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Towards Long Wavelength Absorbing Photodynamic Therapy Photosensitizers via the Extension of a [Ru(bipy)3]2+ Core
A Journal of
Accepted Article
Title: Towards Long Wavelength Absorbing Photodynamic Therapy
Photosensitizers via the Extension of a [Ru(bipy)3]2+ Core
Authors: Johannes Karges, Olivier Blacque, Philippe Goldner, Hui
Chao, and Gilles Gasser
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To be cited as: Eur. J. Inorg. Chem. 10.1002/ejic.201900569
Link to VoR: http://dx.doi.org/10.1002/ejic.201900569
10.1002/ejic.201900569
European Journal of Inorganic Chemistry
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Towards Long Wavelength Absorbing Photodynamic Therapy
Photosensitizers via the Extension of a [Ru(bipy)3]2+ Core
Abstract: Complementary to classical treatment methods used
as a complementary medical technique to these blockbusters.
against cancer, photodynamic therapy (PDT) has received increased
PDT is based on the combination of light, a photoactive
attention over the last years. PDT relies on the generation of reactive
compound called a Photosensitizer (PS) and oxygen. Ideally, the
oxygen species (ROS) upon light irradiation to trigger cell death. As
PS should be nontoxic in the absence of light and generate highly
the wavelength employed during such treatments directly influences
toxic species upon light irradiation. The mechanism of action of
the light penetration depth and therefore the possibility to treat deep
seated tumours or large tumours, research efforts have been made
towards the development of photosensitizers (PS) with an absorption
in the phototherapeutic window (600-900 nm). To tackle this drawback,
we report herein the preparation and characterisation of new Ru(II)containing PDT PSs, that are based on a [Ru(bipy)3]2+ core (1; bipy:
2,2'-bipyridine) and that are extended with methyl groups (2) or vinyl
PDT is based on the generation of reactive oxygen species (ROS).
More specifically, upon light irradiation, the PS is excited to a
singlet state, which can be transformed into an excited triplet state
by an intersystem crossing (ISC) process. From there, the PS is
able to influence its biological environment by two pathways,
namely Type I and Type II. During a Type I reaction, an electron
dimethylamino groups (3). As anticipated with our design, we found a
or proton is transferred to/from the PS from/to its biological
red-shift of 65 nm of the maximum absorption of complex 3 in
surrounding. This leads to the generation of radicals and ROS like
comparison to complex 1. In addition, we report on the in-depth
superoxides or hydroxyl radicals. In a Type II reaction, the energy
photophysical properties as well as (photo-)cytotoxicity against
of the exited triplet state of the PS is transferred to molecular
cervical cancerous HeLa cells of the investigated compounds.
oxygen (3O2) to produce singlet oxygen (1O2). This highly
energetic form of oxygen is highly reactive. Consequently, during
both pathways, ROS or 1O2 react with its biological surrounding,
Introduction
generating cellular damages and therefore ultimately trigger cell
Over the last decades, cancer has emerged to be one of the
death.[2]
deadliest diseases worldwide.[1] Next to the classical treatments
The most commonly used PS for PDT treatments is Photofrin
(e.g., chemotherapy, surgery and radiotherapy), the use of
(Figure 1), which is approved for the treatment of bladder cancer,
Photodynamic Therapy (PDT) has received increased attention
early stage lung cancer, oesophageal cancer and early non-small
cell lung cancer in various countries. To date, the majority of
approved PSs are based on a tetrapyrrolic scaffold. Due to their
[a]
[b]
J. Karges, Dr. G. Gasser
Chimie ParisTech, PSL University, CNRS, Institute of Chemistry for
Life and Health Sciences, Laboratory for Inorganic Chemical Biology,
75005 Paris, France.
gilles.gasser@chimieparistech.psl.eu; www.gassergroup.com
Dr. O. Blacque
Department
of
Chemistry,
University
of
Winterthurerstrasse 190, CH-8057, Zurich, Switzerland.
Zurich,
[c]
Dr. P. Goldner
Chimie ParisTech, PSL University, CNRS, Institut de Recherche de
Chimie Paris, 75005 Paris, France.
[d]
Prof. H. Chao
MOE Key Laboratory of Bioinorganic and Synthetic Chemistry,
School of Chemistry, Sun Yat-sen University, 510275 Guangzhou,
People’s Republic of China.
relatively similar structures, the majority of these compounds
have a tendency to share the same disadvantages, which are 1)
poor water solubility; 2) tedious synthesis; 3) photobleaching
effect
and
4)
slow
clearance
from
the
body
causing
photosensitivity. At this stage, it is important to mention that some
compounds based on a tetrapyrrolic scaffold do not share these
limitations.[2c, 3]
Supporting information for this article is given via a link at the end of
the document.
This article is protected by copyright. All rights reserved.
Accepted Manuscript
Johannes Karges,[a] Olivier Blacque,[b] Philippe Goldner,[c] Hui Chao,[d] and Gilles Gasser[a],*
10.1002/ejic.201900569
European Journal of Inorganic Chemistry
FULL PAPER
have shown that the π-extension of the bipy core of the Ru(II)
a)
(CH 2) 2CO2Na
polypyridine complex caused a red shift of the absorption of the
R
(CH2) 2CO2Na
N
N
N
NaO2C(CH 2)2
NH
NH
O
HN
NH
HN
O
N
HN
differently substituted Ru(II) polypyridine complex have indicated
N
N
O
R
NaO 2C(CH 2)2
R
CH 3
HO
or
n = 0 -6
of 65 nm in comparison to complex 1 and an absorption in the
2+
phototherapeutic window as well as highly increased extinction
S
coefficients. Importantly, we could show the ability of this
S
N
N
H
N
N
N
compound to produce 1O2 at longer wavelengths as well as to
S
Ru
N
compound 3, with an extended π-system and dimethylamine
groups, which was indeed found to have a red-shifted absorption
b)
N
that dialkylamino substituents strongly promote a desired red shift
in absorption.[9] Combining these concepts, we report here
n
R=
resulting complex.[8] In addition, previous systematic studies of
cause phototoxicity at this wavelength in cancerous cells while
N
having no observed dark toxicity.
2+
2+
2+
N
Figure 1. Structure of a) Photofrin and b) TLD-1433. [Ru(dmb)2(IP-TT)]2+
(dmb=4,4′-dimethyl-2,2′-bipyridine,
IP-TT=2-(2′,2″:5″,2′
′′-terthiophene)-
N
imidazol[4,5-f][1,10]phenanthroline).
N
N
N
N
N
N
N
Ru
To tackle these drawbacks, new classes of PDT PSs are currently
N
N
N
N
N
Ru
N
Ru
N
N
N
N
being developed. Among the different classes, Ru(II) polypyridyl
N
complexes seem to be excellent candidates. The majority of these
compounds have generally a high water solubility, long
N
N
N
N
1
2
3
luminescence decay, high 1O2 production as well as a high
chemical and photophysical stability.[4] Therefore, it is not
surprising that the complex TLD-1433 (Figure 1, λex = 525 nm,
Figure 2. Chemical structures of the investigated compounds in this study. The
complexes were isolated as PF6 salts.
εmax = 2000 M-1 cm-1, Φ ~ 0.99 in CH3CN) has just completed
phase I clinical trial as a novel PDT PS for the treatment of bladder
Results and Discussion
cancer.[4h, 5]
To date, most studied Ru(II) polypyridyl complexes lack
significant absorption in the phototherapeutic window (600-900
nm).[6] It is well-established that the wavelength used during
treatments directly correlates with the tissue penetration depth.
Longer wavelengths are able to penetrate deeper in the tissue
and are hence potentially able to treat deeper-seated tumours or
larger tumours. Additionally, as longer wavelengths are less
energetic, less photodamage caused by the light source has been
associated with treatments at longer wavelengths. Based on this,
PSs with an absorption at wavelengths in the phototherapeutic
window are sought after.[7]
With the aim to develop Ru(II) polypyridyl complexes with a redshift absorption in view of applications as PDT PSs, we have
extended the parent complex [Ru(bipy)3]2+ (1) (bipy = 2,2′bipyridine) with methyl groups (2) and the conjugated system with
vinyl dimethylamino groups (3) (Figure 2). Recent investigations
Synthesis and Characterisation
The complexes synthesised in this work are shown in Figure 2.
Compound 1 was obtained from a commercial source whereas 2
was synthesised as previously reported by complexation of
RuCl2dmso4 with 4,4´-dimethyl-2,2´-bipyridine.[10] To the best of
our knowledge, the synthesis the complex 3 has not been yet
reported. The ligand (E,E’)-4,4’-bis(N,N-dimethylaminovinyl)-2,2’bipyridine present in complex 3 was synthesised as previously
reported.[11] However, complexation attempts of this ligand using
similar conditions to complex 2 (e.g. in EtOH or DMF as well as
addition of AgBF4 to remove the Cl ligands by precipitation of
AgCl) were unsuccessful. Based on these findings, the synthetic
procedure was changed (Scheme 1). As the first step, the
complexation of RuCl2dmso4 with 4,4´-dimethyl-2,2´-bipyridine
was performed. In a second step, an enamination reaction with
tert-butoxy bis(dimethylamino)methane (Bredereck reagent) was
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NaO2C(CH2)2
10.1002/ejic.201900569
European Journal of Inorganic Chemistry
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accomplished to obtain the desired compound 3. The signals in
crystal structure of 2 is isostructural with the previously reported
the NMR for 3 were correlated to their protons/carbons in the
crystal structure of the iron complex [Fe(4,4′-dimethyl-2,2′-
structure (numbering of the complex can be found in Figure S1)
dipyridyl)3][PF6]2 (Figure S9).[13] In the crystal, ions and solvent
via 2D-NMR (Figures S4 and S5). The identity of complex 3 was
molecules are linked together through C—H⋯F and C—H⋯O
1
confirmed by H- and
13
C-NMR, ESI-HRMS (Figure S2, S3, S6)
and the purity of all compounds verified by elemental analysis.
interactions. Despite the numerous aromatic rings of complex 2
no π…π or C-H…π interactions are observed.
2+
N
2+
N
N
N
N
a)
N
N
b)
Ru
N
N
Ru
N
N
N
N
N
N
N
N
2
3
Scheme 1. Synthesis of complexes 2 and 3. a) 4,4´-dimethyl-2,2´-bipyridine,
DMF,
reflux,
12
h,
nitrogen
atmosphere,
92%;
b)
tert-butoxy
bis(dimethylamino)methane, DMF, 140°C, 40 h, nitrogen atmosphere, 85%.
X-ray crystallography
The crystal structures of 4,4’-bis(N,N-dimethylaminovinyl)-2,2’bipyridine and 2 have been determined by single crystal X-ray
Figure 3. Molecular structure of compound 2. The thermal ellipsoids are drawn
diffraction studies. Crystal data, structure refinement parameters
at the 30 % probability level and all H atoms and the solvent molecule of diethyl
and molecular structures are presented in Table S1 as well as
ether are omitted for clarity.
Figures 3 and S7.
The crystal structure of 4,4’-bis(N,N-
dimethylaminovinyl)-2,2’-bipyridine presented in the manuscript is
Photophysical properties
a new monoclinic polymorph (a) of the previous structure (b)
The photophysical properties of the complexes were then
reported by Viau et al. in 2003.[12] The asymmetric unit in a
evaluated to assess their potential as PDT PSs. To investigate
contains one and a half molecules: one of the independent
this, we have measured the absorption of complexes 1-3 in
molecules lies on a center of inversion located in the middle of the
CH3CN (Figure 4, Table 1). Typically, Ru(II) polypyridyl
central C – C bond while the second one occupies a general
complexes have, as the lowest energy absorption band, a spin-
position. In both polymorphs the bipyridine derivative exhibits a
allowed metal-to-ligand charge transfer (MLCT) transition, which
classical transoid arrangement due to the repulsion of the nitrogen
occurs for the prototype complex [Ru(bpy)3]2+ 1 at 450 nm. The
lone pairs and a E configuration of the enamine double bonds.
band at 285 nm was assigned to spin-allowed ligand-centered
Polymorphs a and b significantly differ from each other in the
(LC) transition and the shoulders around 350 nm to metal-
relative orientation of the C=C double bonds of the enamine
centered (MC) transitions.[6f] The comparison between complexes
moieties with the central rings. Indeed, in a they adopt a s-trans
1 and 2 shows only small differences indicating that the additional
conformation with respect to C3-C4, C12-C13 and C20-C21
methyl groups in 2 do not significantly influence the absorption
(Figure S7) while in b a s-cis conformation is observed (see
properties. On the contrary, the absorption of complex 3 was
Figure S8). In our crystals the molecules are linked by C—H⋯N
highly modified with a strong increase of the extinction coefficient
and C—H⋯π interactions. In the structure of the trisbipyridyl
as well as a strong red-shift of 65 nm of the maximum of the MLCT
ruthenium(II) complex 2 the central Ru atom is expectedly
transition caused by the extension of the π-system as well as the
coordinated to the six nitrogen atoms of the three substituted
insertion of dimethylamine groups at the terminal end. Importantly,
bipyridines in a distorted octahedral geometry. The complex
the absorption tail of the compound is in the desired
cations crystallized with PF6- counter-ions and solvent molecules
phototherapeutic window (600-900 nm). For further investigation
of diethyl ether in a ration 1/2/1. It is interesting to note that the
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RuCl2dmso4
N
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European Journal of Inorganic Chemistry
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of the excited state, the emission properties of the complexes
Singlet oxygen generation
were investigated upon excitation of the compounds in CH3CN at
As discussed in the photophysical evaluation section, the lifetimes
355 nm. The emission signal was measurable between 550-850
of the excited state of the investigated compounds are drastically
nm (Figure S10) with a maximum at 622 nm for complex 2 and
decreasing in the presence of air indicating that the triplet state of
621 nm for complex 3. Comparison to the standard compound 1
the compound (3PS) is able to interact with molecular oxygen
(Φem = 0.059) shows that complex 2 has an increased emission
(3O2). As the active species for most applied PSs in PDT, the
(Φem = 0.083) whereas complex 3 is only weakly emitting and
production of singlet oxygen (1O2) is responsible for most PDT
could only be detected at the detection limit of our used setup.
effects. To investigate the ability of our compounds to generate
These results fit with those of a recent study which compared
1
different 4,4´-π-conjugated[2,2´]-bipyridines and which found that
measurement of the phosphorescence of 1O2 or 2) indirect by
(E,E’)-4,4’-bis(N,N’-dimethylaminovinyl)-2,2’-bipyridine
itself
measurement of the change in absorbance of a reporter
already had a low fluorescence quantum yield of 0.015 in
molecule.[15] The results presented in Table 2 show that
dichloromethane.[14] As an additional characterisation of the
compounds 1 and 2 are generating 1O2 decently whereas 3
excited state, the luminescence lifetimes in degassed and air
generates 1O2 only poorly.
saturated CH3CN upon excitation at 355 nm were determined
(Figure S11-S13). The measured lifetimes (Table 1) were found
Table 2. Singlet oxygen quantum yields in CH3CN and aqueous solution.
to be in the same range as for other investigated Ru(II)
Average of three independent measurements, ±10%.
poylpyridine complexes.[9] Importantly, the lifetimes of the excited
at room temperature. λem = emission maximum, Φem = luminescence quantum
direct
direct
450 nm
450 nm
D2O
CH3CN
1
54%
21%
2
66%
25%
3
n.d.
n.d.
n.d. = not detectable.
yield, τ = lifetime.
n.d. a) = not detectable due to missing absorbance at this wavelength.
state is strongly decreasing in the presence of air.
Table 1. Spectroscopic properties of the investigated complexes 1-3 in CH3CN
UV/Vis λ / nm
(ε / M-1 cm-1 * 10-3)
1
2
3
285 (80.8), 450
(14.6)
285 (91.8), 325
(13.3), 460 (16.6)
295 (86.2), 385
(149.7), 515 (56.4)
indirect
450 nm
CH3CN
57%
64%
21%
indirect
450 nm
PBS
20%
27%
3%
indirect
540 nm
CH3CN
n.d. a)
n.d. a)
18%
Indirect
540 nm
PBS
n.d. a)
n.d. a)
2%
λem /
nm
Φem
0.059
air
130
degassed
925
(Photo-)stability
610
622
0.083
109
1024
used as a PDT PS, is its (photo-)stability. To investigate this, we
621
<0.001
76
410
τ / ns
An important property of a molecule, which is envisioned to be
have assessed the stability in organic solvents of our complexes
as it was shown in previous works that this could already be
problematic.[16] For this purpose, the compounds were incubated
in CH3CN and, in time intervals (0, 1, 4, 8, 12, 24, 48 h), their
UV/Vis spectra measured. During the incubation in CH3CN, no
change in the spectra for all compounds (Figure S14-S16) could
be detected indicating their stability in this solvent. As a second
experiment, the stability in an aqueous PBS solution was
investigated. Also, here, no decomposition was observed (Figure
S17-S19), proving the stability of these compounds. Finally, as a
third experiment, the complexes were incubated in human plasma
and the stability of the complexes identified by an HPLC analysis.
As an internal standard, caffeine was used, which has already
been shown to be suitable for these experiments.[17] After 48 h
incubation, the compounds were extracted from the plasma and
the HPLC chromatogram before and after the incubation
Figure 4. Absorption spectra of complexes 1-3 in CH3CN.
compared. The analysis showed that no decomposition for the
compounds 1 and 2 (Figure S20-S21) occurred. On the contrary,
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O2, we used two different methods, namely 1) direct by
10.1002/ejic.201900569
European Journal of Inorganic Chemistry
complex 3 was clearly transformed to a mixture of different kinds
Cytotoxicity and Phototoxicity
of unidentified products (Figure S22), clearly proving its
After having assessed the chemical and photophysical properties
decomposition. After investigation of the stability of the
of compounds 1-3, their influence on cell viability in the dark and
compounds in a biological environment, we have tested their
upon light irradiation was investigated. For this purpose, the
stability upon irradiation in an air saturated CH3CN solution while
compounds were incubated in non-cancerous retinal pigment
monitoring their UV/Vis spectra in constant time intervals. The
epithelium (RPE-1) and HeLa cells in the dark as well as upon
stability was compared to the PS Protoporphyrin IX (PpIX), which
light irradiation at 480 nm (10 min, 3.1 J/cm2) and 540 nm (40 min,
is well known to be photodegradating. The comparison between
9.5 J/cm2). The obtained IC50 values were further compared with
the spectra (Figure S23-S26) shows only a small decrease in
the chemotherapeutic drug cisplatin and the PS Protoporphyrin IX
absorption for 1 and 2 indicating only a small photobleaching
(PpIX) (Table 3). For all investigated complexes, no toxicity in the
effect. On the contrary, the bands of 3 strongly decrease and shift,
dark could be observed (IC50 > 200 μM), which is a desired
proving its decomposition upon light exposure. Consequently, the
characteristic for a potential PDT PS. Disappointingly, the
modification caused by the irradiation was investigated by NMR
exposure to light had only a small effect on the cell viability for the
spectroscopy. For this purpose, a solution of 3 in CD3CN was
three compounds. While no toxicity was observed for compounds
2
irradiated at 450 nm (30 min, 36.0 J/cm ) and the change in the
1 and 2, compound 3 showed some phototoxicity. Of note, these
1
H-NMR spectrum monitored. The compound was transformed in
findings are in agreement with a study of the [Ru(bipy)3]2+ complex
an unidentified mixture of different compounds (see Figure S27).
which showed no dark and phototoxic effect in the high
micromolar range.[18] To evaluate the ability of a compound to act
Cellular Uptake
as a PS, the phototoxic index (PI) is calculated as the ratio
A crucial parameter for the bioactivity of a molecule is its cellular
between the IC50 values in the dark and upon light exposure. For
uptake. To investigate this, compounds 1-3 were incubated for 4
compound 3, a PI value of 1.3 at 480 nm and 1.4 at 540 nm for
h in the dark in human cervical carcinoma (HeLa) cells. The
HeLa cells and 1.3 at 480 nm and 1.2 at 540 nm for RPE-1 cells
amount of the metal Ru inside the cells was then determined
was determined. These results demonstrate that 3 is able to have
using inductively coupled plasma mass spectrometry (ICP-MS).
a slight phototoxic effect upon exposure to higher wavelength
The results (Figure 5) show that complex 3 has a much higher
which is a desired characteristic for a PS. However, the obtained
uptake than 1 or 2.
PI values are quite low in comparison to established PSs like PpIX.
The results can be rationalised by the rather poor generation of
singlet oxygen of this complex. One has also to highlight that the
instability of this compound in human plasma and upon irradiation
is extremely problematic and could also explain these poor
biological results. Overall, the biological results obtained in this
section are fitting with the ICP-MS experiments carried out which
showed a much better cellular accumulation of complex 3
compared to complexes 1 and 2.
Table 3. IC50 values in the dark and upon irradiation at 480 (10 min, 3.1 J/cm2)
and 540 nm (40 min, 9.5 J/cm2) for complexes 1-3 in comparison to cisplatin
and Protoporphyrin IX (PpIX) on non-cancerous retinal pigment epithelium
(RPE-1) and human cervical carcinoma (HeLa) cells. Average of three
independent measurements. n.d. = not determinable.
HeLa
Figure 5. Comparison of the cellular uptake of complexes 1-3 after 4 h
incubation in HeLa cells.
1
2
Da
rk
>2
00
>2
00
480
nm
>20
0
>20
0
PI
n.
d.
n.
d.
This article is protected by copyright. All rights reserved.
RPE-1
540
nm
>20
0
>20
0
PI
n.
d.
n.
d.
Da
rk
>2
00
>2
00
480
nm
>20
0
>20
0
PI
n.
d.
n.
d.
540
nm
>20
0
>20
0
PI
n.
d.
n.
d.
Accepted Manuscript
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European Journal of Inorganic Chemistry
FULL PAPER
3
>2
00
PpIX
>1
00
Cispl
atin
152
.4 ±
3.8
2.5
±
0.1
-
10.
5±
0.8
n.d. = not determinable.
1.
3
146
.3 ±
4.2
2.1
±
0.3
-
>4
0
-
1.
4
>2
00
>4
8
>1
00
-
29.
3±
1.4
158
.5 ±
8.1
3.8
±
0.1
-
1.
3
>2
6
-
161
.7 ±
6.2
3.1
±
0.1
-
1.
2
>3
2
-
Overall this study demonstrates how the extension of the
[Ru(bipy)3]2+
core
through
methyl
groups
(2)
or
vinyl
dimethylamino groups (3) effects their photophysical properties
including their absorption. We are currently investigating other
options to synthesise Ru(II) polypyridyl complexes with a stronger
luminescence, higher production of 1O2 as well as stability in a
biological environment as well as upon light exposure.
Conclusions
Experimental Section
MLCT transition of Ru(II) polypyridyl complexes towards the red
Materials
region to enable the use of longer wavelengths during PDT
All chemicals were obtained from commercial sources and used without
treatments. This would allow for deeper tissue penetration and
further purification. Tris(2,2′-bipyridine)ruthenium(II) hexafluorophosphate
therefore the possibility to treat deep-seated tumours and larger
[Ru(bipy)3][PF6]2 (1) was bought from Sigma Aldrich. The Ru(II) precursor
tumours. For this purpose, the [Ru(bipy)3]2+ complex was
extended with methyl groups (2) and the conjugated system
RuCl2dmso4 was synthesised as previously reported.[19] The ligand (E,E’)4,4’-bis(N,N-dimethylaminovinyl)-2,2’-bipyridine present in complex 3 was
synthesised as previously reported.[11]
extended with vinyl dimethylamino groups (3). The compounds
were characterized in-depth including by 2D-NMR techniques and
Instrumentation and methods
single crystal X-ray crystallography. Whereas the photophysical
1
H and 13C NMR spectra were recorded on a Bruker 500 MHz NMR
properties of 2 were found to be in the same range as for the
spectrometer. Chemical shifts (δ) are reported in parts per million (ppm)
standard complex 1, compound 3 showed a highly increased
referenced to tetramethylsilane (δ 0.00) ppm using the residual proton
absorption as shown by the very high extinction coefficients as
well as a strong red shift of 65 nm. Further analysis of the
photophysical properties revealed that this compound was weakly
solvent peaks as internal standards. Coupling constants (J) are reported
in Hertz (Hz) and the multiplicity is abbreviated as follows: s (singlet), d
(doublet), dd (doublet of doublet). ESI-MS experiments were carried out
using a LTQ-Orbitrap XL from Thermo Scientific and operated in positive
emissive and has short excited state lifetimes. We assume that
ionization mode, with a spray voltage at 3.6 kV. No Sheath and auxiliary
these properties are limiting the necessary energy transfer from
gas was used. Applied voltages were 40 and 100 V for the ion transfer
the excited state 3PS to molecular oxygen (3O2) to ultimately
capillary and the tube lens, respectively. The ion transfer capillary was held
produce singlet oxygen ( O2). This probably explains the poor
at 275°C. Detection was achieved in the Orbitrap with a resolution set to
singlet oxygen quantum yield determined in this study for complex
100,000 (at m/z 400) and a m/z range between 150-2000 in profile mode.
1
3. Investigation of the stability of the compounds revealed that
complexes 1 and 2 are stable in CH3CN, PBS and human plasma
Spectrum was analyzed using the acquisition software XCalibur 2.1
(Thermo Fisher Scientific). The automatic gain control (AGC) allowed
accumulation of up to 2*105 ions for FTMS scans, Maximum injection time
whereas 3 decomposes in human plasma as well as upon light
was set to 300 ms and 1 µscan was acquired. 10 µL was injected using a
irradiation over time. Biological evaluation on the cancerous cell
Thermo Finnigan Surveyor HPLC system (Thermo Fisher Scientific) with
line HeLa and the non-cancerous cell line RPE-1 revealed no dark
a continuous infusion of methanol at 100 µL.min-1. Elemental
toxicity for any of the investigated complexes in this study. While
microanalyses were performed on a Thermo Flash 2000 elemental
no toxicity upon light exposure for compounds 1 and 2 could be
analyser. For analytic HPLC the following system has been used: 2 x
observed, complex 3 showed some slight phototoxicity in the high
micromolar range against cervical cancerous HeLa cells and
importantly
no
measurable
dark
cytotoxicity.
Despite
Agilent G1361 1260 Prep Pump system with Agilent G7115A 1260 DAD
WR Detector equipped with an Agilent Pursuit XRs 5C18 (100Å, C18 5 μm
250 x 4.6 mm) Column. The solvents (HPLC grade) were millipore water
(0.1% TFA, solvent A) and acetonitrile (0.1% TFA, solvent B). Inductively
unfavourable photophysical properties, 3 showed a stronger
coupled plasma mass spectrometry (ICP-MS) experiments were carried
cytotoxic effect than the two other complexes 1 and 2.
out on an iCAP RQ ICP-MS instrument (Thermo Fisher).
Quantification of the cellular uptake of the complexes by ICP-MS
experiments rationalized this observation with complex 3 being
Synthesis
much better taken up by HeLa cells than compounds 1 and 2.
[Ru(4,4′-Dimethyl-2,2′-dipyridyl)3][PF6]2 (2)
This article is protected by copyright. All rights reserved.
Accepted Manuscript
In this study, we aimed to shift the absorption wavelength of the
10.1002/ejic.201900569
European Journal of Inorganic Chemistry
FULL PAPER
[Ru(4,4′-Dimethyl-2,2′-dipyridyl)3][PF6]2 (2) was synthesized as previously
The absorption of the samples has been measured with a SpectraMax M2
published[8] using RuCl2dmso4. Experimental data fits with the literature.
Spectrometer (Molecular Devices). The emission was measured by
Purity of the sample was assessed by HPLC and elemental analysis.
irradiation of the sample in fluorescence quartz cuvettes (width 1 cm) using
Elemental analysis calcd for C36H36F12N6P2Ru (%): C 45.82, H 3.85, N
a NT342B Nd-YAG pumped optical parametric oscillator (Ekspla) at 355
nm. Luminescence was focused and collected at right angle to the
8.91; found: C 45.71, H 3.69, N 8.83.
excitation pathway and directed to an Acton SP-2300i monochromator
[Ru((E,E’)-4,4’-Bis(N,N-dimethylaminovinyl)-2,2’-bipyridine)3][PF6]2
(Princeton Instruments). As a detector a PI-Max 4 CCD camera (Princeton
(3)
Instruments) has been used.
(2)
(188 mg,
0.20 mmol,
Luminescence quantum yield measurements
1.0 equiv.) was dissolved in dry DMF (12 mL) under nitrogen atmosphere
For the determination of the luminescence quantum yield, the samples
and
4.36 mmol,
were prepared in a not degassed CH3CN solution with an absorbance of
21.8 equiv.) was added slowly. The mixture was heated at 140 °C for 40
0.1 at 355 nm. This solution was irradiated in fluorescence quartz cuvettes
h. The solution was then cooled down and a sat. aqueous solution of
(width 1 cm) using a NT342B Nd-YAG pumped optical parametric oscillator
NH4PF6 was added. The crude product, which precipitated as a PF6 salt
(Ekspla) at 355 nm. The emission signal was focused and collected at right
was collected by filtration and washed with H2O and Et2O. The product
angle to the excitation pathway and directed to an Acton SP-2300i
was isolated via fractionated precipitation from CH3CN by adding dropwise
monochromator (Princeton Instruments). As a detector a XPI-Max 4 CCD
tert-Butoxy
bis(dimethylamino)methane
(0.9 mL,
1
Et2O. 215 mg of 3 (0.17 mmol, 85 %) were yielded as a dark red solid. H-
camera (Princeton Instruments) has been used. The luminescence
NMR (CD3CN, 500 MHz): 7.98 (d, J = 2.0 Hz, 6H, H3), 7.45 (d, J = 13.4
quantum yields were determined by comparison with the reference
Hz, 6H, H8), 7.24 (d, 3J = 6.2 Hz, 6H, H6), 6.90 (dd, 3,4J = 6.2, 2.0 Hz, 6H,
[Ru(bipy)3]Cl2 in CH3CN (Φem=5.9%)[27] applying the following formula:
4
3
H5), 5.09 (d, 3J = 13.4 Hz, 6H, H7), 2.94 (s, 36H, H10). 13C-NMR (CD3CN,
Φem, sample = Φem, ref * (Fref / Fsample) * (Isample / Iref) * (nsample / nref)2
125 MHz): δ = 157.8 (C2), 150.2 (C4), 150.0 (C6), 147.0 (C8), 120.2 (C5),
F = 1 – 10-A
116.7 (C3), 93.0 (C7), 40.9 (C10). ESI-HRMS (pos. detection mode): calcd
Φem = luminescence quantum yield, F = fraction of light absorbed, I =
for C54H66N12Ru m/z [M]
2+
492.2283; found: 492.2284. Elemental analysis
calcd for C54H66F12N12P2Ru (%): C 50.90, H 5.22, N 13.19; found: C 50.64,
integrated emission intensities, n = refractive index, A = absorbance of the
sample at irradiation wavelength.
H 4.96, N 12.90.
Lifetime measurements
X-ray crystallography
For the determination of the lifetimes, the samples were prepared in an air
X-ray single-crystal data were collected at low temperatures, 160(1) K for
saturated and in a degassed CH3CN solution with an absorbance of 0.2 at
4,4’-bis(N,N-dimethylaminovinyl)-2,2’-bipyridine and at 183(1) K for
355 nm. This solution was irradiated in fluorescence quartz cuvettes (width
compound 2, with an Oxford liquid-nitrogen Cryostream cooler on a Rigaku
1 cm) using a NT342B Nd-YAG pumped optical parametric oscillator
OD XtaLAB Synergy Dualflex (Pilatus 200K detector) diffractometer. A
(Ekspla) at 355 nm. The emission signal was focused and collected at right
single wavelength X-ray source from a micro-focus sealed X-ray tube were
angle to the excitation pathway and directed to an Acton SP-2300i
used with the Cu Kα radiation (λ = 1.54184 Å)
[20]
for both analyses. The
selected single crystals were mounted using polybutene oil on a flexible
monochromator (Princeton Instruments). As a detector a R928
photomultiplier tube (Hamamatsu) has been used.
loop fixed on a goniometer head and transferred to the diffractometer. Preexperiments, data collections, data reductions and analytical absorption
corrections
[21]
were performed with the program suite CrysAlisPro.
[22]
Singlet oxygen measurements
-
Direct evaluation
small
The samples were prepared in an air saturated CH3CN or D2O solution
molecule structure solution program and refined with the SHELXL2018/3
with an absorbance of 0.2 at 450 nm. This solution was irradiated in
program package[25] by full-matrix least-squares minimization on F2.
fluorescence quartz cuvettes (width 1 cm) using a mounted M450LP1 LED
Using Olex2,
[23]
[24]
the structures were solved with the SHELXT
The crystal data
(Thorlabs) whose irradiation, centered at 450 nm, has been focused with
collections and structure refinement parameters are summarized in Table
aspheric condenser lenses. The intensity of the irradiation has been varied
S1. CCDC 1914096 (for 2) and CCDC 1914097 (for 4,4’-bis(N,N-
using a T-Cube LED Driver (Thorlabs) and measured with an optical power
dimethylaminovinyl)-2,2’-bipyridine)
supplementary
and energy meter. The emission signal was focused and collected at right
crystallographic data for these compounds, and can be obtained free of
angle to the excitation pathway and directed to an Acton SP-2300i
charge
monochromator (Princeton Instruments). A longpass glass filter was
Molecular graphics were created using Mercury 4.0.
from
the
Cambridge
contain
the
Crystallographic
www.ccdc.cam.ac.uk/data_request/cif.
[26]
Data
Centre
via
placed in front of the monochromator entrance slit to cut off light at
wavelengths shorter than 850 nm. As a detector an EO-817L IR-sensitive
Spectroscopic measurements
liquid nitrogen cooled germanium diode detector (North Coast Scientific
This article is protected by copyright. All rights reserved.
Accepted Manuscript
[Ru(4,4′-Dimethyl-2,2′-dipyridyl)3][PF6]2
10.1002/ejic.201900569
European Journal of Inorganic Chemistry
FULL PAPER
Corp.) has been used. The singlet oxygen luminesce at 1270 nm was
continuous gentle shaking (ca. 300 rpm). The reaction was stopped after
measured by recording spectra from 1100 to 1400 nm. For the data
the incubation time by addition of 2 mL of methanol. The mixture was
analysis, the singlet oxygen luminescence peaks at different irradiation
centrifuged for 45 min at 650 g at 4 °C. The methanolic solution was filtered
intensities were integrated. The resulting areas were plotted against the
through a 0.2 μm membrane filter. The solvent was evaporated under
percentage of the irradiation intensity and the slope of the linear regression
reduced pressure and the residue was dissolved in 1:1 (v/v) CH3CN/ H2O
calculated. The absorbance of the sample was corrected with an
0.1% TFA solution. The solution was filtered through a 0.2 μm membrane
absorbance correction factor. As reference for the measurement rose
filter and analysed using an HPLC System. Based on the big differences
bengal (Φ = 76 %)[28] was used and the singlet oxygen quantum yields
in lipophilicity, two different HPLC methods have been used. The solvents
were calculated using the following formula:
(HPLC grade) were millipore water (0.1% TFA, solvent A) and acetonitrile
-A
(solvent B). Method M1: 0-3 minutes: isocratic 95% A (5% B); 3- 17
I = I0 * (1 – 10 )
minutes: linear gradient from 95% A (5% B) to 0% A (100% B); 17-23
Φ = singlet oxygen quantum yield, S = slope of the linear regression of the
minutes: isocratic 0% A (100% B). Method M2: 0-3 minutes: isocratic 80%
plot of the areas of the singlet oxygen luminescence peaks against the
A (20% B); 3-17 minutes: linear gradient from 80% A (20% B) to 0% A
irradiation intensity, I = absorbance correction factor, I0 = light intensity of
(100% B); 17-23 minutes: isocratic 0% A (100% B). The flow rate was 1
the irradiation source, A = absorbance of the sample at irradiation
mL/min and the chromatogram was detected at 250 nm.
wavelength.
-
Indirect evaluation
Photostability
For the measurement in CH3CN: The samples were prepared in an air-
The samples were prepared in an air saturated CH3CN solution. To
saturated CH3CN solution containing the complex with an absorbance of
measure the photostability, the samples were irradiated at 450 nm in 96
0.2 at the irradiation wavelength, N,N-dimethyl-4-nitrosoaniline aniline
well plates with an Atlas Photonics LUMOS BIO irradiator during time
(RNO, 24 µM) and imidazole (12 mM). For the measurement in PBS buffer:
intervals from 0-10 min. The absorbance spectrum from 350-700 nm was
The samples were prepared in an air-saturated PBS solution containing
recorded with a SpectraMax M2 Microplate Reader (Molecular Devices)
the complex with an absorbance of 0.1 at the irradiation wavelength, N,N-
after each time interval and compared.
dimethyl-4-nitrosoaniline aniline (RNO, 20 µM) and histidine (10 mM). The
samples were irradiated on 96 well plates with an Atlas Photonics LUMOS
Cell culture
BIO irradiator for different times. The absorbance of the samples was
Human cervical carcinoma (HeLa) cells were cultured using DMEM media
measured during these time intervals with a SpectraMax M2 Microplate
and retinal pigment epithelium (RPE-1) cells using DMEM/F-12 with
Reader (Molecular Devices). The difference in absorbance (A0-A) at 420
addition of 10% FBS and 1% penstrep. The cells were cultivated and
nm for the CH3CN solution or at 440 nm a PBS buffer solution was
maintained at 37 °C in a cell culture incubator at 37 °C with 5% CO2
calculated and plotted against the irradiation times. From the plot the slope
atmosphere. Before an experiment, the cells were passaged three times.
of the linear regression was calculated as well as the absorbance
correction factor determined. The singlet oxygen quantum yields were
Cellular uptake
calculated using the same formulas as used for the direct evaluation.
The cellular uptake of the complex was investigated by the determination
of the Ru content inside the cells. The complex with a final concentration
Stability in CH3CN and PBS
of 25 μM (1% DMSO, v%) was incubated for 4 h at 37 °C in the dark on a
The stability of the compound in CH3CN and PBS was determined by
cell culture dish with a density of ca. 6 . 106 cells in 10 mL of media. After
UV/Vis spectroscopy. The compound was dissolved and stored at room
this time, the media was removed and the cells were washed with cell
temperature in the dark. The absorption spectrum from 250-700 nm was
media.
recorded with a SpectraMax M2 Microplate Reader (Molecular Devices)
resuspended. The number of cells on each dish was accurately counted.
after each time interval (0, 1, 4, 8, 12, 24, 48 h) and compared.
Each sample was the digested using a 60% HNO3 solution overnight. Each
The
cells
were
trypsinised,
harvested,
centrifuged
and
sample was diluted to solution of 2% HNO3 in water. The Ru content was
Stability in human plasma
determined using an ICP-MS apparatus and comparing the results with the
The stability of the complexes was evaluated with caffeine as an internal
Ru references. The Ru content was then associated with the number of
standard, which has already shown to be suitable for these experiments.[17]
cells.
The pooled human plasma was obtained from Biowest and caffeine from
TCI Chemicals. Stock Solutions of the compounds (40 μM) and caffeine
(Photo-)cytotoxicity
(20 μM) were prepared in DMSO. One aliquot of the solutions was added
The cytotoxicity of the compounds was accessed by measuring the cell
to 975 μL of human plasma to a total volume of 1000 μL. Final
viability using a fluorometric resazurin assay. The cultivated cells were
concentrations of the compounds of 0.5 μM n-and caffeine of 0.25 μM
seeded in triplicates in 96 well plates with a density of 4000 cells per well
were achieved. The resulting solution was incubated for 48 h at 37 °C with
in 100 μL of media. After 24 h the medium was removed and the cells were
This article is protected by copyright. All rights reserved.
Accepted Manuscript
Φsample = Φreference * (Ssample / Sreference) * (Ireference / Isample)
10.1002/ejic.201900569
European Journal of Inorganic Chemistry
treated with increasing concentrations of the compound diluted in cell
media achieving a total volume of 200 μL. The cells were incubated with
the compound for 4 h. After this time, the media was removed and replaced
with 200 μL of fresh medium. For the phototoxicity studies, the cells were
exposed to light with an Atlas Photonics LUMOS BIO irradiator. Each well
was constantly illuminated with either a 480 nm or 540 nm irradiation.
During this time, the temperature was maintained constantly at 37 °C. The
cells were grown in the incubator for additional 44 h. For the determination
of the dark cytotoxicity, the cells were not irradiated and after the medium
[5]
exchange directly incubated for 44 h. After this time, the medium was
replaced with fresh medium containing resazurin with a final concentration
of 0.2 mg/mL. After 4 h incubation, the amount of the fluorescent product
[6]
resorufin was determined upon excitation at 540 nm and measurement its
emission at 590 nm using a SpectraMax M2 Microplate Reader (Molecular
Devices). The obtained data was analysed with the GraphPad Prism
software.
Acknowledgments
[7]
Le Conseil Régional d'Ile de France is acknowledged for
providing financial support for the purchase of a 500 MHz NMR
[8]
spectrometer. This work was financially supported by an ERC
Consolidator Grant PhotoMedMet to G.G. (GA 681679) and has
[9]
received support under the program “Investissements d’ Avenir”
launched by the French Government and implemented by the
ANR with the reference ANR-10-IDEX-0001-02 PSL (G.G.), the
[10]
National Science Foundation of China (Nos. 21525105 and
[11]
21778079 for H.C.) and the 973 Program (No. 2015CB856301
for H.C.).
[12]
[13]
Keywords: Anticancer • Bioinorganic Chemistry • Medicinal
Inorganic Chemistry • Metals in Medicine • Photodynamic
[14]
[15]
Therapy
[16]
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10.1002/ejic.201900569
European Journal of Inorganic Chemistry
FULL PAPER
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Accepted Manuscript
[25]
This article is protected by copyright. All rights reserved.
10.1002/ejic.201900569
European Journal of Inorganic Chemistry
FULL PAPER
FULL PAPER
Key Topic: Metals in Medicine •
Photodynamic Therapy
Johannes
Karges,
Olivier
Blacque,
Philippe Goldner and Gilles Gasser *
Towards Long Wavelength Absorbing
Photodynamic
Therapy
Photosensitizers via the Extension of
a [Ru(bipy)3]2+ Core
This article is protected by copyright. All rights reserved.
Accepted Manuscript
The preparation and characterisation
of new Ru(II)-containing photodynamic
therapy, that are based on a
[Ru(bipy)3]2+ core (bipy: 2,2'-bipyridine)
and that are extended with methyl
groups or vinyl dimethylamino groups
is
described.
Their
in-depth
photophysical properties as well as
(photo-)cytotoxicity against cervical
cancerous HeLa cells is also reported.