
nate ligand can be opportunely changed to modify the dis-
tance between the UCNPs and photoactivatable complexes
and perhaps modulate their photochemistry.
156.7 ppm; FTIR (KBr pellet) n˜max =3300, 1580, 1410, 840, 560 cmÀ1
;
ESI-MS: m/z (H2O/MeOH) calcd for [C27H27N3RuPF6]+: 640.09; found:
640.30; elemental analysis calcd (%) for C27H27F12N3P2Ru: C 41.34, H
3.47, N 5.36; found: C 40.94, H 3.12, N 5.08.
[(h6-p-cym)Ru(bpm)(m-CCH-Py)][(PF)6]2 (2): Complex 2 was syn-
thesised by using the procedure described for 1 with the [(h6-p-
cym)Ru(bpm)Cl][PF6] precursor (200 mg, 0.48 mmol). Yield:
(120 mg, 32%); 1H NMR (500 MHz, D2O): d=0.88 (d, 3J(H,H)=
6.9 Hz, 6H), 1.81 (s, 3H), 2.43 (spt, 3J(H,H)=7.0 Hz, 1H), 6.14 (d,
3J(H,H)=6.5 Hz, 2H), 6.44 (d, 3J(H,H)=6.5 Hz, 2H), 7.37 (dd,
3J(H,H)=8.0, 5.8 Hz, 1H), 7.98 (dt, 3J(H,H)=8.0, 1.5 Hz, 1H), 8.09
Our proof of concept study demonstrates that UCNPs are
convenient platforms for the use of deep-penetrating NIR light
in the activation of anticancer metal complexes and the gener-
ation of active species in situ.
3
3
(dd, J(H,H)=5.8, 4.9 Hz, 2H), 8.31 (d, J(H,H)=5.8 Hz, 1H), 8.53 (s,
1H), 9.24 (dd, 3J(H,H)=4.8, 1.9 Hz, 2H), 10.00 ppm (dd, 3J(H,H)=
5.8, 1.9 Hz, 2H); the proton of the alkyne group overlaps the resid-
ual water signal in D2O, whereas this proton resonates at d=
4.72 ppm (s, 1H) in [D6]DMSO; 13C NMR (125 MHz, [D6]DMSO): d=
17.7, 22.2, 30.4, 78.8, 86.1, 87.3, 90.8, 106.3, 107.6, 122.0, 126.0,
126.9, 143.0, 153.4, 156.0, 160.8, 161.5, 164.1 ppm; FTIR (KBr pellet)
n˜max =3300, 1580, 1410, 840, 560 cmÀ1; ESI-MS: m/z (H2O/MeOH)
calcd for [C25H25N5RuPF6]+: 642.08; found: 642.28; elemental analy-
sis calcd (%) for C25H25F12N5P2Ru: C 38.18, H 3.20, N 8.90; found: C
37.59, H 3.04, N 8.71.
Experimental Section
Materials
RuCl3·3H2O (99%) was purchased from Precious Metals Online
(PMO Pty Ltd) and used as received. 2,2’-Bipyrimidine (bpm; 97%),
2,2’-bipyridine (bpy; ꢀ99%), silver nitrate (AgNO3; ꢀ99%), potassi-
um hexafluorophosphate (KPF6; 98%), 3-ethynylpyridine (98%), yt-
trium(III) acetate hydrate (99.9%), ytterbium(III) acetate tetrahy-
drate (99.9%), thulium(III) acetate hydrate (99.9%), 1-octadecene
(technical grade, 90%), oleic acid (technical grade, 90%), sodium
Diethyl-3-azidopropyl phosphonate: The preparation of the azido
ligand for click chemistry is based on the procedure reported by
Chem. Eur. J. 2016, 22, 2801 – 2811
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Tucker-Schwartz and Garrell.[47] (3-Bromopropyl)phosphonic acid di-
ethyl ether (5 mL, 26.02 mmol) was dissolved in acetone (25 mL)
and dried by passing through neutral alumina. NaN3 (2.6 g,
40.4 mmol) was added to the reaction mixture, which was heated
to reflux for 18 h. The reaction mixture was cooled, filtered
through celite, and washed several times with acetone. The solvent
was removed by rotary evaporation to leave a clear yellowish oil
(5.81 g, quantitative yield). 1H NMR (500 MHz, [D6]DMSO): d=1.24
doped NaYF4 protective shell was grown around on the surfaces of
the core NPs by using the same synthetic procedure. Full synthetic
details and characterisation (IR, XPS, TEM, optical proprieties) of
the nanoparticles are reported in the Supporting Information.
Synthesis of the adduct UCNP@4
Both 3 and 4 selectively photodissociate the P-Trz-Py ligand upon
irradiation at l=400 nm. However, 4 was selected for the function-
alisation of UCNPs (Scheme 1B) and NIR photolysis studies because
the bpm derivative 2 displayed a slightly higher photodissociation
yield relative to the bpy derivative 1.
3
3
(t, J(H,H)=7.1 Hz, 6H), 1.78 (m, 4H), 3.41 (t, J(H,H)=6.6 Hz, 2H),
4.00 ppm (m, 4H); 31P NMR (200 MHz, [D6]DMSO): d=30.9 ppm;
FTIR (KBr pellet) n˜max =3400, 3000, 2100, 1240, 1050, 960 cmÀ1
.
[(h6-p-cym)Ru(bpy)(P-Trz-Py)][(PF)6]2
(3):
CuSO4
(8 mg,
Deprotection of [(h6-p-cym)Ru(bpm)(P-Trz-Py)][(PF)6]2 (4): We fol-
lowed a similar procedure to that reported for the deprotection of
diethyl-3-azidopropyl phosphonate to yield 3-azido propyl phos-
phoric acid.[47] An aluminium-foil-covered round-bottom flask was
charged with [(h6-p-cym)Ru(bpm)(P-Trz-Py)][(PF)6]2 (4; 2.5 mg) in
CH2Cl2 (anhydrous; 1 mL) to obtain a yellow solution. Trimethylsilyl
bromide (TMSBr) was added (c.a. 10 drops) to the flask. Instantane-
ously, the reaction solution changed from transparent to cloudy.
After the end of the addition, the reaction mixture was stirred
overnight at ambient temperature. Afterward, the solvent was re-
moved under gentle nitrogen flow to give a yellow precipitate.
0.032 mmol), sodium ascorbate (6.3 mg, 0.032 mmol), and diethyl-
3-azidopropyl phosphonate (14.4 mg, 0.064 mmol) were added to
a
solution of [(h6-p-cym)Ru(bpy)(m-CCH-Py)][(PF)6]2 (1; 50 mg,
0.064 mmol) in THF/H2O (4:1, 5 mL). The reaction mixture was
heated at 608C for 72 h. KF6 (9.5 g, 52 mmol) in H2O (30 mL) was
then added to the reaction mixture, which was placed in a separat-
ing funnel and extracted with dichloromethane (3”10 mL) to give
a reddish solution. This mixture was dried over MgSO4, and the sol-
vent was removed by rotary evaporation to give an oily product,
which was dissolved in methanol. KPF6 (2 g) was added to the so-
lution and the product was precipitated by the addition of diethyl
ether. The solid was collected by decanting the diethyl ether, the
residue was redissolved in dichloromethane, and the precipitated
salt was filtered off to obtain a red solid, which was dried in air
Preparation of oleate-free UCNPs: The preparation was per-
formed following a reported procedure by Bogdan et al.[52] The
core@shell NaYF4:Yb(30%)/Tm(0.5%)@NaYF4 nanoparticles (50 mg)
were suspended in H2O (5 mL) in a round-bottom flask. The mix-
ture was adjusted to pH 4 by using 0.1m HCl solution, and the sus-
pension was stirred for 2 h at ambient temperature. Afterward, the
oleate-free UCNPs were purified from the released oleic acid by ex-
traction with diethyl ether (3”5 mL). The product (ca. 30 mg) was
dried at ambient temperature overnight.
1
3
(25 mg, 41%). H NMR (500 MHz, D2O): d=0.83 (d, J(H,H)=6.9 Hz,
3
6H), 1.19 (t, J(H,H)=7.1 Hz, 6H), 1.82 (m, 5H), 2.15 (m, 2H), 2.44
(stp, J(H,H)=6.8 Hz, 1H), 4.02 (m, 4H), 4.49 (t, J(H,H)=6.7 Hz, 2H),
6.05 (d, J(H,H)=6.3 Hz, 2H), 6.44 (d, J(H,H)=6.3 Hz, 2H), 7.40 (dd,
3J(H,H)=8.1, 5.8 Hz, 1H), 7.89 (t, 3J(H,H)=7.2 Hz, 2H), 8.14 (d,
3J(H,H)=8.0 Hz, 1H), 8.22 (t, J(H,H)=7.9 Hz, 2H), 8.29 (m, 4H), 8.79
3
3
3
3
3
(s, 1H), 9.72 ppm (d, 3J(H,H)=6.0 Hz, 2H); 13C NMR (125 MHz,
[D6]DMSO): d=16.7, 17.9, 21.5, 22.2, 23.8, 30.6, 50.2, 61.6, 85.0,
92.3, 104.0, 108.6, 124.0, 125.1, 127.6, 129.5, 130.0, 136.8, 141.7,
141.8, 148.5, 152.4, 155.3, 156.7 ppm; 31P NMR (200 MHz,
[D6]DMSO): d=30.4 ppm; ESI-MS m/z (H2O/MeOH) calcd for
[C34H43O3N6PRuPF6]+: 861.18; found: 861.45.
Functionalisation of oleate-free UCNPs with 4: Deprotected com-
plex 4 (2.5 mg) was mixed with oleate-free core@shell NaYF4:Yb/
Tm@NaYF4 (10 mg) and suspended in H2O (1.5 mL) in an alumini-
um-foil-covered round-bottom flask. The suspension was stirred
overnight at ambient temperature and then freeze-dried. The ob-
tained yellow powder was washed several times with ethanol and
precipitated by centrifugation (10000 rpm for 5 min) to remove
excess Ru complex. UCNP@4 (ca. 6 mg) was dried at ambient tem-
perature overnight. 1H NMR (500 MHz, D2O): d=0.87 (6H), 1.81
(3H), 2.16 (2H), 2.43 (1H), 4.47 (2H), 6.18 (2H), 6.47 (2H), 7.44 (1H),
8.11 (2H), 8.18 (1H), 8.27 (1H), 8.36 (1H), 8.83 (1H), 9.22 (2H),
10.09 ppm (2H). Note: All the proton signals of UCNP@4 were
broader than those of 4, loss of multiplicity is due to the paramag-
netic nature of the UCNPs, and four aliphatic protons relative to
the propyl chain of the phosphonic acid group gave signals too
broad to be observed (previously falling in at dH =4.03 ppm in 4)
due to their proximity to the surface of UCNPs.
[(h6-p-cym)Ru(bpm)(P-Trz-Py)] [(PF)6]2 (4): Complex 4 was synthes-
ised by using the procedure described above for 3 and starting
from [(h6-p-cym)Ru(bpm)(m-CCH-Py)][(PF)6]2 (50 mg, 0.06 mmol).
Yield: 15 mg, 23%; 1H NMR (500 MHz, D2O): d=0.89 (d, 3J(H,H)=
3
6.9 Hz, 6H), 1.21 (t, J(H,H)=7.1 Hz, 6H), 1.82 (m, 5H), 2.16 (m, 2H),
3
3
2.45 (stp, J(H,H)=6.8 Hz, 1H), 4.03 (m, 4H), 4.50 (t, J(H,H)=6.8 Hz,
3
3
2H), 6.19 (d, J(H,H)=6.4 Hz, 2H), 6.48 (d, J(H,H)=6.4 Hz, 2H), 7.46
(dd, 3J(H,H)=8.1, 5.8 Hz, 1H), 8.11 (dd, 3J(H,H)=5.9, 4.8 Hz, 2H),
8.18 (dt, J(H,H)=8.1, 1.6 Hz, 1H), 8.30 (d, J(H,H)=5.7 Hz, 1H), 8.36
3
3
3
(s, 1H), 8.84 (s, 1H), 9.23 (dd, J(H,H)=4.9, 1.9 Hz, 2H), 10.09 ppm
(dd, 3J(H,H)=5.9, 1.9 Hz, 2H); 13C NMR (125 MHz, [D6]DMSO,) d=
16.7, 17.8, 22.2, 22.7, 23.8, 30.5, 50.3, 61.6, 86.2, 90.6, 106.7, 107.5,
124.0, 126.0, 127.5, 130.1, 137.6, 142.0, 149.5, 152.9, 160.8, 161.5,
164.1 ppm; 31P NMR (200 MHz, [D6]DMSO): d=30.4 ppm. ESI-MS m/
Photolysis experiments
z
(H2O/MeOH), calcd for [C32H41O3N8PRuPF6]+: 863.17; found:
Photoirradiation of Ru complexes at l=395 nm: Aqueous solu-
tions of 1–4 were irradiated at l=395 nm on a Prizmatix LED Mul-
tiwavelength MWLLS-11 source (15 mWcmÀ2) at ambient tempera-
ture. The progress of the photoreaction was followed by using
1H NMR or UV/Vis spectroscopy. Finally, the nature of the photo-
products was also analysed by UPLC-MS.
863.42.
Synthesis of UCNPS
The core@shell NaYF4:Yb(30%)/Tm(0.5%)@NaYF4 nanoparticles
were synthesised in two steps by thermal decomposition, as previ-
ously reported by us[16] and others.[45,46] The oleate-coated core
NaYF4:Yb3+/Tm3+ (30/0.5%) nanoparticles were first synthesised by
employing the acetate salts of Rare Earth elements (i.e., Y, Yb, and
Tm) in solutions of oleic acid and octadecene. Subsequently, an un-
Photoirradiation of 4 and UCNP@4 at l=980 nm: Aqueous solu-
tions of 4 and UCNP@4 were irradiated at l=980 nm with a BWT
diode laser DS3-11312-110. Complex
4 (150 mm, 400 mL) and
UCNP@4 (10 mgmLÀ1, 400 mL) solutions were irradiated for 7 and
5.5 h (8.8 and 8.1 WcmÀ2, respectively). The progress of the photo-
Chem. Eur. J. 2016, 22, 2801 – 2811
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reaction was followed by 1H NMR spectroscopic measurements,
after solutions of UCNP@4 had been centrifuged (6000 rpm, 5 min)
to improve the quality of the NMR spectra. The nature of the pho-
toproducts was also analysed by UPLC-MS. The output-power den-
sity of the light sources employed was measured with an optical
power meter (Ophir Photonics PD300-3W).
was calibrated by using the 3d5/2 line of Ag with a full width at half
maximum (FWHM) of 1.1 eV. All the measurements were made in
an ultrahigh vacuum (UHV) chamber at
a pressure below
8·10À8 mbar. The samples were measured on titanium surfaces.
UPLC-MS
UPLC-MS measurements on 1–4 were performed by using positive-
ion electrospray ionisation mass spectrometry (ESI-MS) on a LCT
Premier XE machine (resolution: 10000 FWHM) from Waters cou-
pled to an ultraperformance liquid chromatograph. Samples were
prepared in a H2O/DMSO (95:5%) mixture. The analysis was ach-
ieved on an Acquity UPLC BEH C18 column (50”2.1 mm) with H2O
(0.1% formic acid)/MeOH as the mobile phase at a flow rate of
0.3 mLminÀ1 and an injection volume of 5 mL. The ESI source was
employed in the W-optics positive-ionisation scan mode with the
capillary voltage at 2.5 kV. The temperatures of the source and de-
solvation were 1208C. The cone and desolvation gas flows were 50
and 600 LhÀ1. The collision gas flow was 0.2 mLminÀ1 and a colli-
sion energy of 15–18 V was applied. Sonicated and ultracentrifuga-
tion (105 rpm, 45 min) was used in the case of UCNP@4 to elimi-
nate possible aggregates and nanoparticles before injection for
UPLC-MS.
NMR spectroscopy
1 H, 13C, and 31P NMR spectra of the various samples in deuterated
solvents (D2O or [D6]DMSO) were acquired by using standard pulse
programs on an Avance III Bruker 500 NMR spectrometer. The
chemical shifts were reported in parts per million (d, ppm) and ref-
erenced to the residual solvent peak.
X-ray crystallography
Diffraction data for 1 and 2 were obtained on an Agilent Super
Nova Mo diffractometer coupled to a CCD area detector at 100 K
using an Agilent 700 Cryosystem Cooler fed with liquid nitrogen.
Full-matrix least-squares refinements based on F2 were performed
by using SHELXL-97, and the structures were solved by direct
methods. The rest of the hydrogen atoms were located by using
a riding model. The crystallographic details of 1 and 2 have been
deposited in the Cambridge Crystallographic Data Centre.
CCDC 1055609 and 1055610 contain the supplementary crystallo-
graphic data for this paper. These data can be obtained free of
charge from .
Ligand-photodissociation quantum yield
The quantum yields (F) of ligand photodissociation were deter-
mined for 1 and 2 in H2O upon excitation at l=395 nm. UV/Vis ab-
sorption spectroscopy was employed to quantify the formation of
the photoproducts as function of irradiation time (nmolsÀ1). At the
same time, ferrioxalate actinometer K3[Fe(C2O4)3] was used to de-
termine the photon flux (mmolsÀ1) on the samples exposed to the
Prizmatix LED Multiwavelength MWLLS-11 at l=395 nm. Full de-
tails are given in the Supporting Information. Complex
K3[Fe(C2O4)3] was obtained by following the procedure described
by Carriazo.[53]
FTIR spectroscopy
FTIR spectra of 1, 2, and core-NaYF4:Yb/Tm, core@shell-NaYF4:Yb/
Tm@NaYF4, and oleate-freecore@shell-NaYF4:Yb/Tm@NaYF4 nano-
particles were recorded on a Nicolet FTIR 6700 spectrometer as KBr
disks.
UV/Vis absorption spectroscopy
Computational details
UV/Vis spectroscopic experiments of the Ru complexes and
UCNP@4 were performed in H2O on a Varian Cary 5000 spectro-
photometer.
All the calculations were performed with the Gaussian 09 (G09)
program package,[54] which employed DFT and TD-DFT meth-
ods,[55,56] the Becke three-parameter hybrid functional,[57] and the
Lee–Yang–Parr gradient-corrected correlation functional (B3LYP).[58]
The solvent effect was included by using the polarizable continu-
um model (PCM)[59,60] with water as the solvent. The LanL2DZ basis
set[61] and effective core potential were used for the Ru atom and
the 6-31G** basis set[62] was used for all the other atoms. The
B3LYP/LanL2DZ/6–31G** combination was selected because it had
previously provided satisfactory results for similar ruthenium–arene
complexes.[35,36]
Emission spectroscopy
The emission spectra of UCNP dispersions in THF or H2O were ob-
tained under excitation at l=980 nm (15 WcmÀ2) with a laser
diode (l=980 nm, CNI, MDL-N-980) coupled to a Fluorometer Flu-
oroLog (Horiba). The laser-power density was measured as de-
scribed above.
TEM
Geometry optimisations of the ground states (S0) and the lowest-
lying triplet states (T0 and T1) for 1 and 2 were carried out without
any symmetry constraints. The nature of all the stationary points
was verified by using harmonic vibrational frequency calculations.
No imaginary frequencies were found, thus indicating we had lo-
cated the minima on the potential-energy surfaces.
TEM images were collected for core-NaYF4:Yb/Tm, core@shell-
NaYF4:Yb/Tm@NaYF4, and UCNP@4 samples on a JEOL JEM-1400
PLUS-HC microscope that operated at 120 kV. A small amount of
the sample was dispersed in H2O or THF (1 mL) as the solvent to
give an approximate 0.5 mgmLÀ1 solution. One drop (3 mL) of the
resulting solution was allowed to evaporate on a carbon film sup-
ported on a 300 mesh copper grid (diameter=3 mm).
The UV/Vis electronic absorption spectra were simulated by using
TD-DFT,[55,56] in which a total of 50 singlet excited states were com-
puted. The electronic distribution and the localisation of the singlet
excited states were visualised by using electron-density difference
maps (EDDMs).
XPS
XPS data of 4 and UCNP@4 were acquired on a SPECS Sage HR
100 spectrometer with a non-monochromatic X-ray source (MgKa
of 1253.6 eV) and an applied power of 250 W. The spectrometer
GaussSum 2.2.5[63] was used to simulate the theoretical UV/Vis
spectra and for extraction of EDDMs.[64,65] Molecular-graphic images
were produced by using the UCSF Chimera package from the Re-
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Acknowledgements
Our work in this area was supported by the Spanish Ministry
of Economy and Competitiveness (grant CTQ2012-39315), the
Department of Industry of the Basque Country (grant ETOR-
TEK). L.S. and E.R. were supported by the MICINN of Spain with
the Ramón y Cajal Fellowship RYC-2011-07787 and by the MC
CIG fellowship UCnanomat4iPACT (grant no. 321791). We
gratefully acknowledge Ikerbasque for the Visiting Professor
Fellowship to A.H. and members of the European COST Actions
CM1105 and CM1403 for stimulating discussions. Marco Moller,
Javier Calvo, Daniel Padró, and Luis Yate are also kindly ac-
knowledged for help in the collection of experimental data.
Moreover, technical and human support provided by SGIker
(UPV/EHU, MINECO, GV/EJ, ERDF, and ESF) is gratefully ac-
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Published online on January 19, 2016
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