Combined effect of caspase-dependent and caspase-independent apoptosis in the anticancer activity of gold complexes with phosphine and benzimidazole derivatives

Article abstract of DOI:10.3390/ph14010010

Since the potential anticancer activity of auranofin was discovered, gold compounds have attracted interest with a view to developing anticancer agents that follow cytotoxic mechanisms other than cisplatin. Two benzimidazole gold(I) derivatives containing triphenylphosphine (Au(pben)(PPh₃ )) (1) or triethylphosphine (Au(pben)(PEt₃ )) (2) were prepared and characterized by standard techniques. X-ray crystal structures for 1 and 2 were solved. The cytotoxicity of 1 and 2 was tested in human neuroblastoma SH-SY5Y cells. Cells were incubated with compounds for 24 h with concentrations ranging from 10 μM to 1 nM, and the half-maximal inhibitory concentration (IC₅₀ ) was determined. 1 and 2 showed an IC₅₀ of 2.7 and 1.6 μM, respectively. In order to better understand the type of cell death induced by compounds, neuroblastoma cells were stained with Annexin-FITC and propidium iodide. The fluorescence analysis revealed that compounds were inducing apoptosis; however, pre-treatment with the caspase inhibitor Z-VAD did not reduce cell death. Analysis of compound effects on caspase-3 activity and reactive oxygen species (ROS) production in SH-SY5Y cells revealed an antiproliferative ability mediated through oxidative stress and both caspase-dependent and caspase-independent mechanisms.

Full text of DOI:10.3390/ph14010010

pharmaceuticals
Article
Combined Effect of Caspase-Dependent and Caspase-
Independent Apoptosis in the Anticancer Activity of Gold
Complexes with Phosphine and Benzimidazole Derivatives
Lara Rouco 1 , Ángeles Sánchez-González 2,*, Rebeca Alvariño 3,
* ,
, Amparo Alfonso 3
Ezequiel M. Vázquez-López 4 , Emilia García-Martínez 4 and Marcelino Maneiro 1,
*
1
Departamento de Química Inorgánica, Facultade de Ciencias, Universidade de Santiago de Compostela,
27002 Lugo, Spain; lara.rouco.mendez@usc.es
2
3
4
Departamento de Química Inorgánica, Facultade de Farmacia, Universidade de Santiago de Compostela,
15782 Santiago de Compostela, Spain
Departamento de Farmacología, Facultade de Veterinaria, Campus Terra,
Universidade de Santiago de Compostela, 27002 Lugo, Spain; amparo.alfonso@usc.es
Departamento de Química Inorgánica, Facultade de Química, Campus Universitario Lagoas-Marcosende,
Universidade de Vigo, 36310 Vigo, Spain; ezequiel@uvigo.es (E.M.V.-L.); emgarcia@uvigo.es (E.G.-M.)
Correspondence: angeles.sanchez@usc.es (Á.S.-G.); rebeca.alvarino@usc.es (R.A.);
marcelino.maneiro@usc.es (M.M.); Tel.: +34-982-824-106 (M.M.)
*
Abstract: Since the potential anticancer activity of auranofin was discovered, gold compounds
have attracted interest with a view to developing anticancer agents that follow cytotoxic mecha-
nisms other than cisplatin. Two benzimidazole gold(I) derivatives containing triphenylphosphine
(Au(pben)(PPh3)) (
standard techniques. X-ray crystal structures for
was tested in human neuroblastoma SH-SY5Y cells. Cells were incubated with compounds for 24 h
with concentrations ranging from 10 M to 1 nM, and the half-maximal inhibitory concentration
(IC50) was determined. and showed an IC50 of 2.7 and 1.6 M, respectively. In order to better
understand the type of cell death induced by compounds, neuroblastoma cells were stained with
Annexin-FITC and propidium iodide. The fluorescence analysis revealed that compounds were in-
ducing apoptosis; however, pre-treatment with the caspase inhibitor Z-VAD did not reduce cell death.
Analysis of compound effects on caspase-3 activity and reactive oxygen species (ROS) production
in SH-SY5Y cells revealed an antiproliferative ability mediated through oxidative stress and both
caspase-dependent and caspase-independent mechanisms.
1
) or triethylphosphine (Au(pben)(PEt3)) (
2
) were prepared and characterized by
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
1
and were solved. The cytotoxicity of
2
1
and
2
Citation: Rouco, L.; Sánchez-González,
Á.; Alvariño, R.; Alfonso, A.; Vázquez-
López, E.M.; García-Martínez, E.;
Maneiro, M. Combined Effect of Caspase-
Dependent and Caspase-Independent
Apoptosis in the Anticancer Activity
of Gold Complexes with Phosphine
and Benzimidazole Derivatives.
Pharmaceuticals 2021, 14, 10.
µ
1
2
µ
https://dx.doi.org/10.3390/ph1401
0010
Keywords: gold(I) compounds; cytotoxic activity; caspase; apoptosis; neuroblastoma SH-SY5Y
Received: 24 November 2020
Accepted: 21 December 2020
Published: 24 December 2020
1. Introduction
Publisher’s Note: MDPI stays neu-
tral with regard to jurisdictional claims
in published maps and institutional
affiliations.
The pharmacological use of gold compounds for the treatment of different diseases
has been documented since ancient times [
the antiarthritic properties for Au(I) thiolates were observed [
fects led to the development of gold complexes with phosphine ligands, which are more
lipophilic and remain in circulation for a longer time [ ]. Auranofin, 2,3,4,6-tetra-O-acetyl-
L-thio- -D-glyco-pyranosato-S-(triethylphosphine) gold(I) is an example of these Au(I)
thiolates-phosphine derivatives (see Scheme 1A), which contain a S-Au-P fragment, used as
1
,2
]. More recently, in the twentieth century,
2], but their adverse ef-
3
β
Copyright: © 2020 by the authors. Li-
censee MDPI, Basel, Switzerland. This
article is an open access article distributed
under the terms and conditions of the
Creative Commons Attribution (CC BY)
license (https://creativecommons.org/
licenses/by/4.0/).
disease-modifying anti-rheumatic drugs [3–5] but also investigated for other potential ther-
apeutic applications, from neurodegenerative disorders [
inhibition of the novel SARS-COV-2 coronavirus [8].
6] or bacterial infections [7] to the
Pharmaceuticals 2021, 14, 10. https://dx.doi.org/10.3390/ph14010010
https://www.mdpi.com/journal/pharmaceuticals

Pharmaceuticals 2021, 14, 10
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(A)
(B)
Scheme 1.
(
A
) Auranofin: 2,3,4,6-tetra-O-acetyl-L-thio-
β
-D-glyco-pyranosato-S-(triethylphosphine) gold(I); (B) Numbering
scheme of 2-(20-pyridyl)benzimidazole (Hpben), which is the N-donor ligand used in this work to coordinate gold atom
instead of the S-glycoside ligand of auranofin.
The anticancer activity of this type of compound has also attracted interest due to its
cytotoxic activity against cells from several tumor cell lines [9–11]. Among them, ovarian
carcinoma [12], colorectal cancer [13], cervical epithelial carcinoma [14], and lung cancer
cells [15]. The mechanism associated with this therapeutic activity has not yet been totally
established, but results point to the ability of the Au(I) complexes to interact with biological
targets such as proteins or nucleic acids [16
,17] and, particularly, with thiol-containing
proteins like the thioredoxin reductase (TrxR) [18
,19], an enzyme involved both in the
defense against oxidative damage and in the redox signaling, being relevant for cell growth
and development [20]. Other studies have clearly shown the proteasome as a primary target
for these complexes [21] or the inhibition of other enzymes such as glutathione reductase or
peroxidase [22]. The anticancer activity of these gold compounds seems to follow different
mechanisms than cisplatin or other Pt(II)/Pd(II) compounds, which mainly act by binding
to DNA and inhibiting its replication. Gold compounds also exhibit cytotoxic activity
against cisplatin-resistant cell lines [9,10].
The increasing interest in developing different types of S-Au-P gold-based anticancer
candidates led to new synthetic drugs rather than the former pyranosidic derivatives.
In this sense, gold complexes incorporating ligands such as thiosemicarbazone [14
,23,24],
thiolate [25 26], dithiocarbamate [27], and sulfanylcarboxylates [28] have been probed
,
to inhibit thioredoxin reductase by binding the selenocysteines or the cysteinyl thiols of
the enzyme.
The replacement of the S-donor ligands by N-donor ligands produced a new family
gold complexes that have revealed interesting anticancer activities. These N-donor ligands
include imidazoles [29], naphthalimides [30], cytosines [31], bipyridines [32], phenanthro-
lines [33], or benzimidazoles [34–36]. The biochemical mechanism of action involved in the
anticancer activity of the gold complexes with N-donor ligands is presumably the same
as that of auranofin, although it is a matter of study interest due to the diversity of re-
sults obtained in different investigations regarding biological targets for auranofin [18–22],
and due to the different complex stabilization by the replacement of the thiolate ligand by
the N-donor ligand.
In the present study, we propose to use 2-(20-pyridyl)benzimidazole (Hpben) as
a ligand with potentially three N-donor atoms (see Scheme 1B), which would lead to
different coordinations with the gold atom [35]. Benzimidazoles are very versatile ligands
that show various pharmacological properties such as antibacterial, antiviral, and anti-
inflammatory [37,38]. Our aim is to obtain N-Au-P complexes, for which Hpben and two
different phosphines are used: triphenylphosphine and triethylphosphine. Human neurob-
lastoma SH-SY5Y is the cell line chosen to study the cytotoxicity, the type of cell death in the
anticancer activity, and the mechanism of antiproliferative action of these gold complexes.

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2. Results
2.1. Analytical and Spectroscopic Characterization of 1 and 2
Complexes
1
and
2
were obtained in high yield as detailed in the experimental sec-
1
tion (Scheme 2). Their structures were fully characterized using H, 13C and 31P NMR
spectroscopy, IR and UV spectroscopy, electrospray ionization mass spectroscopy (ESI-
MS), and single-crystal X-ray crystallography. Elemental analyses establish a formula
(Au(pben)(PR3)), being R=Ph for
mulas for the neutral complexes by showing molecular ion peaks at m/z 644.14 for
and m/z 510.13 for (Figures S1 and S2). UV-Vis spectra showed in DMSO a common
absorption band at 313 nm assigned to * transitions located in the heteroaromatic
benzimidazole rings (Figures S3 and S4). Complex displays an additional band at 325
1 and R=Et for 2. ESI-MS spectra confirmed these for-
1
2
π–π
1
nm, which has also previously been found in gold(I) complexes containing the ancillary
PPh3 ligand [34].
Scheme 2. Synthesis of complexes 1 and 2.
The IR spectra of these complexes (Figures S5 and S6), when compared with the
corresponding spectrum of its free ligand, show the absence of the ν(N–H) band, which is
present in the spectrum of the Hbpen between 2990–2700 cm−1, consistent with the de-
protonation of this group and the N-coordination to the Au atom. In both complexes,
the aromatic tension bands of the pben ligand do not undergo significant changes when
coordination occurs, although rigorous assignment is made difficult by the great intensity
showed by the bands from the phosphines.
1
The H NMR spectra of
1
and
2
showed each one as well-resolved set of signals
(Figures S7 and S8); the H60 proton at
δ 8.57/8.52 was the most deshielded and downfield
shifted of 0.13–0.18 ppm with respect to the free ligand (1H NMR and 13C NMR spectra
for free ligand are collected in Figures S9–S12), while the resonance of the H30 proton was
shifted at about 0.18 ppm, which suggests some involvement of the pyridinic nitrogen in
the Au-pben bonding. In both complexes, the N–H signal of the free ligand disappeared,
confirming the deprotonation of Hbpen. In the spectrum of 1, the signals corresponding to
the co-ligand PPh3 overlap with those of pben−, so the assignment is tentative. Aromatic
carbons from pben− and triphenylphosphine ligands are registered in the 13C NMR spec-
trum of
evidence that the gold complex is not altered in solution (Figure S14). In the case of
1 1 shows a single signal at 30.9 ppm, which is
(Figure S13). 31P NMR spectrum of
2,
the identification and assignment of PEt3 signals were unambiguous with the –CH3 signals
as a doublet of triplets at 1.31 ppm due to the coupling with 31P (coupling constant 3J(H–P)

Pharmaceuticals 2021, 14, 10
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= 18.5 Hz, slightly higher than the corresponding to the free PEt3, 14 Hz). The 13C NMR
spectrum of
and 18.5 (d, CH2, J(C–P) = 37.3 Hz) ppm,
other Au(I)-PEt3 complexes [14 28], and that contrast with the values of non-coordinated
PEt3. 31P NMR spectrum of 2 showed a single signal at 26.9 ppm (Figure S16).
The electrochemical behavior of compounds was investigated in DMSO- tetraethy-
2
showed two signals (Figure S15) corresponding to the phosphine at 9.2 (CH3)
δ
and J values which are like those found for
,
1, 2
lammonium perchlorate 0.1 M solvent system through cyclic voltammetry. Figure 1 shows
the voltammograms at different scan rates. The Au(I) gold complexes undergo one oxida-
tion process (at −0.594 for 1 and at −0.597 V for 2) to form the Au(III) species.
(A)
(B)
Figure 1. Cyclic voltammograms for (
A
)
1
and (
B
)
2
at different scan rates: 0.02 V s−1 (blue), 0.05 V s−1 (green) and
−1
0.09 V s (red).
The redox properties of the two gold(I) complexes showed grossly similar behavior
consisting of a quasi-reversible Au(I)/Au(III) oxidation. The criteria of reversibility were
checked by observing the constancy of peak-peak separation (
for and 214 mV for
at 0.02 V s−1. The reversible character decreased as scan rates
increased. Thus, Ep are 281 mV for and 259 mV for
at 0.05 V s−1, and even 344 mV for
and 297 mV for
at 0.09 V s−1. As is predictable, the intensity of the current also increased
∆Ep = Epa – Epc) of 188 mV
1
2
∆
1
2
1
2
at higher scan rates. Cyclic voltammograms of
complexes in solution.
1,2 endorse the purity and stability of both
2.2. Crystal Structures of Complexes 1 and 2
Single crystals of complex
1 suitable for X-ray diffraction studies were obtained by
slow evaporation of the methanolic solution at room temperature. The molecular structure
is shown in Figure 2 and Figure S17, and the main bond distances and angles are shown in
Table 1.

Pharmaceuticals 2021, 14, 10
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Figure 2. ORTEP representation of one of the molecules presents in the crystal structure of compound
1 (hydrogen atoms have been omitted for clarity).
◦
Table 1. Selected bond lengths (Å) and angles ( ) for 1.
◦
Bond
Bond
Angles ( )
Lengths (Å)
Au(1)-N(1)
Au(1)-P(1)
Au(1)···N(3)
2.058(6)
2.230(2)
2.792(7)
176.40(18)
68.5(2)
113.52(17)
110.5(3)
115.1(3)
112.9(3)
129.6(6)
125.5(5)
106.2(5)
134.2(7)
Au(2)-N(4)
Au(2)-P(2)
Au(2)···N(6)
2.060(6)
2.235(2)
2.782(7)
174.75(18)
68.8(2)
114.48(16)
113.9(3)
108.0(3)
114.9(3)
126.3(5)
128.7(5)
104.7(5)
135.3(6)
N(1)-Au(1)-P(1)
N(1)-Au(1)-N(3)
P(1)-Au(1)-N(3)
C(13)-P(1)-Au(1)
C(25)-P(1)-Au(1)
C(19)-P(1)-Au(1)
C(3)-N(1)-Au(1)
C(1)-N(1)-Au(1)
C(8)-N(3)-Au(1)
C(9)-N(3)-Au(1)
N(4)-Au(2)-P(2)
N(4)-Au(2)-N(6)
P(2)-Au(2)-N(6)
C(43)-P(2)-Au(2)
C(49)-P(2)-Au(2)
C(55)-P(2)-Au(2)
C(31)-N(4)-Au(2)
C(33)-N(4)-Au(2)
C(38)-N(6)-Au(2)
C(39)-N(6)-Au(2)
The asymmetric unit contained two molecules of
1, (Au(pben)PPh3), with slightly
different bond distances and angles values and one disordered methanol molecule. Since it
did not establish any relevant interaction with (Au(pben)PPh3), it was removed using
the SQUEEZE program [39]. In the two molecules of the complex, the metal ion is co-
ordinated to the phosphorous atom of triphenylphosphine and to the nitrogen atom of
the benzimidazolate ring with an almost linear disposition. An additional contact was
established between the nitrogen atom of the pyridine and the gold ion, giving rise to
a 2 + 1 coordination number.
The Au-P Bond lengths (2.230(2) y 2.235(2) Å) were similar to those found in the
cationic complex (Au(Hpben)(PPh3))ClO4 [40]. However, the Au-N bond distances (2.058(6)
and 2.060(6)) were slightly shorter than those found in the (Au(Hpben)(PPh3))+ cation
(2.075(4) Å), consistent with a stronger interaction between the metal ion and the depro-
tonated ligand. The bond length between the gold and the pyridine nitrogen (Au(1)···
N(3) = 2.729(7) Å and Au(2)···N(6) = 2.782(7) Å) was shorter than the sum of the Van
der Waals radii (3.25 Å), and shorter than that observed in (Au(Hpben)(PPh3))ClO4
(2.930 Å) [40]. Bond lengths and angles found in the triphenylphosphine ligands (not
collected in Table 2) showed normal values and similar to those reported for complexes of
Au(I) and PPh3 [40–42].

Pharmaceuticals 2021, 14, 10
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◦
Table 2. Selected bond lengths (Å) and angles ( ) for 2.
◦
Bond
Bond
Angles ( )
Lengths (Å)
Au(1)-N(1)
Au(1)-P(1)
2.050(6)
2.2288(18)
2.975(6)
172.59(16)
66.34(19)
120.75(12)
Au(2)-N(4)
Au(2)-P(2)
2.058(5)
2.224(2)
2.849(6)
174.37(17)
67.38(19)
116.88(13)
Au(1)···N(3)
Au(2)···N(6)
N(1)-Au(1)-P(1)
N(1)-Au(1)-N(3)
P(1)-Au(1)-N(3)
N(4)-Au(2)-P(2)
N(4)-Au(2)-N(6)
P(2)-Au(2)-N(6)
Single crystals of 2 were obtained as detailed in the experimental section; a perspective
view is shown in Figure 3 and Figure S18, and selected bond parameters are collected in
Table 2. The asymmetric unit of the crystal structure of also consists of two chemically
2
equivalent molecules in which the benzimidazolate unit is acting as a monodentate ligand
through the N(1) nitrogen atom. The gold(I) ion was also arranged in the usual almost
linear coordination geometry, coordinated to pben and to the phosphorous atom of tri-
ethylphosphine. In the same way as in 1, an additional contact was established between
the nitrogen atom of the pyridine and the gold ion, giving rise to a 2 + 1 coordination
number. Au-P Bond lengths (2.2288(18) and 2.224(2) Å) and Au-N bond distances (2.050(6)
and 2.058(5)) are values very close to those found in 1.
Figure 3. ORTEP representation of one of the molecules presents in the crystal structure of compound
2 (hydrogen atoms have been omitted for clarity).
2.3. Cytotoxicity Studies and Determination of Cell Death Type
SH-SY5Y neuroblastoma cells were used to study the toxic effect of the Au(I) com-
plexes. Cells were treated with different concentrations of
6 and 24 h. Table 3 shows IC50 values calculated for compounds (see also Figure S19).
Complex was more cytotoxic, presenting lower IC50 values than complex at both times.
1 and 2 at two incubation times,
2
1

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Table 3. IC50
(
µM) of gold compounds in SH-SY5Y cells determined with a 3-(4,5-dimethyl thiazol-
2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. IC50 (half maximal inhibitory concentration),
R2 (coefficient of determination which gives the goodness-of-fit for the regression model).
Compound
1
Incubation Time (h)
IC50 (µM)
R2
95% Confidence Interval
6
24
6
6.2
2.7
3.1
1.6
0.97
0.94
0.93
0.91
3.851 to 8.30
1.534 to 4.684
1.340 to 5.015
0.8094 to 3.018
2
24
Next, to determine the type of cell death produced by gold complexes, their effects on
neuroblastoma cells were evaluated using the fluorescent dyes Annexin V-FITC (fluorescein
isothiocyanate) and propidium iodide (PI), which allow the detection of necrotic and
apoptotic cell populations by flow cytometry. Three populations were discriminated:
viable cells (Annexin V-FITC and PI negative cells), apoptotic cells (including Annexin V-
FITC positive and PI negative cells, and Annexin V-FITC and PI-positive cells), and necrotic
cells (Annexin V-FITC negative and PI-positive cells). In this assay, SH-SY5Y cells were
treated with compounds at IC50 concentrations for 6 and 24 h. Furthermore, to check
if the complexes
incubated with the pan-caspase inhibitor Z-VAD-FMK (carbobenzoxy-valyl-alanyl-aspartyl-
[O-methyl]-fluoromethylketone) at 40 M for 24 h, followed by treatment with compounds
1 and 2 were inducing caspase-dependent apoptosis, cells were pre-
µ
at IC50 concentrations for 6 and 24 h. In order to validate the model, staurosporine (STS)
was used as cell death control (Figure 4).

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Figure 4. Determination of cell death type produced by
with compounds for 6 (A) and 24 h (B) with and without pre-incubation with 40 µ
1
and
2
on SH-SY5Y cells. Human neuroblastoma cells were treated
M Z-VAD. Staurosporine (STS) was used
as a positive control. Cell death type was determined with Annexin V-FITC/PI staining by flow cytometry. Values are mean
SEM of three independent replicates expressed as a percentage of untreated control cells. Statistical differences were
±
determined by Student’s t test. * p < 0.05, comparison of compound treatment and control cells. # p < 0.05, comparison
between treatments with compounds alone and co-treatment with compounds and Z-VAD.
Treatment with compounds
1
and
2
for 6 h produced a significant decrease in vi-
1.4%, respectively (p < 0.05) (Figure 4A).
able cells, with levels of 70.6 3.0% and 74.1
±
±
In this assay, gold compounds produced a lower decrease in cell viability than expected,
which could be due to the differences between the 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl
tetrazolium bromide (MTT) assay and Annexin/PI staining. IC50 values were determined
with MTT, which quantifies cell viability based on mitochondrial activity, so it was more
sensitive than the Annexin/PI staining, which detects changes in the plasmatic mem-
brane [43
–
45]. Both complexes produced an increase in apoptotic cells (15.0
1.3% for ). Moreover, compound significantly augmented necrotic cells
1.0%) compared to control cells. As expected, the addition of 0.1 M STS, a known
±
1.2% for
1
and 13.7
±
2
1
(14.4
±
µ
apoptotic inducer, generated an increase of apoptotic cells (29.3 ± 3.1%).
On the other hand, cells co-treated with Z-VAD and compounds and
sented a significant decrease in viable cells (71.6 2.7% and 74.3 2.1%, respectively).
Interestingly, the percentages of apoptotic cells after pre-treatment with Z-VAD (14.1 ± 0.7%
and 13.0 0.9%, respectively) were similar to those obtained with the compounds alone.
Otherwise, the apoptotic death produced by 0.1 M STS alone was significantly reduced
when neuroblastoma cells were pre-treated with the caspase inhibitor (18.5 1.1%, p < 0.05).
1
2 for 6 h pre-
±
±
±
µ
±
Regarding necrosis, its levels were comparable to those observed with gold complexes
alone, with percentages among 12.7–14.3%. In the case of STS, necrotic cells were signif-
icantly reduced by pre-treatment with Z-VAD. The results obtained with STS validated
the model since its cytotoxic effects were reduced when the caspase inhibitor was added
to SH-SY5Y cells. However, Z-VAD was unable to reduce the cell death produced by
gold compounds.
This assay was repeated after a longer incubation time, 24 h (Figure 4B). A reduction in
cell survival was observed when cells were treated with complexes
59.3
apoptotic cells, with levels of 22.8
when cells were treated with
neuroblastoma cells were pre-treated with 40
complexes and for 24 h. In this assay, both compounds produced a significant decrease
in viable cells (67.5 2.1% and 66.2 3.3%, respectively). In addition, the levels of
1
and
2
(
68.1 ± 0.9% and
±
2.0%, respectively). Once again, compounds produced a significant augmentation of
±
0.6% after the addition of complex
. In order to further confirm the results obtained at 6 h,
1
, and 27.0 ± 0.5%
2
µM Z-VAD, followed by the addition of
1
2
±
±

Pharmaceuticals 2021, 14, 10
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apoptotic cells were between 19.9–23.2%, confirming the inability of Z-VAD to inhibit the
death of neuroblastoma cells. With respect to STS, pre-treatment with Z-VAD did not
produce a significant reduction in the apoptotic death generated by the compound after
24 h. This effect has been previously described and is related to the ability of STS to produce
caspase-dependent and caspase-independent apoptosis. The first one happens at shorter
incubation times, whilst the second one occurs more slowly and can be observed at longer
incubation times [46,47]. In our model, Z-VAD reduced the apoptosis produced by STS
after 6 h of incubation, but no effect was found at 24 h, agreeing with these previous results.
In the case of gold complexes, previous studies have reported that this class of
compounds can also induce apoptosis through both caspase-dependent and caspase-
independent mechanisms [48,49]. In this context, the effect of complexes 1 and 2 on
caspase-3 activity was analyzed. SH-SY5Y cells were treated with compounds at IC50
concentrations for 6 and 24 h, and the enzymatic activity was determined (Figure 5).
Figure 5. Caspase-3 activity in human neuroblastoma cells treated with gold compounds
Compounds were added to the cells for 6 h and 24 h. STS was used as a positive control. Data are
mean SEM of three experiments performed in triplicate, expressed as a percentage of control cells,
and compared control cells by Student’s t test. * p < 0.05, ** p < 0.01, *** p < 0.001.
1 and 2.
±
At 6 h, STS produced an augmentation in caspase-3 activity of 242.5
agreeing with its previously reported effect on caspase-dependent apoptosis at short
incubation times [46 47]. With regard to gold complexes, only showed a significant
increase in enzyme activity (190 10.3%; p < 0.001). The assay was repeated for 24 h and
both complexes augmented caspase-3 activity, 129.0 10.5% (p < 0.05) and 140.8 12.3%
(p < 0.01), respectively. At this time, STS also produced a significant increase in caspase-3
activity (170.5 6.8%, p < 0.001). This rise was smaller than the observed at 6 h, confirming
that the involvement of caspase-3 in STS-induced death is lesser at 24 h. With regard to
gold complexes, compound presented a similar behavior to STS, with a lower increase in
caspase-3 activity at 24 h, whereas complex produced a slight augmentation of caspase-3
±
7.2% (p < 0.001),
,
2
±
±
±
±
2
1
activity. These results could explain the lack of effect of pre-treatment with Z-VAD in our
previous assays, pointing to a combined effect of the compound on caspase-dependent and
caspase-independent apoptosis.
Previously published studies [48
cell death through the induction of oxidative stress. Therefore, the effect of compounds on
ROS production was analyzed. Cells were co-treated with and at nine concentrations
(0.01–10 M) for 24 h, and the levels of these damaging molecules were evaluated with
a fluorometric assay (Figure 6). Treatment with complex significantly augmented ROS
,49] stated that Au(I) and Au(III) complexes produce
1
2
µ
1

Pharmaceuticals 2021, 14, 10
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production, reaching levels of 122.4
Compound
(0.01, 0.5, and 7.5
±
11.9% (p < 0.05) at 5
also produced a significant increase in ROS release at three concentrations
µ
M compared to control cells.
2
µ
M), being the greater augmentation at 7.5 M (129.7 13.8%, p < 0.05).
µ
±
The increase generated by gold complexes was comparable to the rise produced by the
known pro-oxidant tert-butyl hydroperoxide (TBHP) at 75 µM (134.9 ± 7.1%, p < 0.001).
Figure 6. Effect of
were added to the cells for 24 h. The fluorescent dye carboxy-H2DCFDA was used to evaluate reactive
oxygen species (ROS) production. tert-butyl hydroperoxide (TBHP) at 75 M was used as a positive
control. Mean SEM of three independent experiments. Data are expressed as a percentage of
1 and 2 on ROS levels in human neuroblastoma cells. Compounds (0.01–10 µM)
µ
±
control cells. Statistical differences between treatments and control cells were determined with
Student’s t test. * p < 0.05, ** p < 0.01, *** p < 0.001.
3. Discussion
The set of techniques used to characterize 1 and 2 both in solid-state and in solution
has made it possible to establish the high purity of both compounds and their stability in so-
lution. Structural characterization did not allow the differentiation of any property between
compounds that caused a difference in cytotoxicity. However, complex
2, which incorpo-
rates triethylphosphine co-ligand, shows more cytotoxicity than , with a triphenylphos-
1
phine co-ligand. The same influence of substitution of PPh3 for PEt3 on cytotoxicity has
been already reported before for mono phosphinegold(I) sulfanylcarboxylates [28].
Different electrochemical properties have been reported for other anticancer gold
complexes [34–36], but, in the present case, the electrochemical behavior of 1 and 2 is
basically similar, according to both their redox potential values and their quasi-reversible
character, so we can conclude that this should not be the factor that determines the different
toxicity of both compounds. The greatest steric effect of the triphenyl substituents on
phosphine in
1 with respect to the triethyl substituents in 2 arises as a possible explanatory
factor that would explain the different antiproliferative action of these gold complexes.
One of the main interest in anticancer gold complexes is the development of pharmaco-
logical candidates with cytotoxic activity against cells from tumor lines which are resistant
to the chemotherapeutic drug cisplatin or new drugs that may induce apoptosis in cancer
cells through different mechanisms than cisplatin or other Pt(II)/Pd(II) compounds [9,10].
The cytotoxicity of cisplatin and oxaliplatin have been previously reported on the hu-
man neuroblastoma cell line SH-SY5Y [50]. IC50 values for these platinum compounds
of 15–50 µM using the MTT assay at an incubation time of 24 h are significantly higher

Pharmaceuticals 2021, 14, 10
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than values obtained for gold(I) complexes
more cytotoxic.
1
and
2
(IC50 of 1.6–2.7
µM), which behaved as
In the case of the cisplatin derivatives, their cytotoxic activity has been linked to their
ability to crosslink with the purine bases on the DNA. In the present study, the results
obtained in the caspase-3 assay and in the determination of ROS production indicate
that gold complexes could have antitumor behavior, being able to induce apoptotic cell
death through the increase of ROS levels. The redox potentials of
1 and 2 are outside
the biologically accessible redox potential window of 0.4 to +0.8 V, suggesting that
−
these complexes do not directly generate ROS due to the Au(I)/Au(III) process but rather
as a consequence of inhibition of TrxR [48]. This inhibition damages the thioredoxin
system, an important cellular antioxidant defense, increasing ROS levels, and leading
to cell death [51–53]. The apoptotic cell death produced by complexes 1 and 2 seems
to be mediated by two mechanisms, both caspase-dependent and caspase-independent.
This dual behavior has been reported for other gold complexes [49] and could be related to
the increase in ROS levels produced by compounds. Elevated ROS causes mitochondrial
membrane depolarization, which produces a disruption of the electron transport chain,
leading to the release of cytochrome c and the apoptosis-inducing factor (AIF) from the
mitochondria. Cytochrome c release induces caspase-dependent apoptosis, mediated by
caspase-3 and caspase-9 activation, whilst AIF is translocated to the nucleus and produces
caspase-independent apoptosis [48].
4. Materials and Methods
4.1. Materials
Triphenylphosphinegold(I) chloride (99.9%, Strem Chemicals), Triethylphosphine-
gold(I) chloride (99%, ABCR) and 2-(20-pyridyl)benzimidazole (Hpben; 99.8%, Aldrich,
Darmstadt, Germany) were used as supplied.
4.2. Physical Measurements
Elemental analyses of C, H, and N were performed on a Carlo Erba EA-1108 (CE
Instruments, Wigan, UK). IR spectra were recorded as KBr discs on a Bio-Rad FTS135
spectrophotometer (Bio-Rad Laboratories, Hercules, CA, USA) in the range 4000–400 cm–1.
IR data are reported in the experimental section following abbreviations: vs = very strong;
s = strong; m = medium; w = weak; sh = shoulder; br = broad.
1H NMR and 13C NMR spectra were recorded on a Bruker AC-300 spectrometer
(Bruker BioSpin, Rheinstetten, Germany) at 296 K using DMF-d7 or CDCl3 as deuterated
1
solvents. H NMR and 13C NMR were recorded in
δ units relative to deuterated solvent as
an internal reference. 31P NMR spectra were recorded in CDCl3 at 202.46 MHZ on a Bruker
AMX 500 spectrometer (Bruker Analytik, Karlsruhe, Germany) using 5 mm o.d. tubes and
are reported to external H3PO4 (85%). The following abbreviations were used as s = singlet;
d = doublet; t = triplet; m = multiplet.
Positive electrospray ionization (ESI) mass spectra of the ligands and complexes were
recorded on a LC-MSD 1100 Hewlett-Packard instrument (Hewlett-Packard, Palo Alto, CA,
USA) (positive-ion mode, 98:2 CH3OH/HCOOH as the mobile phase, 30–100 V). Electronic
spectra were recorded on a Cary50 spectrometer.
Electrochemical measurements were performed using an Autolab PGSTAT101
(Metrohm Autolab, Kanaalweg, The Netherlands) using a three-electrode configuration.
The working electrode was a Metrohm model 6.1204.300 graphite disc, while a Pt wire
and an Ag/AgCl electrode served as counter and reference electrodes, respectively. Mea-
surements were made with ca. 10−3 M solutions of complexes in DMF using 0.1 M
tetraethylammonium perchlorate as a supporting electrolyte.

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4.3. Synthesis of the Complexes
Complexes were prepared through ligand deprotonation with sodium hydroxide,
previous to the addition of the gold salt in stoichiometric relation [28,35]. Experimental
procedure and characterization data for 1 and 2 are collected below.
4.3.1. (Au(pben)(PPh3)) (1)
To a stirred solution of (AuCl(PPh3)) (200 mg, 0.4 mmol) in methanol (10 mL) was added
0
a methanolic solution (4 mL) of 2-(2 -pyridyl)benzimidazole (Hpben, 79 mg, 0.4 mmol) and
NaOH (16 mg, 0.4 mmol). The yellow solution formed was stirred in absence of light
for 24 h at room temperature. The crystalline white solid formed was filtered, washed
with cold methanol, and dried under vacuum. Yield: 83%. M.P.: 158–160 ◦C. Anal Calcd.
(%) for C30H23N3PAu (653.46 g mol−1): C, 55.1; N, 6.4; H, 3.6. Found: C, 55.0; N, 6.4;
H, 3.7. MS ES (m/z) 654.14 (Au(pben)(PPh3))+. IR (KBr, cm−1):
ν
(C–H) 3050w;
ν(PPh3)
1590 m, 1500 m, 1420 s; 1230 m, 1160 m, 1045 m, 820 m. UV-vis (DMSO): max: 313, 325 nm.
λ
1H NMR (CDCl3, ppm):
δ
8.57 (d, 1H, H60), 8.20 (d, 1H, H30), 7.5–7.9 (m + m + m, 4H,
H50-H7-H4-H40), 7.5–7.7 (m + m + m, 15H, PPh3), 7.20 (m, 2H, H5-H6). 31P NMR (CDCl3,
ppm) 30.93 (s). Eox (at 0.02 V s−1) = −0.594 V; Ered (at 0.02 V s−1) = −0.782 V.
4.3.2. (Au(pben)(PEt3)) (2)
A solution of Hpben (83.5 mg, 0.428 mmol) and NaOH (17.1 mg, 0.428 mmol) in
methanol (4.3 mL) was added to a stirred solution of (AuCl(PEt3)) (150 mg, 0.428 mmol) in
methanol (3 mL). The resulting mixture was stirred in the dark for 48 h at room temperature.
Afterward, the solution was cooled to 4 ◦C, the solvent was evaporated under vacuum,
the oil formed was dissolved in acetonitrile, filtered, and the solvent removed under
vacuum. The white solid formed was dried under vacuum. Yield: 56%. M. P.: 156–158 ◦C.
Anal Calcd. (%) for C18H23N3PAu (509.33 g mol−1): C, 42.5; N, 8.3; H, 4.5. Found: C, 42.2;
N, 8.1; H, 4.6. MS ES (m/z) 510.13 (Au(pben)(PEt3))+. IR (KBr, cm−1): 3049w,
(PEt3), 1587 m, 1499 w, 1420 s; 1275 m, 1140 m, 1044 m, 993 m, 820 m. UV-vis (DMSO):
ν(C-H);
ν
1
λ
max: 313 nm. H NMR (CDCl3, ppm):
3H, H7-H4-H40), 7.23 (d, 1H, H5-H6), 1.31 (dt, 9H, PEt3), 1–93 (m, 6H, PEt3). 31P NMR
(CDCl3, ppm) 26.91 (s). Eox (at 0.02 V s−1) = 0.597 V; Ered (at 0.02 V s−1) =
0.811 V.
from acetonitrile afforded crystals suitable for X-ray crystallography.
δ
8.52 (d, 1H, H60), 8.47 (d, 1H, H30), 7.65–7.85 (m,
−
−
Recrystallization of
2
4.4. Cell Culture
The human neuroblastoma SH-SY5Y cell line used in this study was purchased from
the American Type Culture Collection (ATCC), number CRL2266. Cells were grown in
Dulbecco’s modified Eagle’s medium: Nutrient Mix F-12 (DMEM/F-12) supplemented
with 10% fetal bovine serum (FBS), 1% glutaMAX, 100 U/mL penicillin and 100 µg/mL
streptomycin. Cells were maintained at 37 ◦C in a humidified atmosphere of 5% CO2 and
95% air. Cells were dissociated weekly using 0.05% trypsin/EDTA when they reached
a confluence of 80%. All reagents were provided by Thermo Fisher Scientific (Waltham,
MA, USA).
4.5. Cytotoxic Effects
SH-SY5Y cells were seeded into 96-well plates at a density of 5
and allowed to grow for 24 h. Cells were treated with compounds
×
104 cells per well
and at different
1
2
concentrations (0.01, 0.1, 0.5, 1, 2.5, 5, 7.5, and 10
fect of compounds was evaluated with MTT (3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl
tetrazolium bromide) assay [54 56]. SH-SY5Y cells were rinsed with saline solution and
200 L of 500
µM) for 6 and 24 h. The cytotoxic ef-
–
µ
µg/mL MTT (Merck, Darmstadt, Germany) dissolved in saline buffer were
added to each well. Following 1 h of incubation in an orbital shaker at 37 ◦C and 300 rpm,
SH-SY5Y cells were disaggregated with 5% sodium dodecyl sulfate. The absorbance of
formazan crystals was measured at 595 nm with a spectrophotometer plate reader. Saponin
at 1 mg/mL was used as cell death control. The concentration of compound that produced

Pharmaceuticals 2021, 14, 10
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a 50% inhibition of cell survival (half maximal inhibitory concentration, IC50) was deter-
mined by fitting the data with a log(inhibitor) vs response model using GraphPad Prism
6 software.
4.6. Flow Cytometry Analysis of Cell Death Type
The cell death type induced by compounds was determined with an Annexin V-
FITC Apoptosis detection kit (Immunostep, Salamanca, Spain) following the manufac-
turer’s instructions [56]. Cells were seeded in 12-well plates at 1
treated with compounds at IC50 concentrations for 6 and 24 h. Then, cells were washed,
resuspended in PBS (Phophate-Buffered Saline), and 5 L of Annexin V-FITC and Propid-
×
106 cells per well and
µ
ium Iodide (PI) were added to each tube. Cells were incubated for 15 min in the dark and
analyzed by flow cytometry using the ImageStreamMKII instrument (Amnis Corporation,
LuminexCorp, Austin, TX, USA). The fluorescence of 10,000 events was analyzed with
IDEAS Application 6.0 software (Amnis Corporation, LuminexCorp). The percentages of
apoptotic cells, including early apoptotic cells (Annexin-FITC positive and PI negative)
and late apoptotic cells (Annexin-FITC and PI-positive), and necrotic cells (Annexin-FITC-
negative and PI-positive), were calculated. To further confirm if apoptotic cell death was
occurring, SH-SY5Y cells were preincubated with the pan-caspase inhibitor Z-VAD-FMK
(Merck) for 24 h. Then, the assay was carried out as described above. Staurosporine (STS)
(Merck) was used as a positive control in all the experiments [56].
4.7. Caspase-3 Assay
Analysis of caspase-3 activity in SH-SY5Y cells after exposure to gold complexes was
carried out using the EnzChek Caspase-3 Assay Kit (Thermo Fisher Scientific), follow-
ing the manufacturer’s instructions [56]. Cells were seeded in 12-well plates at 1
cells per well and treated with compounds at IC50 concentrations for 6 and 24 h. Then,
cells were lysed with 50 L lysis buffer (10 mM TRIS, pH 7.5, 0.1 M NaCl, 0.01% CHAPS
(3-[(3-chloramidopropyl)dimethylammonio]-1-propanesulfonate), 1 mM EDTA and 0.01%
TRITON X-100), resuspended and centrifuged. The pellet was resuspended in 50
of reaction buffer (20 mM PIPES, pH 7.4, 4 mM EDTA, 0.2% CHAPS), 10 L of 1 M DTT
(dithiothreitol), and 590 L of H2O. Then, 20 L of 0.2 mM Z-DEVD–AMC substrate
×
106
µ
™
µL
µ
µ
µ
(7-amino-4-methylcoumarin-derived substrate) were added, and lysates were incubated
for 30 min at room temperature. Finally, the fluorescence was monitored with a spec-
trophotometer plate reader (excitation/emission 342/441 nm). Signals were normalized
by protein concentration, which was quantified by the Bradford method. Briefly, 2
µL of
each lysate were added to 200 L of Bradford reagent, and absorbance was measured at
µ
590 nm. Protein concentration in samples was determined using a standard curve with
known concentrations of bovine serum albumin. Experiments were carried out three times
in triplicate, and STS was used as a positive control.
4.8. Determination of ROS Production
Intracellular ROS production was evaluated with the fluorescent dye carboxy-H2DCF
DA [57]. Cells were treated with complexes 1 and 2 at concentrations between 0.01 and
10 µM for 24 h. After this time, cells were washed with medium without serum, and 20 µM
carboxy-H2DCFDA was added. The plate was incubated in an orbital shaker for 1 h
at 37◦C and 300 rpm. Then, PBS was added to each well, and cells were incubated for
30 min before measuring the fluorescence with a spectrophotometer plate reader (495 nm
excitation and 527 nm emission). Experiments were performed at least three times by
triplicate. The known oxidant tert-butyl hydroperoxide (TBHP) at 75
a positive control to validate the assay.
µM was used as
4.9. Statistical Analysis
Data are presented as mean
t-test with Graph Pad Prism 6 software. Statistical significance was considered at p < 0.05.
±
SEM. Statistical differences were evaluated by Student’s

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4.10. Crystallographic Studies
Crystals and were obtained as mentioned above. Data for
at 293 K for and 100 K for
monochromated Mo-K radiation (k = 0.71073 Å) and corrected for absorption effects by
1
2
1
and
2
were collected
1
2 on a BRUKER CCD Smart diffractometer, using graphite-
α
SADABS [58]. The structures were solved by the Patterson method [59], and successive
Fourier synthesis gave the location of heavy atoms. All hydrogen atoms were included
in the model at geometrically calculated positions and refined on F2. Diffuse scatter-
ing reflections due to the disordered methanol solvent molecules in
1 were corrected by
SQUEEZE [39]. One of the PEt3 groups present in the structure of also presents disorder,
2
which was modeled considering two alternative positions (occupancy factors of 64 and 36%)
for the Et groups and using common anisotropic factors for both carbon atoms. Atomic
scattering factors were taken from International Tables for X-ray crystallography [60].
Molecular graphics were generated by ORTEPIII [61] and MERCURY [62]. Crystal
and structure refinement data are reported in Table 4.
Table 4. Crystal data and structure refinement for 1 and 2.
Compound
1
2
Empirical formula
Formula weight
Temperature (K)
Wavelength (Å)
Crystal system
Space group
a (Å)
C30H23AuN3P
653.45
C18H23AuN3P
509.33
100(2)
0.71073
Monoclinic
P21/n
293(2)
0.71073
Monoclinic
P21/c
9.4636(5)
18.3327(10)
32.0984(17)
90
98.2780(10)
90
14.6206(10)
13.0799(9)
20.0179(14)
90
b (Å)
c (Å)
◦
α ( )
◦
◦
β ( )
102.335(2)
◦
γ ( )
90
3739.8(4)
Volume (Å3)
5510.8(5)
Z
8
8
−3
−1
1.575
5.419
2544
1.809
7.956
1968
Density (calculated) (g cm
)
)
Absorption coefficient (mm
F(000)
Crystal size (mm3)
0.35 × 0.15 × 0.09
1.70 to 28.01
−12 ≤ h ≤ 12,
−17 ≤ k ≤ 24,
−39 ≤ l ≤ 42
30301
0.311 × 0.281 × 0.252
2.485 to 28.423
−19 ≤ h ≤ 19,
−17 ≤ k ≤ 17,
−25 ≤ l ≤ 26
89059
◦
Theta range for data collection ( )
Index ranges
Reflections collected
Independent reflections
Data/restraints/parameters
12296
12296/0/631
9338
9338/10/444
Goodness-of-fit on F2
0.805
1.140
−3
1.345 and −1.339
R1 = 0.0440, wR2 = 0.0875
R1 = 0.1218, wR2 = 0.0979
4.100 and −5.096
R1 = 0.0463, wR2 = 0.0881
R1 = 0.0640, wR2 = 0.0997
Largest diff. peak and hole (e Å
Final R indices (I>2sigma(I))
R indices (all data)
)
5. Conclusions
Two new gold(I) complexes containing benzimidazole and two different phosphines
have been prepared and characterized. The complex that incorporates triethylphosphine
ligand (
2) is more cytotoxic against neuroblastoma SH-SY5Y cells than the complex with
triphenylphosphine ligand (
1
). Our studies show that complexes and induce apoptosis
1
2
through both caspase-dependent and caspase-independent mechanisms. In the seek of
novel anticancer agents through different mechanisms than cisplatin or other Pt(II)/Pd(II)
compounds, the pro-apoptotic effects of complexes
1 and 2 in neuroblastoma cells make
them promising molecules for further anticancer studies.

Pharmaceuticals 2021, 14, 10
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Supplementary Materials: The following are available online at https://www.mdpi.com/1424-824
7/14/1/10/s1, Figure S1: ESI MS spectrum for
1; Figure S2: ESI MS spectrum for
2; Figure S3: UV-Vis
spectrum for
1
; Figure S4: UV-Vis spectrum for
2
; Figure S5: IR spectrum for ; Figure S6: IR spectrum
1
for
2
; Figure S7: 1H NMR spectrum for
1
(registered in CDCl3). Inset: details zoom to 7–9 ppm;
1
1
Figure S8: H NMR spectrum for
2
(registered in CDCl3); Figure S9: H NMR spectrum for Hpben.
Inset: details zoom to 7–9 ppm; Figure S10: 13C NMR spectrum for Hpben; Figure S11: Heteronuclear
Multiple Quantum Coherence (HMQC) correlation for Hpben; Figure S12: Heteronuclear Multiple
Bond Correlation (HMBC) for Hpben; Figure S13: 13C NMR spectrum for
1
(registered in CDCl3);
(registered in CDCl3); Figure S15: 13C NMR spectrum for
(registered in CDCl3); Figure S16: 31P NMR spectrum for
(registered in CDCl3); Figure S17:
MERCURY view for ; Figure S18: MERCURY view for ; Figure S19: Cytotoxicity curves for and
Checkcif files for the crystal structures of 1 and 2.
Figure S14: 31P NMR spectrum for
1
2
2
1
2
1
2;
Author Contributions: Conceptualization, Á.S.-G. and M.M.; methodology, L.R., Á.S.-G., R.A.,
and M.M.; validation, Á.S.-G., R.A., A.A., and M.M.; formal analysis, L.R., R.A., E.M.V.-L., E.G.-M.;
investigation, L.R., Á.S.-G., R.A., and M.M.; data curation, L.R., Á.S.-G., R.A., E.M.V.-L., and E.G.-M.;
writing—original draft preparation, L.R., R.A., and M.M.; writing—review and editing, L.R., Á.S.-G.,
R.A., A.A., E.M.V.-L., M.M.; supervision, Á.S.-G., R.A., A.A., and M.M.; project administration, M.M.
funding acquisition, A.A. and M.M. All authors have read and agreed to the published version of
the manuscript.
Funding: The research leading to these results has received funding from the following FEDER
cofunded-grants. From Consellería de Cultura, Educación e Ordenación Universitaria, Xunta de Gali-
cia, 2017 GRC GI-1682 (ED431C 2017/01), 2018 GRC GI-1584 (ED431C 2018/13), MetalBIO Network
(ED431D 2017/01). From CDTI (Centro para el Desarrollo Técnico Industrial) and Technological
Funds, supported by Ministerio de Economía, Industria y Competitividad, CTQ2015-65707-C2-
2-P, AGL2016-78728-R (AEI/FEDER, UE), ISCIII/PI16/01830 and RTC-2016-5507-2, ITC-20161072.
From European Union POCTEP 0161-Nanoeaters -1-E-1, Interreg AlertoxNet EAPA-317-2016, Interreg
Agritox EAPA-998-2018, and H2020 778069-EMERTOX.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data is contained within the article or supplementary material.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design
of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript,
or in the decision to publish the results.
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