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Bioactive Heterobimetallic Re(I)/Au(I) Complexes Containing Bidentate N-Heterocyclic Carbenes
Bioactive heterobimetallic Re(I)/Au(I) complexes containing bidentate N-heterocyclic carbenes
Andrés Luengo,† Vanesa Fernández-Moreira,†* Isabel Marzo‡ and M. Concepción Gimeno†*
†
Departamento de Química Inorgánica, Instituto de Síntesis Química y Catálisis Homogénea (ISQCH), CSIC-Universidad de
Zaragoza Pedro Cerbuna 12, 50009, Zaragoza, Spain
‡
Departamento de Bioquímica y Biología Molecular, Universidad de Zaragoza, Pedro Cerbuna 12, 50009, Zaragoza, Spain.
Supporting Information Placeholder
ABSTRACT: The first cationic heterobimetallic complexes of the type fac-[Re(CO)3(NHC)(LAuPPh3)]+, where NHC is an imidazole pyridine-based carbene and L is either 3-pyridylalkyne, 4-pyridylalkyne or 5-ethynyl-1-methyl-1H-imidazole, have been
synthesized together with their Re(I) precursors. All of them have showed similar emissive properties resulting from the presence of
the NHC system within the Re(I) core. Thus, emission can be ascribed as a phosphorescent process with a mixture of a MLCT from
the Re(dπ) → NHC(π*), LLCT from the imidazolyl/pyridyl to the NHC ligand and LC (NHC derivative) transitions. In all cases,
the emission maximum is blue shifted in comparison with that observed in the typical diimine-Re(I) systems. Only the heterobimetallic species displayed antiproliferative activity against tumor lung A549 cells, which was increased after irradiation at 405 nm up
to nearly five times for complexes 4 and 5. A necrotic process seems to be the preferred cell death mechanism. Fluorescence microscopy showed that only heterobimetallic complexes 4 and 5 were suitable for cell visualization. Their biodistribution pattern
reveals accumulation within the cytoplasm close to the nucleus, and some nucleus permeation. Overall it can be suggested that
whereas the emissive properties are dominated by the NHC-Re(I) fragment, the anticancer activity is mainly dependent on the Au(I)
counterpart.
INTRODUCTION
Cancer is one of the diseases leading the causes of death in the
last decade. For that reason, further research into new anticancer drugs is essential and one of the latest strategies to
tackle this problem is to build bioactive trackable species. 1
The aim of this strategy is the compilation of information
regarding the biodistribution and inner-interplay of drugs with
the biological targets in order to design and deliver a sophisticated generation of anticancer metallodrugs. Within this frame
several metallic structures combining well-known anticancer
metal species such as Cisplatin,2 Auranofin3 or RAPTA4 analogues with either an organic chromophore or an emissive
metallic species have been already described, see Figure 1A.
However, a step forward would be the incorporation of an
emissive species that is able to add extra-antiproliferative
value to the metalodrugs. Luminescent Re(I) complexes of the
type fac-[Re(N^N)(CO)3(X/L)]+/0, where (N^N) represents a
diimine derivative and X is a halogen or L a N-donor ligand,
have been proven to be excellent cellular imaging agents for
fluorescence microscopy.5 Their emission is generally assigned to a 3MLCT (metal-to-ligand-charge-transfer) transition, where the diimine is the main ligand contributor. 6 As
consequence, L can be easily functionalized to introduce the
bioactive fragment with no alterations of the emissive properties. In general, this type of Re(I) complexes do not seem to
affect the antiproliferative properties when they are combined
with a bioactive target (Figure 1B).7 However, it was recently
reported as the coordination of a bidentate N-heterocyclic
carbene instead of the typical diimine ligand confers the Re(I)
complex a remarkable anticancer activity against pancreatic
cancer cells, see Figure 1B.8 This was the first and the only
example in which a Re(I) species containing a bidentate Nheterocyclic carbene has been tested as anticancer agent and
based on the promising result it seems clear that the substitution of the typical diimine ligand for a bidentate NHC scaffold
has been key to achieve an anticancer rhenium drug. Alternatively antimicrobial activity was also recently found for rhenium complexes containing monodentate NHC ligands.9 Therefore, it can be postulated that combination of a NHC-Re(I)
derivative with a metallodrug would be an excellent approach
to maximise the therapeutic potential of a trackable metallodrug. Additionally, further substitution of L, which to the
best of our knowledge has been only reported as a halogen for
this type of Re(I) bidentate N-heterocyclic carbene derivatives10 for a neutral N-donor ligand will deliver highly potential mitochondrial bioprobes as 1) cationic species present a
greater predisposition to permeate those cellular membranes
and 2) the N-donor ligand could be easily used as a linker to
build the theranostic agents. Moreover, new grounds on the
photophysical properties of these type of Re(I) complexes will
be stablished. It is expected that their emissive behavior will
differ from that seen for the reported neutral NHC-Re(I) complexes studied.11
Thus, herein we describe the first approach towards the use of
luminescent N-heterocyclic carbene based-Re(I) species as cell
imaging agents as well as building blocks for trackable anticancer metallodrugs.
Figure 1. A) Examples of the combination of bioactive and
emissive fragments; B) Examples of cytotoxicity displayed by
either diimine or NHC-Re(I) derivatives.
RESULT AND DISCUSSION
Synthetic Approach. In previous studies we have validated
the possibility of visualizing bioactive gold(I) complexes in
cancer cells by combination of the bioactive metallic fragment
with a luminescent Re(I) species, using either functionalized
alkynes or phosphines as linkers.7 In the present work the
focus is set on the development of lumininescent heterobimetallic Re(I)/Au(I) complexes with an improved antiproliferative activity towards cancer cells. For that, substitution of the
typical diimine ligand within the Re(I) scaffold by an analogous N-heteroclyclic carbene (NHC) chelate is proposed.
These systems would deliver the first example of a NHCRe(I)/Au(I) heterobimetallic complex used for cellular imaging and as anticancer agent. Similarly to previous synthetic
procedures the N-methyl-N’-2-pyridylimidazolium salt was
prepared by nucleophilic substitution of imidazole with 2bromo-pyridine followed by an imidazole alkylation reaction
with methyl iodide.12 The N-methyl-N’-2-pyridylimidazolium
salt reacts with silver oxide forming the silver carbene that in
situ transmetalates with Re(CO)5Cl, in acetonitrile, to give A
in a moderate yield.13 It is worth noticing the presence of the
chelated NHC within the fac-{Re(I)CO3} core instead of the
symmetric diimine ligand generate a pair of enantioners.
Therefore, further modification of A leads to subsequent racemic mixtures. Afterwards, abstraction of the chloride ligand
by silver triflate in acetonitrile led to an activated Re(I)-NHC
species B, which can be easily derivatized by further substitution of the labile acetonitrile ligand for the corresponding
alkyne derivatives to obtain complexes 1-3. In order to prepare
the bimetallic Re(I)/Au(I) species, the Re(I) precursor was
stirred in either DCM or acetonitrile in the presence of
Au(acac)PPh3. The acetylacetonate complex facilitates the
deprotonation of the alkyne and the subsequent coordination
of the gold atom to the triple bond, thus affording the bimetallic complexes 4-6. All complexes have been characterized by
1
H, 13C{1H}, 31P{1H} NMR, FT-IR, UV-Vis spectroscopy as
well as mass spectrometry corroborating the accomplishment
on their synthesis. Analysis of the CO stretching frequencies
of all the complexes corroborate the expected facial arrangements as three stretches at c.a. 2020, 1940 and ca. 1890 cm-1
for complexes 1-3 and two stretches for 4-6 around 2022,
1900 cm-1 are observed because of the overlap of A’(2) and
A’’ modes into a single broad band. Similar NHC-rhenium
tricarbonyl derivatives presented analogous values.14 Moreover, the heterobimetallic complexes lack of υ(H-C≡C) band,
which had been observed for their Re(I) precursors, indicating
the coordination of the gold fragment, see Table 1. Additionally, in all cases, 1H-NMR spectra were well defined and
showed the typical patterns for the N-methyl-N’-2pyridylimidazolium and the correspondent pyridyl or imidazolyl carbene derivative coordinated to a fac-Re(CO)3 core.
Specifically, it was observed the disappearance of the acidic
imidazolium proton signal in the 10-12 ppm region of the 1HNMR spectrum. Consequently, a new carbenoic carbon signal
appears between 189 and 197 ppm in the 13C NMR spectra
together with the carbonyl carbons, confirming the coordination of NHC as a bidentate ligand to the rhenium core.15
Moreover, as a result of the gold triphenylphosphine coordination, the heterobimetallic complexes (4-6) revealed in their 1HNMR spectra a new multiplet resonance in the region between
7.49 and 7.55 ppm due to the presence of the new fragment, as
well as the disappearance of the terminal alkyne protons present in their precursors at 3.70, 3.62 and 3.50 ppm respectively, in concordance with the IR results. On top of that, the
downfield shift of the alkynyl carbons seen by 13C-NMR spectroscopy upon the coordination of -AuPPh3 and the single peak
c.a. 41 ppm observed by 31P NMR spectroscopy is consistent
with those values obtained for similar species. 7a Further analytical data provided by mass spectrometry corroborated the
accomplishment on the synthesis.
Figure 2. Schematic synthetic procedure and depiction of final
complexes (1-6). i) K2CO3, 190 ºC, 18 h; ii) MeI, THF, rt, 24 h;
iii) Ag2O, Re(CO)5Cl, CH3CN; iv) AgOTf, CH3CN, reflux 5 h; v)
alkynylpyridine derivative, THF, reflux; vi) Au(acac)PPh3, DCM.
Table 1. IR Stretching bands (cm-1) and 31P{1H} and 13C{1H}
NMR (ppm) chemical shifts (CD2Cl2) of 1−6.
IR(υ(CO))
IR(υ (HC≡C))
31
P{1H}NMR
13
CNMR(C≡C-
H/Au)
1
2022s, 1926m,
1895s
3238w
-
78.0
2
2019s,
1955m,1908s
3236w
-
79.5
3
2023s,
1945m,1887s
3229w
-
69.2
4
2023s,
1900s,br
-
41.5
98.0
5
2021s, 1897br
-
41.5
99.2
6
2019s, 1893br
-
41.8
no
a
w: weak, m: medium, s: strong, br: broad, a (CD3CN). no: Not
Observable.
Crystal Structures. Complexes B, 1, 3 are chiral complexes
and have been crystalized as racemic mixtures by slow diffusion of Et2O in CH2Cl2 (1, 3) or CH3CN (B). Molecular structures together with selected bond distances and angles are
presented in Table 2 and Figure 3. The three complexes presented a single molecule per asymmetric unit and their space
groups are Pn, P21/n or P21/c, respectively. On the contrary,
the enantiomers of all the species can be observed in the packing of the species, see Figure S1. In all cases, the rhenium
coordination sphere could be described as a distorted octahedron, where the three carbonyls are arranged in a facial disposition. Thus, the NHC system lies on the equatorial plane
together with two carbonyl ligands, whereas the remaining
carbonyl and the correspondent N-donor ligand are located in
the axial positions of the octahedron. The Re-Ccarbene distances
are 2.125(5), 2.130(4), 2.135(6) Å for complexes B, 1 and 3
respectively, which are very close values to those reported for
similar carbene Re species.16 Moreover, bond distances of ReCO trans to the NHC unit were much longer (c.a. 1.970 Å) in
comparison with that of the other Re-CO distances (c.a. 1.915
Å) due to the well-known trans influence of NHCs species.
Consequently, it was observed a shortening of the C-O bond
length of the correspondent carbonyl trans to the NHC as a
result of the lower π back-bonding character from the metal to
the carbonyl unit, see caption in Figure 3. These bond length
differences are in agreement with the published crystallographic data of similar NHC-rhenium complexes.11b It is also
worth mentioning that the NHC chelate provides a similar
constriction to the complex than their analogues bipy17 or
phen18 chelates, with narrow C-Re-N angles at c.a. 74°, comparable to those of the cited bidentate ligands.
Table 2. The most relevant bond lengths (Å) and angles (°) of
Complex B, Complex 1 and Complex 3.
Complex B
Complex 1
Complex 3
Re(1)-C(1): 1.911(5)
Re(1)-C(1): 1.968(4)
Re(1)-C(1): 1.921(4)
Re(1)-C(2):1.972(6)
Re(1)-C(2): 1.911(4)
Re(1)-C(2): 1.968(6)
Re(1)-C(3): 1.906(6)
Re(1)-C(3): 1.935(4)
Re(1)-C(3): 1.906(4)
Re(1)-C(4): 2.125(5)
Re(1)-C(9): 2.130(4)
Re(1)-C(4): 2.135(6)
Re(1)-N(3): 2.202(4),
Re(1)-N(1): 2.212(3)
Re(1)-N(3): 2.192(4)
Re(1)-N(4): 2.144(5)
Re(1)-N(4): 2.225(3)
Re(1)-N(4): 2.188(3)
C(1)-O(1): 1.156(6)
C(1)-O(1): 1.144(4)
C(1)-O(1): 1.141(5),
C(2)-O(2): 1.140(7)
C(2)-O(2): 1.149(5)
C(2)-O(2): 1.146(7),
C(3)-O(3): 1.1569(7),
C(3)-O(3): 1.149(5)
C(3)-O(3): 1.155(6)
N(4)-Re(1)-C(3):
175.7(3)
N(4)-Re(1)-C(3):
178.7(1)
N(4)-Re(1)-C(3):
174.3(2),
C(4)-Re(1)-N(3):
74.6(2)
C(9)-Re(1)-N(1):
74.5(1)
C(9)-Re(1)-N(3):
74.9(2)
Figure 3. Pov-ray representation of complexes B, 1 and 3 (the triflate counter ions were omitted for clarity). Note that all the selected
species are of the same chirality.
Optical Properties. The photophysical properties of complexes 1-6 were analyzed by UV-Vis absorption and emission
spectroscopy in dimethyl sulfoxide solution at room temperature. The most significant data are collected in Table 3 and
Figures 4 and S2-S3. All the complexes showed a similar
absorption pattern, with an intense absorption band at c.a. 290
nm that can be attributed to π→π* transitions within the NHC
ligand and also within the phenyl groups in the case of the
heterobimetallic complexes. Additionally, all of them presented a lower energy band of relatively small molar absorptivity
above 300 nm. This band could be attributed to a mixture of
MLCT (Re→NHC) and LLCT (pyridyl/imidazolyl→NHC)
charge transfer transitions, and thus, it is best described as a
MLLCT.13,14 Regarding the emissive properties, these complexes presented blue shifted emission maxima in comparison
with those of the typical [Re(CO)3(N^N)L]0/+.6 Such blue shift
is result of the strongly σ-donating NHC ligands, which ensures strong ligand fields with high lying d–d excited states.
Specifically complexes 1, 2, 4 and 5 exhibit a structured emission at c.a. 481 nm, whereas complexes 3 and 6 presented a
slightly red shifted broad emission band at 488 and 490 nm,
respectively, see Figures 4 and S3. The fact that the imidazolyl
derivatives 3 and 6 presented emission maxima red shifted in
comparison with their analogues 1, 2, 4 and 5 species could be
associated to the higher electron donating character of the
imidazole vs pyridyl derivative present in axial position of the
Re(I) metal center. In consequence, the HOMO orbitals, which
are primary located in the Re(I), get slightly destabilized and a
red shift on the emission maximum is seen. 15a The emissions
could be therefore attributed to a MLLCT transition, i.e. a
MLCT from the Re(dπ)→NHC(π*) mixed with a LLCT transitions from the imidazolyl/pyridyl to the NHC ligand. On top
of that, it is also worth mentioning that the structured emission
profile observed especially for complexes 1, 2 and 4 and 5
suggest that the MLLCT could be partially mixed with a 3LC
state.13 Demas and DeGraff already postulated such behavior
in the cases where MLLCT and LC states were energetically
similar.19 The significant LC state contribution to the emission
was also deducible from the similarities between the excitation
and emission profile of the imidazolium salt itself with that of
the complexes, see Fig S3. Lifetime values at room temperature ranges from tens to hundreds of nanoseconds, which are
typical of phosphorescent nature in this family of complexes. 15
Additionally, the presence of the gold fragment grafted in the
axial ligand does not seem to affect the photophysical behavior of the species, which is in concordance with other heterobimetallic species reported.7a
Figure 4. Emission spectra of complexes 5 and 6 in degassed
DMSO at 298 K.
Table 3. Photophysical properties of 1-6 in degassed DMSO
solution.
λabs/nm (ε / L×mol-1×cm-1)
λem/nm (λem/nm)
τ / ns
1
288 (14000), 320 (8000)
440, 460(s) (400)
-
2
291 (14800), 317 (10900)
456, 481(s), 514
(413)
212
3
286 (10100), 323 (5800)
488 (370)
10, 136
4
285 (36700), 316 (14600)
460, 485(s), 514
(414)
31, 205
5
288 (30300), 310 (28170)
465, 483(s), 512
(412)
207
6
280 (25400), 339 (4000)
493(377)
15, 123
Aerated DMSO: τ(2) = 175 ns. Degassed DMSO Imidazolium
salt: λem= 440 nm (λexc 295 nm); A λem= 506 nm (λexc 401 nm) (s)
strong
Biological Properties. The cytotoxic activity of complexes 1
to 6 as well as their rhenium precursors A and B was determined by an MTT assay in the tumor lung A549 cell line, see
Table 4. Only the heterobimetallic complexes presented a
significant antiproliferative character, c.a. 11 µM. On the
contrary, the monometallic rhenium complexes showed in all
cases cytotoxicity values over 50 µM, which point towards the
gold fragment as the bioactive entity for complexes 4-6. As
similar NHC rhenium derivatives have been proven to dissociate CO under photochemical conditions,15b complexes A, B
and 1 to 3 were tested as photocytotoxic agents using the same
cell line. However, irradiation at 405 nm for 10 minutes did
not showed the expected increase of toxicity. Once again all of
them presented IC50 values over 50 µM. In addition to the
monometallic species, the heterobimetallic complexes were
also analyzed under photochemical conditions. All of them
showed a slightly improvement of their antiproliferative character, see Table 4, being complex 6 the less affected by the
application of the irradiation. Despite the promising result,
these complexes could not be considered as effective photocytotoxic agents as their level of toxicity in absence of light is
already high (c.a. 11 µM). Cell death mechanism of heterobimetallic complexes was investigated by flow cytometry, see
Fig. S4. Complex 4 was incubated at different concentrations
with A549 cells for 24 h. Annexin V-DY634 and 7-AAD were
used as fluorescent markers. Annexin V-DY634 binds to
phosphatidylserine in the cell membrane and 7-AAD is a vital
dye that only enters through damaged cell membrane. Moreover, Z-VAD-fmk, a cell permeant pan caspase inhibitor, was
used as an indicator of the participation of caspases in the cell
death. Since the cells incubated with both markers seemed to
have a positive response by flow cytometry together with the
speed of cell death observed, it can be concluded that the cell
death mechanism is likely to be by a necrotic process. Moreover, the fact that the cells incubated with Z-VAD-fmk behave
in the same way that the ones lacking of such caspase inhibitor
corroborates that the cellular death is following a pathway
independent of caspases, see Figure S5. Additionally, a similar
experiment was performed but this time irradiating the samples for ten minutes at 405 nm during the incubation period in
order to assess whether the irradiation process was affecting
the cellular death pathway somehow. Once again the result
was similar to the non-irradiated experiment; a necrosis seems
to be the preferred cellular death. Investigations regarding the
biological targets for these complexes were also undertaken.
Thus, inhibition of the thioredoxin reductase was performed
using complex 4 as model. Specifically, A549 cells were treated with complex 4 or vehicle (DMSO) for 4 h. Then, total
protein extracts were prepared and used for the determination
of thioredoxin activity. Thereafter, the artificial substrate 5,5'dithiobis-(2-nitrobenzoic acid) (DTNB) was added, which
would rapidly evolve to two molecules of 2-nitro-5thiobenzoaye anion (TNB) if thioredoxin redutase is in the
presence of NADPH. The reduction of DTNB to TNB affords
a yellow color that can be detected at 412 nm by UV-Vis
absorption.20 The evolution of the TNB formation was recorded for 5 min and its comparison with that of a control assay
did not show inhibition of the thioredoxin, see figure 5. This
result was further confirmed by a preliminary analysis on the
production of reactive oxygen species (ROS) in colorectal
adenocarcinoma cells (CACO cells). Once again, incubation
of complex 4 with CACO cells did not promote the production
of ROS, which suggest a different biological target than that of
thioredoxin.
Table 4. IC50 values of 1-6 in A549 cells after incubation for
24 h in absence of light and after irradiation for 10 min at 405
nm.
IC50
IC50(irradiated)
1
>50
>50
2
>50
>50
3
>50
>50
4
12.18 ± 1.19
4.48 ± 0.71
5
10.82 ± 1.63
2.66 ± 0.56
6
12.65 ± 2.10
9.97 ± 3.04
A
>50
>50
NHC
>50
>50
Figure 5. Inhibition of thioredoxin assay for complex 4. Representation of the evolution of absorbance intensity of TNB
during 5 minutes.
In an attempt to elucidate the biodistribution of the complexes
1-6, fluorescence microscopy was used. The species were
incubated with A549 cells for 24 h at a concentration lower
than their IC50 value in order to prevent cell death during the
experiment. In addition to them, Draq5, a nuclear dye with an
excitation wavelength of 647 nm, was used as internal standard to ascertain their localization. Cellular images were taken
after exciting the cells at 405 nm, where the emissive complexes could be visualized. Superimposition of those with the
correspondent images taken after exciting the samples at 647
nm, allowed elucidating whether the complexes 1-6 were
within the cell and/or inside the nucleus. Unexpectedly, none
of the monometallic rhenium complexes were visible within
this assay. In contrast, two out of the three heterobimetallic
complexes showed emission within the cytoplasm surrounding
of the nucleus, and in a smaller amount inside the nucleus,
Figure 6 and 7. Specifically, 4 and 5 were the complexes that
were able to be visualized within the cell. A closer look to
other heterobimetallic Re(I)/Au(I) complexes reported in the
literature as possible cell visualization or theranostic agents
indicates that typically those containing in their structure the
fragment AuPPh3 appear to have more permeability than their
rhenium monometallic analogues.7 The present case seems to
follow the same trend except for complex 6, which despite
having a –AuPPh3 fragment was not observable. A plausible
explanation for this result might relay on the photophysical
properties of complex 6. Contrary to complexes 4 and 5,
whose maximum excitation wavelengths were laying around
410 nm in both cases, in complex 6 the maximum excitation
was at 377 nm, see Table 3 and Fig. S3. Therefore, irradiation
of the cells at 405 nm would not be suitable to excite complex
6, and in consequence emission will not be observed. This
result goes in line with the lower photocytotoxicity seen in the
case of complex 6 in comparison with complexes 4 and 5, see
Table 4. It is possible that the irradiation used is no equally
effective.
As previously commented, complexes 1-3 were neither suitable for cell imaging within these conditions. To assess whether
a possible disruption of the molecules under biological conditions was taking place and thus, preventing their visualization,
a stability assay was performed. Specifically, complexes 1 and
4 were chosen as representative species and they were dissolved in a mixture of DMSO:PBS (< 0.5% DMSO). Stability
of the complexes was analyzed by UV-Vis absorption spectroscopy over a period of 24 h. Both of them seem to remain
stable under those conditions, see Figure S6. Therefore, it
seems clear that decomposition of the complexes could not be
the origin of the lack of response in the localization experiment for the monometallic species 1-3. The main difference
between these Re(I) species and the ones already studied by
fluorescence microscopy is the nature of the chelated ligand. 21
Thus, changing the typical diimine ligand for a pyridylcarbene derivative (NHC) seems to negatively affect the internalization process.
Figure 6. Images of complexes 4 (top row) and 5 (bottom row)
incubated with A459 cells and Draq5 at 37 C for 4 h. (A) After
irradiation at 405 nm. (B) After irradiation at 647 nm. (C) Superimposition image.
Figure 7. Image of complexes 5 (yellow color) incubated with
A459 cells and Draq5 (blue color) at 37 C for 4 h superimposed
with the bright field image.
CONCLUSIONS
In summary, the first bioactive and luminescent heterobimetallic Re(I)/Au(I) complexes containing a pyridyl N-heterocyclic
carbene derivative instead of the typical diimine ligand were
reported, as well as their monometallic Re(I) precursors. Either pyridyl or imidazolyl alkyne derivatives were used as
bridge ligand between both metals, being specifically bounded
to the Re(I) center through the nitrogen atom and to the Au(I)
center through the alkynyl carbon. The photophysical properties of these complexes showed that all of them have a similar
behavior, where the presence of the Au(I) fragment was not
implicated in the emissive process. Thus, the emission was
attributed to a mixture of 3MLCT from the Re(dπ) →
NHC(π*), 3LLCT from the imidazolyl/pyridyl to the NHC
ligand as well as 3LC (NHC derivative) transitions. The similar structured emission profile seen for NHC, 1, 2, 4, 5 was
conclusive on the implication of LC transitions. Moreover,
such LC contribution seems to be less marked in the case of
complexes with axial ligands like a chloride (A) or imidazolyl
derivatives (3 and 6), whose electron donating character is
higher, and presumably the contribution of the LLCT over the
LC transition too. As expected, only the heterobimetallic complexes showed antiproliferative character against A549 cells
(IC50 ≈ 11 μM). Therefore, at this point it can be stated that we
have developed heterobimetallic species able to combine their
intrinsic emissive (Re(I) fragment) and bioactive (Au(I) fragment) properties into a new single molecular structure. Additionally, irradiation of the cells incubated with the heterobimetallic complexes at 405 nm incremented the cytotoxicity up to
five times in some cases. In contrast, monometallic Re(I)
species did not show any extra antiproliferative activity. The
cell death mechanism studied by flow cytometry concluded
that a necrotic process seems to take place for the irradiated
and no-irradiated assay. Moreover, fluorescence microscopy
analysis showed that only 4 and 5 were able to accumulate
within the cells. Specifically, their emission was within the
cytoplasm surrounding of the nucleus, and in a smallest
amount inside the nucleus. The fact that none of the monometallic Re complexes were detected by this technique could be a
combination of several factors. Both photophysical (lower
intensity or quenching processes in the biological media)
and/or the different lipophilic properties (lack of the gold
triphenyl fragment and the presence of pyridyl-NHC derivative instead of the typical diimine) could be implicated. Additionally, the different photophysical properties seem to be the
origin for not being able to detect 6 by fluorescence microscopy. In this particular case we believe that it is possible that the
complexes have entered the cells as it is known that the fragment AuPPh3 renders a higher lipophilicity of the probe and
thus, a better cell permeability. However, this complex has a
maximum excitation wavelength of c.a. 370 nm, which is quite
far away from the excitation wavelength used within the visualization experiment (405 nm).
To conclude, the development of heterobimetallic Re(I)/Au(I)
complexes as emissive and bioactive species could be
achieved attending to the different functionalization of a) the
chelate ligand NHC to modulate the desired photophysical
properties and b) the bridging and ancillary gold ligand in
order to endow specific bioactivity.
EXPERIMENTAL SECTION
General Measurements and Analysis Instrumentation.
Mass spectra were recorded on a BRUKER ESQUIRE 3000
PLUS, with the electrospray (ESI) technique and on a
BRUKER (MALDI-TOF). 1H, 13C{1H} and 31P{1H} NMR,
including 2D experiments, were recorded at room temperature
on a BRUKER AVANCE 400 spectrometer (1H, 400 MHz,
13
C, 100.6 MHz, 31P, 162 MHz) with chemical shifts (δ, ppm)
reported relative to the solvent peaks of the deuterated solvent.
Infrared spectra were recorded in the range 4000–250 cm-1 on
a Perkin-Elmer Spectrum 100 FTIR spectrometer. Room temperature steady-state emission and excitation spectra were
recorded with a Jobin-Yvon-Horiba fluorolog FL3-11 spectrometer fitted with a JY TBX picosecond detection module.
Nanosecond lifetimes were recorded with a Datastation HUBB with a nanoLED controller and software DAS6. The
nanoLEDs employed for lifetime measurements were of 370
and 390 nm. The lifetime data were fitted using the JobinYvon software package and the Origin Pro 8 program. UV-Vis
spectra were recorded with a 1 cm quartz cells on an Evolution
600 spectrophotometer. The quantum yields were measured in
a Hamamatsu Photonics Quantaurus-QY 300-950 nm.
Crystal Structure Determinations. Crystals were mounted
in inert oil on glass fibers and transferred to the cold gas
stream of an Xcalibur Oxford Diffraction diffractometer
equipped with a low-temperature attachment. Data were collected using monochromated MoKα radiation (λ= 0.71073 Å).
Scan type ω. Absorption correction based on multiple scans
were applied using spherical harmonics implemented in
SCALE3 ABSPACK scaling algorithm. The structures were
solved by direct methods and refined on F2 using the program
SHELXL-97.22All non-hydrogen atoms were refined anisotropically, with the exception of complex 7. CCDC deposition
numbers 1858777 (B) 185878 (1) and 1858779 (3) contain the
supplementary crystallographic data. These data can be obtained free of charge by The Cambridge Crystallography Data
Center.
Antiproliferative studies: MTT assay. Exponentially
growing cells (A549) were seeded at a density of approximately 104 cells per well in 96-well flat-bottomed microplates and
allowed to attach for 24 h prior to addition of compounds. The
complexes were dissolved in DMSO and added to cells in
concentrations ranging from 10 to 200 µM in quadruplicate.
Cells were incubated with our compounds for 24 h at 37 °C.
Ten microliters of MTT (5 mg ml-1) were added to each well
and plates were incubated for 2 h at 37 °C. Finally, the growth
media was eliminated and DMSO (100 μl per well) was added
to dissolve the formazan precipitates. The optical density was
measured at 550 nm using a 96-well multiscanner autoreader
(ELISA). The IC50 was calculated by nonlinear regression
analysis.
Flow cytometry assay. The cells were treated for 24 h with
compound 4 at 5, 10 and 25 μM. Then, they were trypsinised
and incubated at 37 °C for 5 minutes in ABB (140 mM NaCl,
2.5 mM CaCl2, 10 mM Hepes/NaOH, pH 7.4) containing 0.5
mg ml−1 of either annexin V-DY634 or 7-AAD. Finally, the
cells were diluted to 0.5 ml with ABB and analysed by flow
cytometry (FACScan, BD Biosciences, Spain).
Thioredoxin inhibition assay. For determination of the
thioredoxin reductase activity, A549 cells were incubated for 9
h with our compound at different concentrations near IC50
values. Cells were collected and washed with PBS and 150 μl
buffer (1% Triton X-100, 150 mM NaCl, 50 mM Tris/HCl pH
7.6, 10% v/v glycerol, 1 mM EDTA, 1 mM sodium orthovanadate, 10 mM sodium pyrophosphate, 10 μg ml−1 leupeptin, 10
mM NaF, 1 mM sodium methylsulphonium) for 30 min at 0
°C, and centrifuged at 200 g for 10 min at 4 °C. The protein
was quantified using the BCA protein assay (Thermo Scientific) and 80 μg was added in each assay. Kinetic studies were
performed in a buffer containing 0.2 M NaCl, H-phosphate pH
7.4, 2 mM EDTA, 0.25 mM NADPH and 3 mM DTNB. The
increase in the absorbance was measured at 412 nm for 5 min
at 25 °C.
Intracellular peroxides (ROS) formation. The production
of ROS (Reactive Oxygen Species) was assessed using the
dichlorofluorescein (DCF) assay.23 Caco-2/TC7 cells were
plated in 96-well plates at a density of 4000 cells perwell and
incubated for 24 h under standard cell culture conditions. For
treatment, 4 was added to cells within its IC50 concentration
that of IC50 and incubated 24 h; mock treated cells were just
incubated with DMSO at the same concentration than treated
cells. Then, cells were washed twice with PBS and 100 μL of
20 mM DCFH-DA (dichloro-dihydrofluoresceindiacetate)
were added to each well. Cells were incubated 1 h at 37 °C
and washed twice with PBS; finally, 100 μL of PBS were
added and fluorescence was analyzed with DTX-880 (Beckman Counter). Excitation and emission settings were 485 and
535 nm, respectively. The intensity of fluorescence is considered as a reflection of total intracellular ROS.
Cell fluorescence microscopy study. European Collection
of Cell Cultures, were maintained in Hepes modified minimum essential medium (DMEM) supplemented with 5% fetal
bovine serum, penicillin, and streptomycin. A549 cells were
detached from the plastic flask using trypsin-EDTA solution
and suspended in an excess volume of growth medium. The
homogeneous cell suspension was then distributed into 24well flat-bottomed microplates over a cover slip placed inside
each well and they were allowed to attach for 24 h prior to
addition of compounds. Complexes were added (10 l) to the
cells up to a final concentration of 25M. After incubation for
4 h at 37 °C, the growth medium was removed and 0.5 ml of
PBS was added for a washing step (3 times). Thereafter, 0.5
ml of paraformaldehyde (4%) was added and allowed to stand
for 15 min at room temperature. Eventually the paraformaldehyde was removed and further washings with PBS were performed (3 × 0.5 ml). The cover slips were collected from the
24 well plate, immersed for 1 or 2 seconds in distilled water
and let them to drip the water. Then, they are placed over a
microscope slide where a drop of fluoromount with 2M
DRAQ5 was previously placed. Preparations were viewed
using an Olympus FV10-i Oil type compact confocal laser
microscope using a ×10 or ×60 objective, with excitation
wavelength at 405 and 650 nm.
Materials and Procedures. The starting material N-methylN’-2-pyridylimidazolium,12 Au(acac)(PPh3)24 and complex A13
were prepared according to literature procedures and their
experimental data agrees with that reported somewhere else.
All other starting materials and solvents were purchased from
commercial suppliers and used as received unless otherwise
stated.
Synthesis of complex B. To a stirred solution of A (83 mg,
0.178 mmol) in CH3CN (20 mL) was added AgOTf (50.4 mg,
0.196 mmol) and refluxed over 5 h and 30 minutes in the dark.
The suspension was filtered over celite and the solvent reduced to minimum volume. The addition of cold ether afforded an oil which was to afford the product as a pale yellow
solid. (83 mg, 79 %). 1H NMR (400 MHz, CD2Cl2) δ 8.76
(ddd, J = 5.6, 1.6, 0.7 Hz, 1H, H6), 8.25 (ddd, J = 8.4, 7.6, 1.6
Hz, 1H, H4), 8.14 (d, J = 2.2 Hz, 1H, H8 or H9), 8.13 – 8.10
(ddd, J = 8.4, 1.2, 0.7 Hz, 1H, H3), 7.44 (ddd, J = 7.5, 5.6, 1.2
Hz, 1H, H5), 7.29 (d, J = 2.2 Hz, 1H, H8 or H9).13C NMR (101
MHz, CD2Cl2) δ 195.0 (s, CO), 194.7 (s, CO), 189.2 (s, CO),
187.0 (s, C7), 154.3 (s, C2), 153.8 (s, C6), 143.7 (s, C4), 125.8
(s, C8 or C9), 124.8 (s, C5), 118.90 (s, C8 or C9), 114.32 (s, C3),
39.78 (s, C10), 4.32 (s, C12). HRMS (m/z): 471.0470 [M-OTf],
C14H12N4O3Re (471.0462). IR (cm-1): υ 3155 (CAr-H), υ 2027,
1882 (CO), υ 1621 (CAr=N)
General procedure for coordination synthesis of 1-3. To a
stirred solution of B in THF was added the corresponding
alkynylpyridine ligand and the reaction mixture was heated
several hours until consumption of the starting material. The
solvent was then evaporated and the crude mixture redissolved in DCM. Filtration over celite followed by addition
of ether afforded the desired product as a pale yellow solid.
Specifically:
Synthesis of complex 1: Complex B (30 mg, 0.048 mmol)
and 3-ethynylpyridine (25 mg, 0.242 mmol) were reacted in
THF (5 mL) at 40 ºC during 32 h. (23.7 mg, 72 %). 1H NMR
(400 MHz, CD3CN) δ 8.99 (d, J = 5.6 Hz, 1H, H6), 8.39 (d, J =
1.7 Hz, 1H, H15), 8.26-8.20 (m, 2H, H4 + H11), 7.93 – 7.89
(dtap, J = 8.0, 1.6 Hz, 1H, H13), 7.89 (d, J = 2.2 Hz, 1H, H9/H8),
7.85 (dm, J = 8.4 Hz, 1H, H3), 7.52 (ddd, J = 7.5, 5.6, 1.1 Hz,
1H, H5), 7.39 (d, J = 2.2 Hz, 1H, H9/H8), 7.29 (ddd, J = 8.0,
5.7, 0.6 Hz, 1H, H12), 4.10 (s, 3H, H10), 3.70 (s, 1H, H17). 13C
NMR (101 MHz, CD3CN) δ 197.6 (s, CO), 191.3 (s, CO),
190.3 (s, C7), 157.1 (s, C15), 154.8 (s, C11), 154.7 (s, C6), 154.6
(s, C2), 144.1 (s, C4), 143.0 (s, C13), 127.3 (s, C12), 126.7 (s,
C9), 125.8 (s, C5), 123.0 (s, C14), 119.0 (s, C8), 114.5 (s, C13),
84.8 (s, C17, (CC-H)), 78.0 (s, C16, (CC-H)), 39.9 (s, C10).
HRMS (m/z): 533.0620 [M-OTf], C19H14AuN4O3Re
(533.0618). IR (cm-1): υ 3238 (CC-H), υ 3238 (CAr-H), υ 2113
(C≡C), υ 2022, 1926, 1895 (CO), υ 1618 (CAr=N)
Synthesis of complex 2. 4-ethynylpyridine hydrochloride
(85 mg, 0.609 mmol) was dissolved in a saturated aqueous
solution of NaHCO3 and extracted with DCM. The organic
phase was dried with anhydrous sodium sulfate and the solvent evaporated to dryness to afford 4-ethynylpyridine. Then
the alkynylpyridine was reacted with complex B following the
same procedure described for complex 1 (30 mg, 0.0484
mmol) in THF (5 mL), (29.2 mg, 88 %). 1H NMR (400 MHz,
CD2Cl2) δ 8.88 (ddd, J = 5.6, 1.6, 0.7 Hz, 1H, H6), 8.26 (ddd, J
= 8.4, 7.5, 1.7 Hz, 1H, H4), 8.21 (dd, J = 5.1, 1.5 Hz, 2H, H11),
8.19 (d, J = 2.2 Hz, 1H, H9), 8.16 (ddd, J = 8.4, 1.7, 1.2 Hz
1H, H3), 7.50 (ddd, J = 7.5, 5.6, 1.2 Hz, 1H, H5), 7.35 (d, J =
2.2 Hz, 1H, H8), 7.33 (dd, J = 5.1, 1.5 Hz, 2H, H12), 4.12 (s,
3H, H10), 3.62 (s, 1H, H15). 13C NMR (101 MHz, CD2Cl2) δ
197.4 (s, CO), 197.0 (s, CO), 190.3 (s, CO or C 7), 190.3 (s,
CO or C7), 154.1 (s, C12), 154.1 (s, C2), 153.8 (s, C6), 144.1 (s,
C4), 134.2 (s, C13), 129.7 (s, C12), 126.3 (s, C9), 125.6 (s, C3),
119.1 (s, C8), 114.7 (s, C5), 87.6 (s, C15, (CC-H)), 79.5 (s, C14,
(CC-H)), 39.8 (s, C10). HRMS (m/z): 533.0644 [M-OTf],
C19H14AuN4O3Re (533.0618). IR (cm-1): υ 3236 (CC-H), υ
3236 (CAr-H), υ 2117 (C≡C), υ 2019, 1955, 1908 (CO), υ 1620
(CAr=N)
Synthesis of complex 3. Complex B (56.6 mg, 0.091 mmol)
and 5-ethynyl-1-methyl-1H-imidazole (9.6 µL, 0.091 mmol)
were reacted in THF (5 mL) at 40 ºC for 24 h. Purification was
carried out by recrystallization in acetone/ether. (23.1 mg, 70
%). 1H NMR (400 MHz, CD2Cl2) δ 8.81 (ddd, J = 5.6, 1.6, 0.6
Hz, 1H, H6), 8.20 (ddd, J = 8.4, 7.6, 1.6 Hz, 1H, H4), 8.07 (d, J
= 2.2 Hz, 1H, H8), 8.03 (ddd, J = 8.4, 1.1, 0.6 Hz, 1H, H3),
7.42 (ddd, J = 7.5, 5.6, 1.1 Hz, 1H, H5), 7.32 (d, J = 2.2 Hz,
1H, H9), 7.30 (sap, 1H, H16), 6.96 (d, J = 1.3 Hz, 1H, H11), 4.06
(s, 4H, H10), 3.59 (s, 3H, H15), 3.56 (s, 1H, H14). 13C NMR
(101 MHz, CD2Cl2) δ 197.4 (s, CO), 197.1 (s, CO), 190.8 (s,
CO or C7), 189.7 (s, CO or C7), 153.9 (s, C2), 153.5 (s, C6),
143.4 (s, C4), 142.6 (s, C16), 136.8 (s, C11), 126.0 (s, C9), 125.1
(s, C5), 118.7 (s, C8), 118.2 (s, C12), 114.4 (s, C3), 87.2 (s, C14,
(CC-H)), 69.2 (s, C13, (CC-H)), 39.9 (s, C10), 33.6 (s, C15).
HRMS (m/z): 536.0738 [M-OTf], C18H15N5O3Re (536.0727).
IR (cm-1): υ 3229 (CC-H), υ 3129 (CAr-H), υ 2125 (C≡C), υ
2023, 1945, 1887 (CO), υ 1617 (CAr=N)
General procedure for gold addition. To a stirred solution
of the rhenium precursor (1, 2 or 3) in acetonitrile or dichloromethane (5 mL) was added Au(acac)PPh3. After 5 hours
stirring at r.t. in the dark, the solution was filtered over celite
and concentrated to dryness. The solid was redissolved in
DCM and addition of ether afforded the desired product as a
solid.
Synthesis of complex 4. Complex 4 was obtained following
the general procedure for the gold addition. Specifically, compound 1 (58 mg, 0.085 mmol) and Au(acac)PPh 3 (47.5 mg,
0.085 mmol) were stirred in DCM (5 mL) affording the desired product as an orange solid (32.6 mg, 34 %). 1H NMR
(400 MHz, CD2Cl2) δ 8.89 (dd, J = 5.6, 1.6 Hz, 1H, H6), 8.37
– 8.36 (m, 1H, H14), 8.26 (ddd, J = 8.5, 7.6, 1.6 Hz, 1H, H4),
8.21 (d, J = 2.2 Hz, 1H, H8), 8.17 (dbr, J = 8.4 Hz, 1H, H3),
7.87 (dd, J = 5.6, 1.4 Hz, 1H, H11), 7.76 (dd, J = 8.0, 1.4 Hz,
1H, H13), 7.60 – 7.46 (m, 20H, HAr+H5), 7.34 (d, J = 2.2 Hz,
1H, H9), 7.16 (ddd, J = 8.0, 5.7, 0.6 Hz, 1H, H12), 4.13 (s, 3H,
H10). 13C NMR (101 MHz, CD2Cl2) δ 197.6 (s, CO), 197.1 (s,
CO), 190.6 (s, C7), 190.2 (s, CO), 157.0 (s, C14), 154.1 (s, C2),
153.4 (s, C6), 150.6 (s, C11), 144.1 (s, C4), 142.3 (s, C13), 134.8
(d, 2J P-C = 13.8 Hz, 6C, o-C, Ph), 132.4 (d, 4J P-C = 2.2 Hz, 3C,
p-C, Ph), 129.9 (d, 1J P-C = 56.3 Hz, 3C, i-C, Ph), 129.9 (d, 2J PC = 11.4 Hz, 6C, m-C, Ph), 126.7 (s, C 12), 126.5 (s, C 15), 126.2
(s, C9), 125.51 (s, C5), 119.2 (s, C8), 98.0 (brs, C16, (CC-H)),
114.7 (s, C3), 39.8 (s, C10). C17 (CC-Au) no observed. 31P NMR
(162 MHz, CD3CN) δ 41.49. HRMS (m/z): 991.1122 [MOTf], C37H28AuN4O3PRe (991.1118). IR (cm-1): υ 3124 (CArH), υ 2125 (C≡C), υ 2023, 1900 (CO), υ 1617 (CAr=N)
Synthesis of complex 5. Complex 5 was obtained following
the general procedure for the gold addition. Specifically, compound 2 (30 mg, 0.044 mmol) and Au(acac)PPh 3 (24.6 mg,
0.044 mmol) were stirred in acetonitrile (5 mL) affording the
desired product as a red solid (31.2 mg, 62 %). 1H NMR (400
MHz, CD2Cl2) δ 8.89 (dd, J = 5.6, 0.9 Hz, 1H, H6), 8.37 – 8.35
(m, 1H, H14), 8.29 – 8.23 (m, 1H, H4), 8.21 (d, J = 2.2 Hz, 1H,
H8), 8.17 (dm, J = 8.3 Hz, 1H, H3), 7.88 – 7.85 (dm, J = 5.7
Hz, 1H, H11), 7.76 (dm, J = 8.0 Hz, 1H, H13), 7.61 – 7.46 (m,
15H, HAr), 7.34 (d, J = 2.2 Hz, 1H, H9), 7.17 (ddd, J = 8.0, 5.7,
0.7 Hz, 1H, H12), 4.13 (s, 3H, H10). 13C NMR (101 MHz,
CD2Cl2) δ 197.7 (s, CO), 197.2 (s, CO), 190.6 (s, C 7), 190.5 (s,
CO), 154.1 (s, C6), 153.3 (s, 3C, C11+C2), 144.0 (s, C4), 137.5
(s, C13), 134.7 (d, 2J P-C = 13.8 Hz, 6C, o-C, Ph), 132.35 (d, 4J
3
P-C = 2.4 Hz, 3C, p-C, Ph), 129.8 (d, J P-C = 11.4 Hz, 6C, m-C,
1
Ph), 129.7 (d, J P-C = 56.9 Hz, 3C, i-C, Ph), 129.7 (s, 2C, C12),
126.2 (s, C9), 125.4 (s, C3), 119.2 (s, C8), 114.6 (s, C5), 99.2
(br s, C14, (CC-Au)), 39.8 (s, C10). C15(CC-Au) no observed.
31
P NMR (162 MHz, CD3CN) δ 41.49. HRMS (m/z): 991.1121
[M-OTf], C37H28AuN4O3PRe (991.1118) IR (cm-1): υ 3124
(CAr-H), υ 2117 (C≡C), υ 2021, 1897 (CO), υ 1602 (CAr=N).
Synthesis of complex 6. Complex 6 was obtained following
the general procedure for the gold addition. Specifically, compound 3 (1 eq, 17.8 mg, 0.026 mmol) and Au(acac)PPh 3 (1 eq,
14.6 mg, 0.026 mmol) were stirred in acetonitrile (5 mL)
affording the desired product as a beige solid (16.8 mg, 52 %).
1
H NMR (300 MHz, CD2Cl2) δ 8.81 (ddd, J = 5.6, 1.6, 0.7 Hz,
1H, H6), 8.20 (ddd, J = 8.4, 7.6, 1.7 Hz, 1H, H4), 8.09 (d, J =
2.2 Hz, 1H, H8), 8.04 (ddd, J = 8.4, 1.2, 0.7 Hz, 1H, H3), 7.60
– 7.45 (m, 16H, HAr), 7.41 (ddd, J = 7.5, 5.6, 1.2 Hz, 1H, H5),
7.31 (d, J = 2.2 Hz, 1H, H9), 7.15 (d, J =1.4 Hz, 1H, H11), 6.59
(d, J = 1.4 Hz, 1H, H12), 4.06 (s, 3H, H10), 3.57 (s, 3H, H14).
13
C NMR (75 MHz, CD2Cl2) δ 197.5 (s, CO), 190.1 (s, CO or
C7), 153.8 (s, C2), 153.5 (s, C6), 143.4 (s, C4), 140.6 (s, C11),
134.74 (d, 2J = 14.4 Hz, 6C, o-C, Ph), 133.93 (s, C12), 132.35
(s, p-C, Ph), 129.86 (d, 1J P-C = 57.3 Hz, 3C, i-C, Ph), 129.73
(d, 3J P-C = 12.0 Hz, 6C, m-C, Ph), 125.94 (s, C9), 125.00 (s,
C5), 120.97 (s, C13), 118.64 (s, C8), 114.16 (s, C3), 39.81 (s,
C2), 33.25 (s, C2). HRMS (m/z): 994.1238 [M-OTf],
C36H29AuN5O3PRe (994.1227). IR (cm-1): υ 3127 (CAr-H), υ
2019, 1893 (CO), υ 1617 (CAr=N)
ASSOCIATED CONTENT
The Supporting Information is available free of charge on the
ACS Publications website.
UV-Vis absorption spectra of 1-6 in DMSO solution (5·10-5
M), Normalized excitation and emission spectra of complexes
1-6, imidazolium salt and A in DMSO solution at 298 K, Flow
cytometry diagram of complex 4 incubated with A549 cell at
different concentrations. Incubation of A549 with complex 4
in presence and absence of Z-VAD-fmk, a caspase inhibitor,
superposition of UV-Vis absorption graphs of complex 1
(5.10-5 M DMSO:PBS, < 0.5 % DMSO) taken over a period of
24 h and superposition of UV-Vis absorption graphs of complex 4 (5.10-5 M DMSO:PBS, < 0.5 % DMSO) taken over a
period of 24 h.
AUTHOR INFORMATION
Corresponding Author
* vanesa@unizar.es and gimeno@unizar.es
ORCID
Vanesa Fernández-Moreira: 0000-0002-1218-7218
M Concepción Gimeno: 0000-0003-0553-0695
Author contributions
The manuscript was written thought contributions of all authors. All the authors have given approval to the final version
of the manuscript.
Notes
the authors declare no competing financial interest.
ACKNOWLEDGMENT
The authors thank the Ministerio de Economía y Competitividad
(MINECO-FEDER CTQ2016-75816-C2-1-P and CTQ201570371-REDT) and Gobierno de Aragón-Fondo Social Europeo
(E07_17R) for financial support. Andrés Luengo thanks Gobierno
de Aragón for a predoctoral fellowship.
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