Photoactivatable RuII Complex Bearing 2,9-Diphenyl-1,10-phenanthroline: Unusual Photochemistry and Significant Potency on Cisplatin-Resistant Cell Lines

Article abstract of DOI:10.1002/ejic.201800194

The current study investigates [Ru(bipy)₂(dpphen)]Cl₂ [where bipy = 2,2′-bipyridine and dpphen = 2,9-diphenyl-1,10-phenanthroline] (complex 1) for photoactivatable chemotherapy (PACT) application on five cancer cell lines. [Ru(bipy)₂(phen)]Cl₂ [where phen = 1,10-phenanthroline] (complex 2) was included as an unstrained control. Upon excitation with visible light, complex 2 proved to be photostable while complex 1 underwent a quantitative dissociation of the bipy ligand and formation of a Ru^II polypyridyl aqua complex in water. Complex 1 demonstrated only marginal activity in the dark; its cytotoxicity increased significantly upon photoactivation with a high phototoxicity index (PI = [IC₅₀ dark]/[IC₅₀ light]) ranging from 39.2-fold in A549 to over 100-fold in MDA-MB-231. Complex 2, on the other hand, did not show much difference in anticancer activity between dark and light conditions. Importantly, the IC₅₀ of the photoproduct of complex 1 was several folds lower than that of cisplatin in all tested cell lines. Furthermore, the dissociating ligand (bipy) was biologically inert in almost all cell lines investigated confirming that phototoxicity was mediated primarily by the Ru aqua complex that is released upon irradiation. In conclusion, the Ru-centered complex 1 could represent a potential photoactivatable chemotherapeutic drug that increases selectivity to tumors and offers alternative treatment in the light of increasing cisplatin resistance.

Full text of DOI:10.1002/ejic.201800194

A Journal of
Accepted Article
Title: Photoactivatable Ru(II) complex bearing 2,9-diphenyl-1,10-
phenanthroline: unusual photochemistry and significant potency
on cisplatin-resistant cell lines
Authors: Najwa Mansour, Stephanie Mehanna, Mohamad A. Mroueh,
Hassib Audi, Kikki Bodman-Smith, Costantine F. Daher,
Robin I. Taleb, Mirvat El-Sibai, and Rony S. Khnayzer
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content of this Accepted Article.
To be cited as: Eur. J. Inorg. Chem. 10.1002/ejic.201800194
Link to VoR: http://dx.doi.org/10.1002/ejic.201800194

10.1002/ejic.201800194
European Journal of Inorganic Chemistry
Photoactivatable Ru(II) complex bearing 2,9-diphenyl-1,10-phenanthroline: unusual
photochemistry and significant potency on cisplatin-resistant cell lines
Najwa Mansour,a,b Stephanie Mehanna,a,b Mohamad A. Mroueh,c Hassib Audi,a Kikki Bodman-
Smith,b Costantine F. Daher,a Robin I. Taleb,a Mirvat El-Sibai,a and Rony S. Khnayzera,*
a Department of Natural Sciences, Lebanese American University, Beirut 1102-2801, Lebanon.
b Faculty of Health and Medical Sciences, Department of Microbial and Cellular Sciences, University of Surrey.
c School of Pharmacy, Department of Pharmaceutical Sciences, Lebanese American University.
KEYWORDS cancer, photoactivatable chemotherapy, ruthenium polypyridyl complexes.
*Corresponding author: e-mail: rony.khnayzer@lau.edu.lb, URL: http://sas.lau.edu.lb/natural-
sciences/people/rony-khnayzer.php
Abstract
The current study investigates [Ru(bipy)2(dpphen)]Cl2 [where bipy= 2,2’-bipyridine and dpphen =
2,9-diphenyl-1,10-phenanthroline] (complex 1) for photoactivatable chemotherapy (PACT)
application on five cancer cell lines. [Ru(bipy)2(phen)]Cl2 [where phen = 1,10-phenanthroline]
(complex 2) was included as an unstrained control. Upon excitation with visible light, complex 2
proved to be photostable while complex 1 underwent a quantitative dissociation of the bipy ligand
and formation of a Ru(II) polypyridyl aqua complex in water. Complex 1 demonstrated only
marginal activity in the dark; its cytotoxicity increased significantly upon photoactivation with a
high phototoxicity index (PI = [IC50 dark] / [IC50 light]) ranging from 39.2-fold in A549 to over
100-fold in MDA-MB-231. Complex 2, on the other hand, did not show much difference in
anticancer activity between dark and light conditions. Importantly, the IC50 of the photoproduct of
complex 1 was several folds lower than that of cisplatin in all tested cell lines. Furthermore, the
dissociating ligand (bipy) was biologically inert in almost all cell lines investigated confirming
that phototoxicity was mediated primarily by the Ru aqua complex that is released upon irradiation.
In conclusion, the Ru-centered complex 1 could represent a potential photoactivatable
chemotherapeutic drug that increases selectivity to tumors and offers alternative treatment in the
light of increasing cisplatin resistance.
1
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10.1002/ejic.201800194
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Introduction
Platinum-based complexes have been extensively used as chemotherapeutic agents since the
discovery of cisplatin, which was proved to treat a variety of cancers.1 However, due to acquired
resistance and dose-limiting side effects, other metals have been investigated.2 Among these
metals, Ru has gained interest due to the tunable physical and chemical properties of the associated
complexes. Furthermore, variation in ligand framework allows Ru compounds to target specific
organelles within cancer cells.3 Moreover, the Ru(II) state was found to be the most active form in
the hypoxic environment of tumors thus conferring selectivity to cancer cells.4 The cellular uptake
of some Ru complexes by transferrin receptors which are overexpressed in cancer cells further
increases tumor selectivity and accordingly limits systemic toxicity.5 Once inside the cell, Ru
prodrugs can undergo light-mediated activation classified as photodynamic therapy (PDT) or
photoactivated chemotherapy (PACT). The latter uses light to change the chemical and physical
properties of the Ru complex by altering its electronic state thus generating a form of the molecule
that mediates cytotoxicity through DNA binding, ligand ejection or caging mechanisms. Unlike
PDT, PACT is not dependent on the presence of oxygen and thus offers a greater advantage in
targeting cancer cells which are hypoxic.6,7 Strained Ru complexes are particularly attractive as
light-activated agents because they absorb in the visible range and are kinetically inert and
photochemically labile. Although most Ru(II) polypyridyl complexes with pseudo-octahedral
geometry are rather stable upon irradiation, those having a distorted octahedral geometry undergo
a rapid deactivation of the excited state due to the decreased ligand-field splitting. This induces
ligand dissociation resulting from the population of the 3d-d state which is thermally accessed from
3
the MLCT excited state. There are many approaches for the use of Ru complexes in PACT
depending on the photoactivation strategy adopted. Ru(II) polypyridyl complexes with distorted
octahedral geometry can undergo ligand dissociation upon irradiation and possibly bind DNA in a
similar fashion to cisplatin.6,8 Using this approach, Glazer and coworkers prepared a series of
Ru(II) complexes bearing methyl substituents near the coordination sphere.8
[Ru(bipy)2(dmbipy)]2+ [where dmbipy = 6,6’-dimethyl-2,2’-bypiridyl] was tested on leukemia and
lung cancer cell lines. As expected, photoejection of the dimethyl substituted ligands upon
irradiation resulted in the formation [Ru(bipy)2(H2O)2]2+ and the free dmbipy photoproducts in
aqueous solution. When added to pUC19 plasmid, the light-activated complex induced DNA
photobinding as evidenced by delayed migration of the supercoiled DNA on the gel and decreased
2
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10.1002/ejic.201800194
European Journal of Inorganic Chemistry
EtBr intercalation. DNA damage occurred only upon photoactivation which was correlated with a
decreased cell viability in leukemia and lung cancer cells, as compared to dark conditions.8
Another methylated derivative, [Ru(bipy)2(dmdpq)]2+ [where dmdpq = 2,9’-dimethyl-
dipirido[3,2-f:2’,3’-h]-quinoxaline] formed the same aquated complex as above following the
photoejection of the straining ligand. However, the photoactivated complex exhibited a dual effect
on DNA, inducing both photobinding and photocleavage.8 Later, the same group used a similar
strategy to explore [Ru(bipy)2(dmdop)]2+ [where dmdop
=
2,3-dihydro-1,4-dioxino
[2,3-f]-2,9-dimethyl-1,10-phenanthroline] and [Ru(dmphen)2(dop)]2+ [where dmphen = 2,9-
dimethyl-1,10-phenanthroline].9 The latter exhibited photoinduced DNA damage via covalent
binding whereas the former induced both single-stranded breaks (SSBs) and covalent binding to
DNA. However, phototoxicity against leukemia cells did not linearly correlate with the degree of
DNA damage.9 Turro and coworkers investigated the photoinduced binding of cis-
[Ru(bipy)2(NH3)2]2+ to DNA to mimic the mode of action of cisplatin. The resulting complex, cis-
[Ru(bipy)2(H2O)(OH)]+, was shown to bind covalently to 9-methyl and 9-ethyl guanine as well as
to single-stranded and double-stranded DNA.10 In another study, Turro and coworkers evaluated
four [Ru(bipy)2(L)]2+ complexes where L corresponded to bisthioether (3,6-dithiaoctane or 1,2-
bis(phenylthio)ethane) or nitrogen bidentate (ethylenediamine or 1,2-dianilinoethane) ligands. The
bis-aqua complexes resulting from ligand exchange of bisthioether were shown to efficiently bind
guanosine 5-monophosphate as well as linear double-stranded DNA.11 Gasser and coworkers
investigated [Ru(II)(bipy)2dppz]2+ derivatives [where dppz = dipyrido[3,2‐a:2′,3′‐c] phenazine].
When irradiated, all complexes produced O2 as efficiently as photosensitizers available in the
market and were shown to intercalate DNA. Complexes with the highest nuclear accumulation and
intercalative potential induced efficient DNA photocleavage which reflected in higher
phototoxicity in HeLa cells. 12 Other ligand frameworks were also tested and showed potential use
for PACT with DNA being the main biological target. MacFarland and coworkers explored three
strained Ru(II) metal-organic dyads bearing the crowded 6,6’-dmb ligand (6,6′-dimethyl-2,2′-
bipyridine) and imidazo[4,5-f] [1,10]phenanthroline (IP) appended with n-thiophene units (nT; n
= 1 to 3). The three complexes were shown to have a dual mode of action; as the number of
thiophene units decreased, the dissociation rate of the 6,6’-dmb ligand increased which correlated
with the formation of cisplatin-like DNA adducts, whereas the complex with the highest n number
had the highest singlet oxygen yield which translated in more DNA single-stranded breaks. All
3
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10.1002/ejic.201800194
European Journal of Inorganic Chemistry
three complexes exhibited a significantly higher phototoxicity than cisplatin in tested cancer cell
lines.13 Bonnet and coworkers were the first to develop Ru(II) prodrugs with a trans configuration
that allows photoactivation with green light. [Ru(biqbipy)(dmso)Cl]Cl [where biqbipy = 6,6′-
bis[N-(isoquinolyl)1-amino]-2,2′-bipyridine)] and [Ru(biqbipy)(Amet)(HAmet)]PF6 [where
HAmet = N-acetyl-L-methionine, Amet = deprotonated N-acetyl-L-methionine] were shown to
eject the trans ligands and become bis-aquated. The latter interacted more strongly with DNA than
the prodrug, however, their confirmed apoptotic activity could not be conclusively attributed to
this interaction.14 Glazer’s group investigated Ru(II) polypyridyl complexes with one or two
bisquinoline ligands. Upon photoactivation with high wavelengths (700 or 800 nm), the resulting
bis-aqua complexes were found to bind DNA and proved to be cytotoxic against leukemia cells.15
While most studies focused on the role of Ru(II) aqua complexesin inducing cytotoxicity, we have
investigated the possible contribution of the dissociating ligand to the biological activity of
[Ru(bipy)2(dmphen)]Cl2 [where dmphen = 2,9-dimethyl-1,10-phenanthroline] on ML-2 Acute
Myeloid Leukemia cell line. Results of cell viability assays showed that the toxicity of dmphen
was significantly higher than the aqua complex formed and thus proved that the Ru(II) center can
act as a cage for the cytotoxic ligand. These findings uncovered the important role of the
coordinating ligands in the biological activity of strained Ru(II) polypyridyl complexes.16
Similarly, another report by Bonnet and coworkers illustrated that the photoreleased 6,6’-dimethyl-
2,2’-bipyridine (dmbipy) ligand is responsible for the phototoxicity of [Ru(bipy)2(dmbipy)]2+,
whereas the Ru bis-aqua photoproduct was found to be biologically inert.17 Turro and coworkers
previously reported similar results on the pseudo-octahedral complex [Ru(tpy)(5CNU)3]2+ (3)
[where tpy = 2,2′:6′,2″-terpyridine and 5CNU = 5-cyanouracil].18 Upon excitation with visible
light, the ligand 5CNU was ejected from the caging Ru(II) complex and the photoproduct exerted
a dual cytotoxic activity; 5CNU induced cell death through the inhibition of pyrimidine catabolism
whereas the Ru(II) center was capable of binding DNA18. In the present study, two Ru(II)
polypyridyl complexes were investigated: [Ru(bipy)2(dpphen)]Cl2 (complex 1) which is sterically
strained by the phenyl groups on the 2,9-positions of dpphen, along with the unstrained control
[Ru(bipy)2(phen)]Cl2 (complex 2), Figure 1.
4
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European Journal of Inorganic Chemistry
1
2
Figure 1. Structures of complexes 1 and 2 where bipy = 2,2’-bipyridine.
The photochemistry of [Ru(bipy)2(dpphen)](PF6)2 was previously studied by Sauvage and
coworkers who reported the quantitative ejection of bipy in acetonitrile instead of the hindering
dpphen ligand thus generating the photoproduct [Ru(bipy)(dpphen)(CH3CN)2]2+.19 Unlike
phenanthroline derevatives, bipy was shown to be inactive in cancer cells since it only forms
electrostatic interactions with DNA and is unable to intercalate.20 In the present work, the
photochemistry of [Ru(bipy)2(dpphen)](PF6)2 was also re-evaluated in acetonitrile for comparison
with reported results and the chloride salt of complex 1 was explored as a potential
photochemotherapeutic candidate. The photochemistry of the chloride complex was examined in
water to determine if bipy photorelease would occur and the phototoxicity of the new product was
evaluated on a panel of cancer cell lines to probe the PACT of complex 1.
Results
In all photochemical and photobiological experiments, the light output from a home-built blue
LED setup 21 (460 nm, ~100 mW/cm2) was used to provide the excitation source. This light source
provides a direct metal-to-ligand charge transfer (MLCT) excitation. The extinction coefficient of
the MLCT transition for complex 1 was 14,000 ± 1,000 M-1cm-1 at 448 nm in both acetonitrile
([Ru(bipy)2(dpphen)](PF6)2) and water ([Ru(bipy)2(dpphen)]Cl2), Figure 2. The peak at 285 nm
can be assigned to intra-ligand ππ* transition.
5
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10.1002/ejic.201800194
European Journal of Inorganic Chemistry
8.0x104
6.0x104
4.0x104
[Ru(bpy)2(dpphen)](PF6)2 in ACN
[Ru(bpy)2(dpphen)]Cl2 in H2O
2.0x104
0.0
200
300
400
500
600
700
800
Wavelength (nm)
Figure 2. UV-vis spectra of complex 1 as hexafluorophosphate and chloride salts in acetonitrile
and water, respectively.
To determine the chemical composition of the photochemical products, 1H NMR was performed
on [Ru(bipy)2(dpphen)](PF6)2 (before and after photoactivation) along with the free ligands
dpphen and bipy for comparison. As shown in Figure 3, the 1H NMR spectrum in CD3CN of the
sample after irradiation showed a quantitative conversion of the [Ru(bipy)2(dpphen)](PF6)2 into
photoproducts. The resulting solution contained peaks assigned to the free bipy (labeled with
circles) while dpphen could not be detected among the photolysis products. A broadening of the
proton signals assigned to the hydrogen atoms at the ortho and meta positions of the phenyl groups
of [Ru(bipy)2(dpphen)](PF6)2 was observed before photoactivation and disappeared in the
photoproducts.
6
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10.1002/ejic.201800194
European Journal of Inorganic Chemistry
1
Figure 3. H NMR spectra (500 MHz) in CD3CN of complex 1 [Ru(bipy)2(dpphen)](PF6)2 before
photolysis (black), after 6 h of photolysis (green) as well as the free ligands 2,9-diphenyl-1,10-
phenanthroline (dpphen) (purple) and 2,2’-bipyridine (bipy) (light blue). The full spectra are found
in Figure S9. Peaks marked with a circle are for the bipy free ligand, the ones marked with stars
are assigned to the ortho and meta hydrogens of the phenyl groups in complex 1. The remaining
assigned peaks were done based on the expected chemical shift of the bipy (red) and dpphen
(violet) ligands. A or D peaks and C or B peaks could not be distinguished in complex 1.
The photochemistry of [Ru(bipy)2(dpphen)]Cl2 in water was confirmed by LC/MS. While only the
peak assigned to [Ru(bipy)2(dpphen)]2+ appeared at m/z = 373.20 [M-2Cl]2+ (Figure 4 left) before
irradiation, bipy was identified at m/z = 157.13 [M+H]+ after irradiation along with the aquated
form of the photoproduct, [Ru(bipy)(dpphen)(H2O)]2+ assigned at m/z = 303.60 [M-2Cl]2+, Figure
4. Dpphen, which produced a signal at m/z = 333.33 [M+H]+ (Figure S10) was absent in the
photoproduct in water. LC/MS spectra of complex 2 before and after irradiation presented a peak
7
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10.1002/ejic.201800194
European Journal of Inorganic Chemistry
at m/z = 297.07 [M-2Cl]2+ (Figure S11), and therefore demonstrate that complex 2 did not undergo
any detectable ligand dissociation upon blue light irradiation.
Before irradiation
After irradiation
157.13
100
95
90
85
80
75
70
65
60
55
50
45
40
35
30
25
20
15
10
5
373.20
100
95
90
85
80
75
70
65
60
55
50
45
40
35
30
25
20
15
10
5
303.60
606.13
310.60
319.40
224.20
411.07
294.60
224.20
240.13
621.20
621.13
214.07
113.13
137.20
387.13
400
467.13
525.07 585.80
500 600
677.13
700
278.93
300
419.67
7
171.07
200
355.00
484.00 516.87
500
588.27
631.20 697.13
0
0
100
100
200
300
400
600
700
m/z
m/z
Figure 4. ESI-MS spectra of [Ru(bipy)2(dpphen)]Cl2 (15 µM) in water before (left) and after
(right) photolysis with blue light for 30 min at an output of 100 mW/cm2 . The peak at m/z = 606.1
depicts the singly charged [Ru(bipy)(dpphen)(OH)]+ and at 303.6 for [Ru(bipy)(dpphen)(H2O)]2+.
The UV-vis spectra of [Ru(bipy)2(dpphen)]Cl2 (45 µM) were measured as a function of irradiation
time (460 nm, 100 mW/cm2), Figure S12. The half-life (t1/2) in water was found to be 4.3 min
whereas it was 2.2 min in acetonitrile. As expected, t1/2 in water was larger than in acetonitrile
since the latter provides enhanced solvation of the photoproducts.16 However, it is important to
note that the rate of photoejection is dependent on the concentration and volume of the solution as
well as the incident power density. In the NMR experiment, the concentration was >100-folds
larger than the ESI-MS, hence the time required to complete photolysis was 6 h in Figure 3 and 30
min in Figure 4 even though photolysis under identical concentration is faster in acetonitrile than
water.
The quantification of cellular uptake of complex 1 was performed using ICP-MS. The optimum
uptake time was used to set the incubation period prior to irradiation in order to maximize cellular
concentration of prodrug. While at t = 0 h no Ru could be detected (C < 2 ng/106 cells), the uptake
started within the first hour (2.73 ng/106 cells) then increased until it reached a maximum between
8
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10.1002/ejic.201800194
European Journal of Inorganic Chemistry
6 and 24 h of approximately 11 ng/106 cells, Figure 5. WST-1 kit was used to assess the anticancer
activity of complexes 1 and 2 and controls on five cell lines: murine melanoma B16-F10, human
alveolar adenocarcinoma A549, triple negative human breast adenocarcinoma MDA-MB-231,
human colorectal adenocarcinoma HT29 and human colon adenocarcinoma Caco-2. The cell
survival assay of complex 1 was performed to assess its biological activity, before and after
photoactivation. Cisplatin was used as a positive control and complex 2 as an unstrained control.
While complex 2 showed no significant activity in both dark and light conditions (IC50 > 100 µM)
(Figure 6 and Table 1), complex 1 exhibited different cytotoxicity profile. In the dark, its IC50
values ranged from 55.1 µM in B16-F10 to higher than 100 µM in the other cells lines. But, upon
irradiation, the cytoxicity of complex 1 increased dramatically with IC50 values ranging from 0.82
µM in B16-F10 cells to a maximum of 2.55 µM in A549 cells. Calculation of the phototoxicity
index (PI = [IC50 dark] / [IC50 light]) showed PI values ranging from 39.2-fold in A549 to greater
than 100-fold in MDA-MB-231 (Table 1). According to Table 1, the IC50 values of cisplatin ranged
between 7.70 µM in B16-F10 cells and greater than 10 µM in HT-29 cells. Also, the dissociating
ligand bipy was found to be biologically inert on all cell lines (IC50 over 100 µM) except on MDA-
MB-231 where an IC50 of 37.5 µM was recorded (Figure 6 and Table 1).
12
10
8
6
4
2
0
24
3
6
0
1
Time (h)
Figure 5. ICP-MS analysis using the EPA 200-7/8 M method for compound 1 uptake by A549
cells at different time points. Results are expressed in ng/106 cells.
9
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Figure 6. Cytotoxic effect of complexes 1, 2, cisplatin and bipy on A549, B16-F10, Caco-2, HT29
and MDA-MB-231. Cells were treated with 3-fold dilutions of all agents starting at 120 µM.
Complexes 1 and 2 were irradiated with blue LED at a power of 100 mW/cm2. The effect of bipy
and cisplatin was only evaluated in the dark. IC50 values are reported in Table 1.
10
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10.1002/ejic.201800194
European Journal of Inorganic Chemistry
Table 1. IC50 values in dark/light and phototoxicity indices (PI) of complexes 1 and 2 on various
cell lines. Data on the free bipy ligand and cisplatin are reported only in the dark. Numbers are
averages of three separate experiments and each experiment included samples in triplicates.
Numbers are expressed as mean ± SEM.
IC50
Cell Line
A549
Compound 1
Compound 2
bipy free ligand
Cisplatin
Dark: >100a
Light: 2.55b ± 1.74
PI: >39.2
Dark: >100a
Light: >100a
PI: ---
>100a
7.74b ± 1.00
Dark: 55.1a ± 5.84
Light: 0.82b ± 0.83
PI: >68.8
Dark: >100c
Light: >100c
PI: ---
>100c
>100a
7.70d ± 0.19
21.8c ± 0.53
B16
Dark: >100a
Light: 1.48b ± 0.22
PI: >67.6
Dark: >100a
Light: >100a
PI: ---
Caco-2
Dark: >100a
Light: 1.98b ± 2.35
PI: >50.5
Dark: >100a
Light: >100a
PI: ---
>100a
>100a
HT-29
Dark: >100a
Light: 1.00b ± 0.78
PI: >100
Dark: >100a
Light: >100a
PI: ---
37.5c± 0.14
30.9c ± 0.82
MDA-MB-231
a, b, c, or d In each row, values that are not sharing the same letter are significantly different (p < 0.05).
Discussion
The use of prodrugs in anticancer therapy has recently become a very attractive approach mainly
because of its potential selectivity to tumors which could reduce systemic toxicity and acquired
resistance induced by conventional chemotherapy.6 Ru(II) polypyridyl complexes having a
distorted octahedral geometry have gained interest as prodrugs due to their chemical stability in
the dark and their ability to undergo ligand dissociation upon visible light excitation.8 In the present
study, we have exploited these features to synthesize the complex [Ru(bipy)2(dpphen)]Cl2
(complex 1) and explore its cytotoxic potential in vitro upon photoactivation. The complex is
sterically strained by virtue of introducing a phenyl group on the 2,9-positions of the
phenanthroline group. It is intuitive to think that the sterically hindering ligand (dpphen) is
expected to dissociate from the complex upon photoactivation, however results showed that the
ligand bipy was ejected when the complex was irradiated in acetonitrile thus generating the
photoproduct [Ru(bipy)(dpphen)(CH3CN)2]2+. Such behavior could be related to the distorted
11
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European Journal of Inorganic Chemistry
geometry of the complex, an effect that might be attributed to the twisted bipy moieties or
prolonged Ru-N(bipy) distances. Therefore, the ejection of bipy would be easier since the rotation
of the pyridine ring about the C2-C2’ bond becomes possible after the first Ru-N bond is broken.19
This is not the case with the dpphen whose plane is resistant to distortion, thus rendering the ligand
less labile. This unexpected ligand substitution is perhaps due to the asymmetrical distortion of the
1
complex.19 To confirm these results, we have conducted H NMR and ESI-MS studies on the
photochemistry of the complex in deuterated acetonitrile and water respectively. The MLCT
absorption of complex 1 was measured by UV/vis spectrophotometry and recorded at around 448
nm (blue range). This wavelength was the basis for choosing blue LED (460 nm) to induce
photolysis for characterization and biological studies. When performed on the irradiated complex,
1H NMR suggested that only bipy was photolabilized in CD3CN which is consistent with
Sauvage’s results.19 At room temperature, broadening of the proton signals was observed for
hydrogen atoms assigned to the ortho and meta positions of the phenyl groups of
[Ru(bipy)2(dpphen)](PF6)2 before photoactivation. This 1H NMR broadening22 could be attributed
to the intramolecular π-π stacking23 of the phenyl groups at 2,9 positions of the 1,10-
phenanthroline and the pyridine ring of the ancilliary ligands. In this perspective, the lack of
dissociation of the dpphen ligand could be due to two main factor: (1) the rigidity of the 1,10-
phenanthroline ligand as compared to 2,2’-bipyridine which possesses a free rotation around C2-
C2’ (2) possible intramolecular π-π stacking of the phenyl groups which likely increases the
activation energy for dissociation of this ligand. LC-MS analysis confirmed that
[Ru(bipy)2(dpphen)]Cl2 has a similar photochemical behavior in water with a quantitative release
of bipy upon irradiation. Similarly to cisplatin, complex 1 is expected to undergo aquation and this
was also verified by LC-MS which identified another photolysis product
[Ru(bipy)(dpphen)(H2O)]2+. However, the electrospray ionization used by the LC-MS may affect
the aquation process as exemplified by the detection of ligand deficient [Ru(bipy)2]2+,
[Ru(bipy)2(H2O)2]2+, and [Ru(bipy)2(H2O)(OH)]+ for similar photoproducts in water.8,10,17
Therefore, the results obtained above do not necessarily translate the actual number of aqua ligands
bound to the Ru center in solution. Moreover, the complex was highly stable in the solid state as
1
well as in water solution under dark conditions, as verified by unchanged H NMR and LC-MS
spectra respectively after months of storage. This constitutes a practical convenience since when
protected from light, the complex can be stored for a long period with no signs of degradation.
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Unlike complex 1, complex 2 did not undergo photolysis and was found to be photostable as
1
confirmed by H NMR and LC-MS data (Figure S11) which were identical before and after
irradiation of the complex. Complex 2, with distorted octahedral geometry, therefore exhibits
completely different photochemical properties as compared to complex 1.
The main purpose of photochemotherapy is to mediate phototoxicity in cancer cells while
minimizing damage to healthy tissues that are not irradiated.24 To be considered for
photochemotherapeutic applications, complex 1 should be primarily active upon photolysis and
thus exhibit a large difference in cytotoxicity between dark and light conditions, as measured by
the phototoxicity index (PI). Cellular uptake of complex 1 was maximized to about 11 ng/106 cells
after 6-24 h of incubation, which compares well with other Ru anticancer drug uptake.25 PI values
measured by cell survival assays were consistently high among all cell lines tested (they ranged
from >39-fold in A549 to >100-fold in MDA-MB-231). This was the result of high difference in
toxicity between dark and light conditions; while complex 1 showed little effect in the dark (with
a minimum IC50 value of 55 µM in murine melanoma cells, B16-F10), cytotoxicity increased
considerably upon irradiation (IC50 between 0.83 µM in B16-F10 cells to 2.6 µM in A549 cells).
As compared to cisplatin, the photoactivated form of complex 1 induced higher toxicity to all cell
lines tested and the difference was mostly noticeable in human colorectal adenocarcinoma cells
(HT-29) which were found to be resistant to cisplatin (IC50 > 100 µM). This finding is consistent
with previous studies that confirmed the refractory response of colon cancer cells to cisplatin due
to their ability to induce DNA repair. 26 The free bipy ligand was included in cell survival assays
to test whether the dissociating ligands contribute to phototoxicity of complex 1. Results showed
that the bipy ligand was not potent (IC50 >100 µM) in all cell lines tested except in MDA-MB-231
where IC50 was still high (37.5 µM). These results were comparable to the data obtained by our
group on ML-2 Acute Myeloid Leukemia cell line16 and in line with previous results that showed
moderate to no potency of bipy on K562, MDA-MB-231 and MCF-7 cells.27,28 This favors the
hypothesis that phototoxicity of complex 1 is predominantly mediated by the Ru-aqua
photoproduct rather than the liberated ligand. According to Lincoln and Nordén, dark toxicity can
be reduced by altering the structure of the Ru complex in a way to avoid DNA binding or
intercalation. This can be achieved by replacing phenanthroline ligands by bipyridine.29 Our results
suggest that complex 1 and complex 2 had reduced DNA interaction in vitro which significantly
reduced their dark toxicity. However, upon irradiation, bipy was photoejected from complex 1
13
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European Journal of Inorganic Chemistry
leading to product [Ru(bipy)(dpphen)(H2O)]2+ as a cytotoxic agent. Complex 2, on the other side
proved to be inactive in both dark and light conditions on all cell lines tested (IC50 > 100 µM)
which is in accordance with the results found by Glazer and coworkers.8 pUC8 plasmid gel
electrophoresis experiments30 using Ru(II) polypyridine complexes have previously demonstrated
potential DNA single and double-stranded breaks11. However, these findings do not necessary
correlate with the mechanism of compound 1 in living cells. A thorough study is currently
underway to elucidate the in vitro mechanistic details of this compound.
Conclusions
Ru(II) polypyridyl complexes with distorted octahedral geometry are potential candidates for
photochemotherapy due to their tunable photochemical properties. [Ru(bipy)2(dpphen)]Cl2
(complex 1) was able to mediate cytotoxicity selectively upon photoejection of the inert bipy
ligand and release of a highly toxic Ru(II) aqua complex. [Ru(bipy)2(phen)]Cl2 (complex 2) control
was shown to be photostable and biologically inert in the dark and upon photoexcitation. Complex
1 exhibited superior cytotoxicity and selectivity upon photoexcitation when compared with
cisplatin, and may be considered for potential application in PACT. Further in vitro and in vivo
investigations are underway to elucidate the biological mechanism of action.
Experimental Section
Instrumentation
Elemental analysis was provided by Atlantic Microlab and HR-ESI MS data were acquired at
Michigan State University Mass Spectrometry Core Facility. MALDI-TOF MS spectra were
collected on a 4800 MALDI TOF/TOF MS/MS Analyzer instrument (Applied Biosystems)
operated by a 4000 Series Explorer software. Briefly, aqueous solutions of Ru(II) complexes (120
µM) were premixed with CHCA matrix (saturated α-cyano-4-hydroxy-cinnamic acid in a mixture
containing 1:1 of 50% acetonitrile and 0.1% TFA. The resulting samples were then spotted on a
stainless-steel plate (Opti-TOF TM 384 Well Insert), dried by vacuum and inserted for data
acquisition. 13 calibration points were included on each plate (4700 Proteomics Analyzer
Calibration mixture). UV/vis absorption was measured by a Cary 60 spectrophotometer from
Agilent Technologies). NMR spectra were acquired on an AC500 Bruker spectrometer (1H NMR
at 500 MHz). Chemical shifts were recorded in delta (δ) units and expressed as ppm values
14
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10.1002/ejic.201800194
European Journal of Inorganic Chemistry
relatively to the internal standard TMS. LC-MS/MS data were recorded by Ultimate 3000
RSLC/TSQ Endura system (ThermoScientific). Briefly, aqueous solutions of complexes 1 and 2
(20 µM) were prepared and infused by direct injection into the heated ESI probe [(2 µL injection
at a flow rate of 100 µL/min of LC-MS grade water containing 0.05% formic acid, sheath gas (2.71
L/min), auxilliary gas (5.78 L/min), sweep gas (1.5 L/min), ion transfer tube temperature (325 °C),
vaporizer temperature (200 °C), positive ion spray voltage (3700 V)]. ICP-MS was conducted at
the American University of Beirut (environment core lab) on Agilent 7500 ICP-MS at 0.3 seconds
(integration time/mass) with 3 repetitions and an uptake speed of 1 rotation per second. Blue LED
was obtained from LED Engin (LZ4-40B208-000) and operated at 50% of its full power (130
lumen). The lamp was regulated by an LED dimmer (home-built) using a Philips Xitanium LED
driver LEDINTA0700C210FO and light was condensed using a focusing lens with narrow beam
output (LLNS-1T06-H). Star LED heat sink was used to cool the system.
Materials
Cis-bis(2,2′-bipyridine)dichlororuthenium(II) hydrate (Ru(bipy)2Cl2·2H2O), 1,10-phenanthroline,
silica gel, sephadex LH 20, Dowex 22 chloride, and all other chemicals and solvents were
purchased from Aldrich and used without any further purification. LC-MS grade water and formic
acid were from Fisher Chemical. DMEM medium, FBS and penicillin G sodium salt were
purchased from Aldrich and WST-1 reagent (tetrazolium salt) was obtained from Roche©. All cell
lines used were from the American Type Culture Collection (ATCC).
Synthesis of [Ru(bipy)2(dpphen)](PF6)2 (Complex 1)
2,9-diphenyl-1,10-phenanthroline was synthesized according to a published procedure.31
Synthesis of the complex was performed according to a modified procedure.19 Ru(bipy)2Cl2·2H2O
(100 mg, 0.2 mmol) and 2,9-dpphen (69 mg, 0.19 mmol) were mixed with ethylene glycol (8 mL)
in a pressure vessel. The solution was degassed for 1 h under argon pressure then refluxed for six
h. After cooling at room temperature, filtration was carried out using micropores (0.45 µm). The
product was precipitated by adding a saturated solution of KPF6.2 The precipitate was filtered then
purified by column chromatography (silica gel, 92% acetonitrile, 7% H2O, 1% KNO3). Yield:
153.5 mg, 72%. 1H NMR (CD3CN, 500 MHz): δ = 8.66 (d, J = 8 Hz, 2H), 8.34 (s, 1H), 8.24 (d, J
= 8 Hz, 2H), 8.06 (td, J = 7.73, 1.47 Hz, 2H), 7.89 (d, J = 6.22 Hz, 2H), 7.84 (d, J = 7.98 Hz, 2H),
15
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10.1002/ejic.201800194
European Journal of Inorganic Chemistry
7.54 (d, J = 8.29 Hz, 2H), 7.45 (td, J = 7.71 Hz, 1.46 Hz, 2H), 7.35 (dq, J = 6.02 Hz, 1.3 Hz, 2H),
7.14 (br. s, 2H), 7.02 (t, J = 7.56 Hz, 2 H), 6.87 (br. s, 2H), 6.74 (br. s, 2H), 6.66 (d, J = 4.93 Hz,
2 H), 6.56 (td, J = 6.09, 1.3 Hz, 2H), 6.13 (br. s, 2 H). 13C NMR (CD3CN, 500 MHz): δ = 168.91,
158.99, 157.05, 154.02, 151.96, 149.55, 139.96, 138.65, 138.16, 137.06, 131.48, 129.96, 129.63,
129.02, 127.60, 127.57, 125.33, 124.14. Elemental Anal. Calcd for C44H32F12N6P2Ru.H2O: C,
50.15; H, 3.25; N, 7.98. Found: C, 50.32; H, 3.17; N, 7.98. HRMS (ESI/QTOF) m/z [M]2+ Calcd
for C44H30N6Ru 373.0872; found 373.0903. MS (MALDI/TOF) m/z, [M]+ 746.15. UV/Vis (ACN):
λmax(ɛ M-1cm-1) 448 nm (14,000). For biological testing the hexafluorophosphate salt was
converted to chloride salt using Dowex chloride ion exchange resins to promote the solubility of
the complex in water.
Synthesis of [Ru(bipy)2(phen)]Cl2 (Complex 2)
Synthesis was performed according to a modified procedure.32 Ru(bipy)2Cl2·2H2O (100 mg, 0.20
mmol) and 1,10-phenanthroline (39.64 mg, 0.22 mmol) were added to a solution of 1:1
EthOH:H2O (8 mL) in a round bottom flask. The mixture was stirred under argon for 1 h then
refluxed for three h. After cooling at room temperature, solvents were evaporated, and the residue
was dissolved in methanol (2 mL) and added dropwise to rapidly stirred diethyl ether (300 mL)
producing an orange precipitate that was collected by vacuum filtration and washed thoroughly
with ether. The compound was then purified on a sephadex LH-20 column with methanol as eluent
and finally dried under reduced pressure. Yield: 121.4 mg; 91 %. 1H NMR (CD3CN, 500 MHz): δ
= 8.63 (dd, J = 8.27 Hz, 1.26 Hz, 2H), 8.60 (d, J = 7.99 Hz, 2H), 8.55 (d, J = 8.11 Hz, 2H), 8.26
(s, 2H), 8.10 (m, 4H), 7.99 (td, J = 7.63 Hz, 1.49 Hz, 2H), 7.85 (d, J = 6.4 Hz, 2H), 7.74 (dd, J =
8.26 Hz, 5.25 Hz, 2H), 7.53 (d, J = 5.64 Hz, 2H), 7.45 (dq, J = 6.31 Hz, 1.33 Hz, 2H), 7.22 (dq, J
= 6.31 Hz, 1.33 Hz, 2H). 13C NMR (CD3CN, 500 MHz): δ = 158.26, 157.99, 153.45, 152.96, 152.81,
148.49, 138.79, 138.66, 137.80, 132.00, 129.05, 128.47, 128.35, 127.03, 125.30, 125.23. Elemental Anal.
Calcd for C32H24Cl2N6Ru.3H2O: C, 53.49; H, 4.21; N, 11.7. Found: C, 53.25; H, 4.53; N, 11.42. HRMS
(ESI/QTOF) m/z [M]2+ Calcd for C32H22N6Ru 297.0557; found 297.0568. MS (MALDI/TOF) m/z, [M]+
594.07. UV/Vis (H2O): λmax(ɛ M-1cm-1) 448 nm (16,000).20
1H NMR and LC-MS/MS of complexes 1 and 2 to evaluate their photochemistry
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European Journal of Inorganic Chemistry
The photochemistry of complex 1 (PF6 salt) was determined by 1H NMR in deuterated acetonitrile
(CD3CN), after which the complex was converted to a chloride salt and subjected an LCMS
analysis in order to determine whether the complex exhibits the same photochemical
transformation in water. Complex 2 was evaluated by LC-MS and all measurements were
1
performed before and after irradiation. For H NMR studies, samples were irradiated for 6 h as
opposed to 35 min for LC-MS (at an output of 100 mW/cm2) and the ligands dpphen and bipy
were included as controls to allow their detection in photolysis products.
Determination of the rate of ligand dissociation of complex 1 by UV-vis spectroscopy
The chloride form of complex 1, [Ru(bipy)2(dpphen)]Cl2 was irradiated with blue LED light (460
nm, 100 mW/cm2) in water and acetonitrile for different durations. UV-vis absorption spectra were
then acquired to determine the half-life of the complex under identical conditions.
Quantification of cellular uptake by ICP-MS
Quantification of cellular uptake was carried out by ICP-MS following a modified procedure.33
Briefly, 1.0 x 106 cells (A549) were plated in 60 mm petri dishes and allowed to adhere for 12 h
at 37° with 5% CO2. Complexes 1 (30 µM) were then added and the plates were incubated for 1,
3, 6, 12 and 24 h. Cells were then harvested by scraping, precipitated by centrifugation at 800 RCF
for 20 minutes at 4° and washed 3 times with PBS. The pellets were resuspended in milli-Q water,
transferred to a glass vial containing 65% HNO3 and completely digested at 120 °. The product
obtained was then dissolved in 1 mL milli-Q water containing 2% HNO3. Quantification of Ru
was performed using Agilent 7500 inductively coupled plasma mass spectrometry (ICP-MS): EPA
200-7/8 M method and data were analyzed using ICP-MS Mass Hunter B.01.01 software.
Cell survival assay
Murine melanoma cell line B16-F10, human alveolar adenocarcinoma cell line A549, triple
negative human breast adenocarcinoma cell line MDA-MB-231, human colorectal
adenocarcinoma cell line HT29 and human colon adenocarcinoma Caco-2 cell line were
maintained in DMEM containing 10% FBS and 1.5% pen-strep at 37° with 5% CO2. Cells were
seeded in a 96 well-plate at a concentration of 104 cells/well and allowed to adhere for 6 h at 37°
with 5% CO2. Treatment with serial dilutions of complexes 1 and 2, cisplatin and the ligands
17
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10.1002/ejic.201800194
European Journal of Inorganic Chemistry
dpphen and bipy was performed, followed by a 12 h incubation period. Cells were then irradiated
with blue LED purchased from LED Engin possessing 460 nm peak wavelength and operated at
50% of its 130 lumen full power which was controlled by a home-built LED dimmer (Light output
~ 100 mW/cm2). The light was focused on the plate using a focusing lens with narrow beam output.
Irradiation was typically done for 35 minutes for “light conditions” or else the plates were left in
the dark for all other experiments. Cell proliferation was assessed after 72 h using WST-1 reagent
(Roche©) according to the manufacturer’s instructions and data were analyzed using GraphPad
Prism 6 software (GraphPad Software). Cell viability was expressed as percent survival relatively
to the negative control (non-treated cells). IC50 was calculated from 3 separate experiments using
a non-linear regression (curve fit) on GraphPad Prism 6 software (GraphPad Software) and
expressed as mean ± SEM, calculated using Microsoft Excel (version 1705, 2016). Two-way
analysis of variance (ANOVA) was used to determine statistical significance (GraphPad) and
differences between all groups were considered significant if p < 0.05 (Computed by Tukey's
multiple comparisons test).
Acknowledgements
We would like to acknowledge financial support from the School Research and Development
Council at the Lebanese American University and the Lebanese National Council for Scientific
Research (Ref: 05-06-14).
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In the present study, [Ru(bipy)2(dpphen)]Cl2 [where bipy= 2,2’-bipyridine and dpphen = 2,9-
diphenyl-1,10-phenanthroline] (complex 1) was shown to eject the bipy ligand upon light
activation resulting in the formation of an aqua complex in water. The complex exhibited a
significantly higher toxicity upon irradiation (up to over 100-fold increase) on five cancer cell lines
including cisplatin-resistant ones. The free bipy ligand displayed minimal potency and therefore
phototoxicity was attributed to the formation of the aqua complex.
21
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