One- and Two-Photon Phototherapeutic Effects of RuII Polypyridine Complexes in the Hypoxic Centre of Large Multicellular Tumor Spheroids and Tumor-Bearing Mice**

Article abstract of DOI:10.1002/chem.202003486

During the last decades, photodynamic therapy (PDT), an approved medical technique, has received increasing attention to treat certain types of cancer. Despite recent improvements, the treatment of large tumors remains a major clinical challenge due to th

Full text of DOI:10.1002/chem.202003486

Accepted Article
Accepted Article
Title: 1- and 2-Photon Phototherapeutic Effects of Ru(II) Polypyridine
Complexes in the Hypoxic Centre of Large Multicellular Tumour
Spheroids and Tumour-Bearing Mice
Authors: Johannes Karges, Shi Kuang, Yih Ching Ong, Hui Chao, and
Gilles Gasser
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To be cited as: Chem. Eur. J. 10.1002/chem.202003486
Link to VoR: https://doi.org/10.1002/chem.202003486
01/2020

10.1002/chem.202003486
Chemistry - A European Journal
RESEARCH ARTICLE
1- and 2-Photon Phototherapeutic Effects of Ru(II) Polypyridine
Complexes in the Hypoxic Centre of Large Multicellular Tumour
Spheroids and Tumour-Bearing Mice
Johannes Karges,[a] Shi Kuang,[b] Yih Ching Ong,[a] Hui Chao,*[b] and Gilles Gasser*[a]
Abstract: During the last decades, photodynamic therapy (PDT), an
approved medical technique, has received increasing attention to
treat certain types of cancer. Despite recent improvements, the
treatment of large tumors remains a major clinical challenge due to
the low ability of the photosensitizer (PS) to penetrate a 3D cellular
architecture and the low oxygen concentrations present in the tumour
centre. To mimic the conditions found in clinical tumors, exceptionally
large 3D multicellular tumour spheroids (MCTSs) with a diameter of
800 µm were used in this work to test a series of new Ru(II)
polypyridine complexes as 1-Photon and 2-Photon PSs. These metal
complexes were found to fully penetrate the 3D cellular architecture
and to generate singlet oxygen in the hypoxic centre upon light
irradiation. While having no observed dark toxicity, the lead compound
of this study showed an impressive phototoxicity upon clinically
relevant 1-Photon (595 nm) or 2-Photon (800 nm) excitation with a full
eradication of the hypoxic centre of the MCTSs. Importantly, this
efficacy was also demonstrated on mice bearing an adenocarcinomic
human alveolar basal epithelial tumour.
the PS is not able to penetrate a 3D cellular architecture; 2) the
oxygen concentration in the centre of the tumour is low (i.e.,
hypoxia), which is hampering the production of the therapeutically
1
necessary ROS and O2; 3) light is not delivered to the tumour
centre.[3] Consequently, there is a need for the evaluation of new
PSs in large tumour models that can better simulate the
conditions found in the tumours of patients.
Multicellular tumour spheroids (MCTSs) can mimic the
pathological conditions found in clinically-relevant tumours.
MCTSs with a diameter of 200 μm are frequently used to simulate
3D cell-cell as well as cell-matrix interactions. They can therefore
be utilised to model the drug delivery of a compound.[4] This is
important since many investigated drug candidates have failed
the translation from promising results in a 2D monolayer model to
a 3D or an in vivo model due to compromised drug delivery.[5]
Furthermore, MCTSs are also able to simulate hypoxia and
proliferation gradients to the centre. Since the vast majority of
PDT agents act by an oxygen-dependent mechanism, the
treatment of hypoxic tumors remains a major medical challenge.[6]
Despite these limitations, the successful treatment of the hypoxic
regions of melanoma MCTSs with a diameter of 280 μm was
recently reported.[7] Studies have shown that the oxygen diffusion
inside of MCTSs is limited to approximately 200 μm.[8] Therefore,
larger MCTS are able to generate a hypoxic core, which can
better simulate the pathological conditions found in clinically-
relevant tumours. Despite these benefits, studies of the antitumor
effects of compounds in large MCTSs remain very rare.[9] To
overcome this limitation and promote a better understanding of
the biological effects of new compounds, there is an urgent need
for in-depth studies of the ability of new compounds in large
MCTSs.
Ru(II) polypyridine complexes are gaining increasing attention as
PDT PSs due to their attractive chemical and photophysical
properties (e.g., high water solubility, chemical stability and
photostability).[10] Despite their remarkable characteristics, the
majority of PSs are activated using either UV or blue light, limiting
the light penetration inside the tissue and therefore their
application to treat deep seated or large tumours. To overcome
this limitation, there is a need for the development of Ru(II)
polypyridine complexes with an absorption in the biological
spectral window (600-900 nm).[11] This aim could be achieved by
a red-shift of the 1-Photon (1P) absorption or the use of a 2-
Photon (2P) process, in which the compound absorbs two
photons of low energy/high wavelengths simultaneously. Next to
a deeper tissue penetration, a 2P excitation correlates with a
reduced photodamage as well as enhanced spatial resolution.
However, the Ru(II) polypyridine complexes reported so far were
found to have relatively poor 2P photophysical properties[7, 9a, 12]
compared to porphyrins, porphyrin oligomers or expanded
porphyrinoids,[13] limiting the application of this technique with
Introduction
Cancer has emerged as one of the deadliest diseases worldwide.
Photodynamic Therapy (PDT) is a minimal invasive medical
technique to treat this disease, often in combination with other
methods (i.e., surgery, chemotherapy, radiation therapy or
immunotherapy). In PDT, a preferably non-toxic photosensitizer
(PS) is activated at a specific wavelength to generate reactive
oxygen species (ROS). The majority of clinically approved PSs
act by an energy transfer of the PS to molecular oxygen (3O2) to
generate singlet oxygen (1O2). As ROS and 1O2 are highly reactive,
they can rapidly interact with their biological surroundings to
trigger cell death.[1] Despite recent research advances, the clinical
applications of PDT remains sometimes unsatisfactory. Many
tumours and especially large tumours show, after a PDT
treatment, a regression of their outer spheres while their core
remain intact, causing, after a certain time, a cancer relapse.[2]
This incomplete tumour eradication is due to several factors: 1)
[a]
[b]
Dr. J. Karges, Dr. Y. C. Ong, 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
S. Kuang, 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.
ceschh@mail.sysu.edu.cn
Supporting information for this article is given via a link at the end of
the document.
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10.1002/chem.202003486
Chemistry - A European Journal
RESEARCH ARTICLE
Previously studied compounds:
2+
2+
2+
R
R
R
R
R
N
Ru
N
N
Ru
N
N
N
N
N
N
N
N
N
N
N
N
N
N
Ru
N
R
R
R
R
R
R
R
NMe2
OMe
R = NMe2
OMe
R =
R = NMe2
OMe
New compounds:
2+
2+
R
R
N
N
N
N
N
N
N
N
N
Ru
Ru
N
N
N
R
R
R = NMe2
OMe
1
2
R = NMe2
OMe
3
4
Figure 1. Chemical structures of previously studied and new compounds investigated in this work. The followed letter a corresponds to the compounds as a
hexafluorophosphate salt whereas the letter b indicated the compound as a chloride salt.
these complexes. In this context, we have recently reported the
an adenocarcinomic human alveolar basal epithelial tumour
inside a mouse model.
2+
rational design of the complexes [Ru(bipy)n(L-NMe2/L-OMe)3-n
]
(bipy 2,2´-bipyridine, L-NMe2 (E,E’)-4,4´-bis[p-(N,N-
=
=
dimethylamino)styryl]-2,2´-bipyridine, L-OMe = (E,E’)-4,4´-bis[p-
methoxystyryl]-2,2´-bipyridine, n = 0, 1, 2, Figure 1) as effective
PSs for 1P (540 nm) and 2P (800 nm) PDT.[14] Worthy of note, the
use of (E,E’)-4,4´-bisstyryl-2,2´-bipyridine type Ru(II) polypyridine
complexes were previously reported as effective 2P absorbing
chromophores.[15] The lead compound of our study, namely
[Ru(bipy)2(L-OMe)]2+ was found to be active in vivo.[14] Despite the
impressive biological property of this complex, its lack of 1P
absorption in the biological spectral window and poor 2P
absorption is limiting the application for deep-seated or large
tumors.
With this in mind, in this work, the analogous complexes that bear
different ancillary ligands [Ru(phen/bphen)2(L-NMe2/L-OMe)]2+
(1-4, phen = 1,10-phenenthroline, bphen = 4,7-diphenyl-1,10-
phenanthroline) are reported as effective PSs. The lead
compound of this new study was found to have a red-shifted 1P
and strong 2P absorption, enabling a phototherapeutic effect
upon 1P (595 nm) or 2P (800 nm) excitation. An in-depth analysis
of their ability to treat the hypoxic core in extraordinary large
MCTS is presented. Further this complex was found to eradicate
Results and Discussion
To the best of our knowledge, the synthesis of the complexes 1-4
(Figure 1) has not been reported yet. The ligands L-NMe2 and L-
OMe were synthesized as previously reported by our group.[14]
The precursors Ru(phen)2Cl2 and Ru(bphen)2Cl2 were prepared
as described in the literature.[16] Finally, the chloride substituents
of the respective precursors were substituted with L-NMe2 or L-
OMe, yielding the desired complexes 1a-4a. All compounds were
characterized using 1H-, 13C-NMR, HR-MS analysis and the purity
of the compounds verified by HPLC and elemental analysis.
Details on the synthesis and characterization of the complexes
can be found in the SI (Scheme S1-S2, Figure S1-S12).
To evaluate the potential of the compounds, their photophysical
properties (Table S1) were determined. All complexes were found
to have a strong red-shifted 1P absorption as indicated by their
high extinction coefficients and absorption tail towards longer
wavelengths (Figure S13a). Strikingly, the complexes have an
exceptionally high 2P absorption up to ~1600 GM (Figure S13b),
which is an order of magnitude higher than the majority of
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10.1002/chem.202003486
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RESEARCH ARTICLE
previously reported Ru(II) polypyridine complexes (~40-250
GM).[7, 9a, 12] Importantly, the compounds have a 1P absorption tail
towards and 2P absorption within the biological spectral window
(600-900 nm), potentially allowing them to be utilized for the
treatment of deep-seated or large tumours. The maximum of the
emission of the complexes (Figure S14) was determined to be at
698 nm for the L-NMe2-coordinated complexes (1a, 3a) and at
663 nm for the L-OMe-coordinated complexes (2a, 4a), resulting
in a large Stokes shift for all investigated compounds. The
comparison of the excitation and absorption spectra of all
compounds showed no significant differences. The L-NMe2 or L-
OMe ligands play a significant role in the absorption and emission
characteristics of the complexes since complexes 1 and 3 as well
as 2a and 4a have similar absorption and emission profiles. The
analysis of the excited states of the analogous bipy derivatives
suggested that these transitions stem from metal-to-ligand charge
transfer/ligand-to-metal charge transfer excitations.[14] The
comparison between the complexes revealed that the L-OMe-
coordinated analogous (2a: 1.9 %, 4a: 2.7 %) have a significantly
higher luminescence quantum yield than the L-NMe2-coordinated
compounds (1a: 0.4 %, 3a: 0.6 %). Worthy of note, other
previously published Ru(II) polypyridine complexes with terminal
[Ru(phen)3]2+ and [Ru(bphen)3]2+ [17] as well as their analogous
bipy derivatives.[14]
The stability of a compound is a crucial factor in PDT applications.
Previous studied have shown some metal complexes are not
stable under physiological conditions, leading to undesired side
effects.[19] The compounds were incubated in human plasma at
37 °C and extracted after 48 h. No significant differences between
the chromatograms (Figure S23-S26) were observed, indicating
that all complexes were stable in a biological environment.
Following this, the stability upon light exposure at 540 nm was
investigated by monitoring their absorption spectra. Previous
studies have shown that this could be an issue for some Ru(II)
complexes.[20] It is important to note that clinically used PSs,
represented here by protoporphyrin IX (PpIX), are generally
associated with a fast photodegradation.[21] The compounds
investigated in this study were found to be highly photostable
(Figure S27-S30) whereas PpIX completely decomposed (Figure
S31) under identical experimental conditions.
To study the effect that these complexes have on cells, we first
assessed the time they need to be taken up by human cervical
carcinoma (HeLa) cells using inductively coupled plasma mass
spectrometry (ICP-MS). Within 6 h, the asymptotic maximum
concentration (Figure S32-S35) was reached. The comparison
between the complexes (Figure S36) shows that the bphen-
coordinated complexes (3a, 4a) are slightly better taken up by
cells than the phen coordinated complexes (1a, 2a). This result is
in agreement with the determination of the logP values of the
complexes (Table S3), which demonstrate their strong lipophilic
character. Following this, the uptake mechanism of the
compounds was investigated by blocking different pathways
(Figure S37-S40) by preincubation with metabolic (2-deoxy-D-
dimethylamine groups were also found to be poorly
23d]
luminescent.[11c,
Theoretical
calculations
for
the
[Ru(bphen)2((E,E’)-4,4’-bis(N,N’-dimethylaminovinyl)-2,2’-
bipyridine)]2+ complex indicated that the energetically lowest lying
excitations are based on non-luminescent ligand-centred
transitions.[23d] The bphen coordinated compounds (3a, 4a)
showed a stronger luminescence than the phen coordinated
compounds (1a, 2a). This observation is in agreement with the
comparison between the corresponding parent complexes
[Ru(phen)3]2+ and [Ru(bphen)3]2+.[17] All complexes were found to
have excited state lifetimes in the nanosecond range (Figure S15-
S18) in an aerated environment (i.e., 72-193 ns) and in a
degassed environment (i.e., 334-981 ns). These values are in the
same range than for other reported Ru(II) polypyridine
complexes.[18]
glucose
and
oligomycin),
cationic
transporter
(tetraethylammonium chloride) and endocytotic (ammonium
chloride or chloroquine) inhibitors as well as incubation at lower
temperature (4 °C).[22] As the incubation with tetraethylammonium
chloride had only a negligible effect on the uptake, the
internalization by this pathway was ruled out. The change to lower
temperatures as well as the incubation with metabolic inhibitors
significantly decreased the uptake, indicating that the mechanism
is energy dependent. As the preincubation with ammonium
chloride or chloroquine strongly decreased the internalization of
the complexes 1a-4a, it indicates that all compounds are taken up
through an energy dependent endocytosis pathway. The
subcellular localization was then examined using confocal laser
scanning microscopy (Figure S41). The compounds were
incubated in HeLa cells with commercial dyes for the major
cellular organelles (i.e., nucleus, mitochondria, lysosomes, Golgi
apparatus, endoplasmic reticulum) and their colocalization in the
cells compared. As no significant overlap was observed, it
suggests that the compounds do not majorly localize in these
organelles. To further investigate the localization, the cell
organelles were separately extracted and the amount of Ru
determined by ICP-MS (Figure S42). All compounds were mainly
found in the cytoplasm with small amounts of unselective
accumulation. The structurally related bipy derivatives previously
studied were also found to majorly localize in this organelle.[14]
To study the (photo-)cytotoxic effects of 1a-4a, these compounds
were incubated in the dark as well as upon light exposure at 480
nm (10 min, 3.1 J/cm2) and 540 nm (40 min, 9.5 J/cm2) in non-
As the lifetimes drastically decrease in the presence of air, it
indicates that the excited state can interact with a component of
air. Capitalizing on this, the type of ROS generated upon light
irradiation was then investigated by electron spin resonance
spectroscopy using 2,2,6,6–tetramethylpiperidine as a singlet
oxygen (1O2) scavenger and 5,5-dimethyl-1-pyrroline N-oxide as
a •OOH or •OH radical scavenger. In contrast to the characteristic
1O2-induced triplet signal of 2,2,6,6-tetramethylpiperidinyloxyl in
CH3CN and PBS for all complexes (Figure S19-S22), no
significant signal was observed for the formation of •OOH or •OH
radicals. The 1O2 quantum yields upon light exposure were
determined using two complementary methods: 1) directly by
measuring the phosphorescence signal of 1O2 upon excitation at
450 nm; 2) indirectly by capturing 1O2 with a reporter molecule and
monitoring its change by absorbance spectroscopy upon
excitation at 450 or 540 nm. The compounds (Table S2) were
1
found to have O2 quantum yields in CH3CN between 43-92 %
and 2-14 % in an aqueous solution. The comparison between the
compounds revealed that the 1O2 production of 2a and 4a is
drastically higher than of 1a and 3a, which is expected due to their
superior photophysical properties. The 1O2 generation of 2a and
4a is superior than their structurally related parent complexes
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cancerous retinal pigment epithelium (RPE-1), HeLa, mouse
colon carcinoma (CT-26) and human glioblastoma astrocytoma
(U373) cells. PpIX, the anticancer drug cisplatin and the parent
complexes [Ru(phen)3]2+ and [Ru(bphen)3]2+ were used as
controls. The light doses and irradiation times used in this work
were optimized to the survival of the cells when treated only with
the light source. All cell lines were tested identically to investigate
the influence of the compounds on different types of cancer. The
results (Table S4-S5) show that the phen-based compounds (1a,
2a) as well as [Ru(phen)3]2+ are non-toxic in the dark (IC50 > 100
mM), which is a crucial requirement for PDT applications. On the
contrary, the bphen-coordinated complexes (3a, 4a) were found
to be cytotoxic in the low micromolar range (IC50 = 5.2 - 20.8 μM)
in all tested cell lines. The observed cytotoxicity in the dark for the
bphen-coordinated Ru(II) complexes could also be observed for
[Ru(bphen)3]2+ and is in agreement with recent studies of
structurally related bphen-coordinated compounds.[23] Upon light
irradiation, all compounds were able to generate 1O2, causing cell
death in all investigated cell lines. The phen-coordinated
compounds (1a, 2a) were found to have a phototoxic effect in the
low micromolar range (IC50 = 0.9 – 15.6 μM), while [Ru(phen)3]2+
only had a negligible effect. The bphen-coordinated complexes
(3a, 4a) as well as [Ru(bphen)3]2+ have, next to a cytotoxic effect
in the dark, a phototoxic effect, as demonstrated by their IC50
values in the high nanomolar to low micromolar range (IC50 = 0.5
– 2.1 μM). The L-OMe-based compounds (2a, 4a) were more
phototoxic than the L-NMe2-based compounds (1a, 3a). This
observation is attributed to their superior photophysical properties
(Table S1-S2), notably their better 1O2 production. Overall, 2a was
identified as the best compound of the series with no observed
toxicity in the dark and a phototoxicity in the high nanomolar range
in CT-26 cells (IC50,540nm = 0.9 ± 0.4 μM, PI540nm > 111). These
highly promising results of 2a are an order of magnitude lower
compared to the IC50 values of the PS PpIX, the parent complex
[Ru(phen)3]2+ and cisplatin tested under identical experimental
conditions.
at 480 nm (10 min, 3.1 J/cm2). The results show highly increased
caspase levels upon irradiation (Figure S44), indicating that the
phototoxic effect of all compounds is caused by a caspase 3/7
pathway. Some previously studied Ru(II) polypyridine complexes
were found to exert their phototoxic effect by the same
mechanism.[26]
The phototoxic profile of the lead compound 2a was further
explored using longer wavelengths. 2a was also active upon
irradiation at 595 nm (60 min, 11.2ꢀJ/cm2) in CT-26 cells with an
IC50 value in the low micromolar range (IC50,595nm = 2.4 ± 0.3 μM,
PI595nm > 41.7). In contrast, no phototoxicity was observed upon
exposure to light at 620 nm (30 min, 3.3ꢀJ/cm2). The cell death
mechanism caused upon irradiation at 480 nm (10 min, 3.1 J/cm2)
was investigated by determining the cell viability upon
preincubation with autophagy (3-methyladenine), apoptosis (Z-
VAD-FMK), paraptosis (cycloheximide) and necrosis (necrostatin-
1) inhibitors (Figure S43). Since preincubation with autophagy
and necrosis inhibitors did not significantly influence the cell
survival, these pathways were ruled out for all compounds. The
preincubation with a paraptosis inhibitor slightly increased cell
survival, while the preincubation with an apoptosis inhibitor highly
increased cell survival. This indicates that the cell death is mainly
caused by the apoptosis pathway, with minor contribution from
the paraptosis pathway. Some previously studied PS were
triggering cell death by the same mechanism.[24] To further
investigate the apoptosis mechanism, its dependency on
caspases 3/7 was studied. Caspases 3/7 are well known
executers of the intrinsic and extrinsic apoptosis mechanism.[25]
The caspase activity was measured in HeLa cells upon irradiation
Figure 2. 1P (λex = 458 nm, λem = 600 – 750 nm) and 2P (λex = 800 nm, λem
=
600 - 750 nm) excited Z-stack images in HeLa MCTS after incubation of 2a (10
μM) for 12 h. a/c) Z-axis images scanning from the top to the bottom of an intact
spheroid. b/d) 3D z-stack of an intact spheroid.
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Figure 4. Tumour growth inhibition assay. Change of the volume in HeLa MCTSs in correlation to treatment time. The MCTSs were treated with the compounds
1a-4a (20 μM), H2TPP (20 μM) and cisplatin (10 μM and 30 μM). The MCTSs were a) strictly kept in the dark b) exposed to a 1P irradiation (500 nm, 10 J/cm2) c)
exposed to a 2P irradiation (800 nm, 10 J/cm2, section interval of 5 μm) on day 1. Representative pictures of the MCTSs can be found in Figure S48-S50. The error
bars correspond to the standard deviation of the three replicates.
As a closer model to clinically treated cancer tumors, the
biological effects of the compounds were evaluated in-depth in
exceptionally large 3D multicellular tumour spheroids (MCTSs)
with a diameter of 800 μm. In addition to the irradiation with a 1P
light source, we have also studied their biological effect using a
2P light source. To investigate the penetration of the compounds
inside the tumour model, HeLa MCTSs were incubated with 1a-
4a (10 μM) for 12 h and their 1P and 2P luminescence images by
laser scanning confocal z-stack microscopy taken. Importantly, all
compounds were able to fully penetrate the MCTSs up to their
central cores, as demonstrated by the strong luminescence signal
at every section depth (Figure 2, Figure S45-S47). A stronger
luminescence signal following the 2P excitation in comparison to
the 1P excitation was observed due to the deeper light penetration
inside the MCTSs. Following this, the capacity of the compounds
also observed in the centre of the MCTSs, suggesting the
generation of 1O2 despite the hypoxic conditions, which are
typically observed in MCTS.
To study the (photo-)cytotoxic effect in MCTSs, 1a-4a (20 μM),
cisplatin (10 μM and 30 μM) and the well-characterized PS
tetraphenylporphyrin (H2TPP, 20 μM) were exposed to 1P (500
nm, 10 J/cm2) and 2P (800 nm, 10 J/cm2, section interval of 5 μm)
irradiation and their tumour growth monitored (Figure 4). As
observed in 2D monolayer cells, 3a and 4a showed a weak
tumour growth inhibition effect in the dark similar to cisplatin at 10
μM while 1a and 2a did not significantly influence the tumour
growth in the dark – the MTCSs grew in similar manner as the
control. Upon 1P or 2P light treatment, all compounds 1a-4a were
able to cause a phototoxic effect inside the MCTSs leading to a
decrease of the volume of the MCTSs. In particular, the
compounds 2a and 4a drastically decreased the tumour growth
upon light exposure, demonstrating their ability to act efficiently
as a PS inside a 3D cellular architecture. Under identical
conditions, the MCTSs treated with H2TPP did not show any
significant effects, highlighting the superior ability of these
compounds.
1
to generate O2 upon irradiation inside the hypoxic centre of the
MCTSs was investigated. For this purpose, HeLa MCTSs were
incubated with 2´,7´-dichlorofluorescein diacetate (DCFH-DA),
which is converted into the highly fluorescent 2´,7´-
dichlorofluorescein in the presence of ROS. While no green
fluorescence signal was observed in the dark, upon 2P irradiation
(800 nm, 2 J/cm2, section interval of 5 μm), a strong signal in the
whole MCTSs was detected (Figure 3). Strikingly, the signal was
To perform a deeper investigation of the phototoxic effect caused
by the Ru(II) polypyridine complexes, the treated MCTSs were
stained with calcein AM and propidium iodide to differentiate
between living and dead cells. While the non-fluorescent calcein
AM is converted to the highly green fluorescent calcein in living
cells, propidium iodide is only able to penetrate dead cells with a
damaged membrane integrity. It can then intercalate into DNA,
causing a strong red fluorescence. As observed during the 2D
monolayer screening, a significant dark cytotoxicity was detected
for compounds 3a and 4a, while 1a and 2a showed no signs of
cell death. Upon 1P or 2P irradiation, the vast majority of the
MCTSs consisted of dead cells, as indicated by the strong red
fluorescence signal of propidium iodide inside the MCTSs (Figure
5). Cellular damage was also caused in the large hypoxic centre,
which remains so far a major challenge during a PDT treatment.
To further study and quantify the (photo-)cytotoxic effect of the
complexes, their IC50 values in MCTSs in the dark as well as upon
1P (500 nm, 10 J/cm2) and 2P (800 nm, 10 J/cm2, section interval
of 5 μm) excitation were determined by measuring their ATP
concentration. The obtained results (Table 1) are in agreement
Figure 3. Confocal fluorescent images of HeLa MCTSs incubated with DCFH-
DA and the compounds 1a-4a (2 μM) kept in the dark and after a 2P irradiation
(800 nm, 2 J/cm2, section interval of 5 μm).
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Figure 5. Representative image of the viability assay with HeLa MCTSs kept in the dark and exposed to light. MCTS were treated with the compounds 1-4 (20 μM)
in the dark for 12 h. After this time, MCTSs were exposed to a 1P (500 nm, 10 J/cm2) or a 2P irradiation (800 nm, 10 J/cm2, section interval of 5 μm). After 2 days,
the cell survival was assessed by measurement of the fluorescence of calcein (λex = 495 nm, λem = 515 nm) and cell death by measurement of the fluorescence of
propidium iodide (λex = 536 nm, λem = 617 nm).
with the tumour growth inhibition assay, showing that the bphen-
coordinated complexes (3a, 4a) have a cytotoxic effect in the low
micromolar range in the dark while the phen-coordinated
complexes (1a, 2a) showed no dark toxicity even up to high
micromolar range (IC50 > 300 μM). Upon exposure to 1P or 2P
irradiation, all compounds were able to cause a phototoxic effect
in the micromolar range (IC50,1P = 3.8 – 32.6 μM, IC50,2P = 0.8 –
27.8 μM). As the lead compound of this study, 2a demonstrated
its remarkable ability as a PS with PI values of >40 upon 1P and
>250 upon 2P irradiation. Further, 2a was also found to be
phototoxic in HeLa MCTSs upon irradiation at 595 nm (60 min,
11.2ꢀJ/cm2) in the low micromolar range (IC50,595nm = 16.7 ± 1.2
μM, PI595nm > 18). Overall, the lead compound 2 was found to have
a two order of magnitudes higher phototoxicity in comparison to
H2TPP, indicating that the compounds reported here can act
using very low drug and light doses.
and the amount of Ru inside each organ determined by ICP-MS
(Figure S51). The compound was rapidly absorbed from the blood
stream, accumulating mainly within the liver and kidney. The
compound also accumulated inside the tumour with
a
concentration maximum 2 h after the injection. Capitalizing on
these results, in vivo PDT experiments were performed using a
1P (500 nm, 10 mW/cm2, 15 min) or 2P irradiation (800 nm, 50
mW, 1 kHz, pulse width 35 fs, 5 s/mm) light treatment on days 1,
4 and 7. Encouragingly, the PDT treated tumours drastically
shrank until they were nearly eradicated. It is important to mention
that while the tumours treated with a 2P light source did not show
a significant growth after the treatment, the tumours treated with
a 1P light source did indicate a small tumour growth. We assume
that this could be caused by the deeper light tissue penetration of
2P irradiation compared to 1P. In contrast, the tumours only
treated by light or with the compound did not show any tumour
inhibition effect (Figures 6a and 6c). The animals treated with the
compound behaved normally, without signs of pain, stress or
discomfort and did not lose or gain weight (Figure 6b). After 17
days (representative picture: Figure S52), all mice were sacrificed
and the tumour and organs separated. As excepted, using a
histological examination with an H&E stain the tumour tissue
showed noticeable pathological alterations (Figure S53) while all
major organs did not show any pathological effect (Figure S54).
Table 1. IC50 values in the dark as well as upon 1P (500 nm, 10 J/cm2) and 2P
(800 nm, 10 J/cm2, section interval of 5 μm) irradiation of 1a-4a as well as of
cisplatin and H2TPP in HeLa 3D MCTS. Average of three independent
measurements. n.d. = not determinable.
dark
>300
1P
32.6 ± 2.5
7.5 ± 0.2
8.9 ± 0.7
3.8 ± 0.4
>100
PI
>9.2
>40.0
3.1
2P
27.8 ± 3.1
1.2 ± 0.3
3.1 ± 0.6
0.8 ± 0.5
>100
PI
>10.8
>250.0
9.0
1
2
>300
3
27.8 ± 3.6
29.3 ± 2.9
>100
4
7.7
36.6
n.d.
H2TPP
cisplatin
n.d.
-
18.6 ± 1.3
-
-
-
For a further investigation, the biological properties of 2 inside a
mouse model was investigated. As the counter ion of a metal
complex can have a significant effect on the overall ability of the
compound including its water solubility or toxicity[27], 2a was
converted into the chloride salt 2b using a counter ion exchange
resin. The biodistribution of the compound inside of nude mice
bearing an adenocarcinomic human alveolar basal epithelial
(A549) tumour upon intravenous tail-injection was time-
dependently (0.5, 1, 2, 4 h) studied. At each time point, the mice
were sacrificed, all major organs collected (i.e., blood, spleen,
intestine, gastric, liver, kidney, lung, heart, brain, tumour), ground
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RESEARCH ARTICLE
Photon excited PDT and is a suitable candidate for further clinical
investigations.
Acknowledgements
We thank Dr. Philippe Goldner for access to state-of-the-art laser
apparatus. This work was financially supported by an ERC
Consolidator Grant PhotoMedMet to G.G. (GA 681679), has
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
National Science Foundation of China (Nos. 21525105 and
21778079 for H.C.) and the 973 Program (No. 2015CB856301 for
H.C.).
Author Contributions: J. K., S. K., H. C. and G. G. were involved
with the design and interpretation of experiments and with the
writing of the manuscript. Chemical, photophysical and biological
cellular experiments were carried out by J.K. Animal testing was
carried out by S. K. Comments on the English writing style were
provided by Y. C. O. The work was supervised by H. C. and G. G.
All authors have given approval to the final version of the
manuscript.
Figure 6. In vivo PDT study of 2b using 1P (500 nm, 10 mW/cm2, 15 min) or 2P
(800 nm, 50 mW, 1 kHz, pulse width 35 fs, 5 s/mm) excitation on nude mice
bearing an adenocarcinomic human alveolar basal epithelial cancer tumour. a)
Tumour growth inhibition curves upon treatment. b) Average body weights of
the tumour-bearing mice. c) Representative photographs of the tumour after
different treatments on day 17. *p<0.05, **p<0.01.
Conclusion
Keywords: Bioinorganic Chemistry
• Medicinal Inorganic
Chemistry • Metals in Medicine • Photodynamic Therapy •
Photosensitizers
In summary, a series of new asymmetric substituted 1,10-
phenenthroline and 4,7-diphenyl-1,10-phenanthroline Ru(II)
polypyridine complexes is presented as efficient PSs for 1- and 2-
Photon PDT. The complexes showed a red shifted 1-Photon
absorption towards and exceptionally strong 2-Photon absorption
in the biological spectral window. They were found to enter cells
by an energy dependent endocytosis mechanism, where the
complexes accumulated primarily in the cytoplasm. Upon
irradiation in various 2D monolayer cancer cell lines, the
complexes caused cell death in the high nanomolar/low
micromolar range by apoptosis utilising the caspase 3/7 pathway.
Following this, the efficiency of the compounds was investigated
in exceptionally large MCTSs with a diameter of 800 μm, which
can better simulate the pathological conditions found in clinically-
relevant tumors. All complexes were able to fully penetrate the
MCTSs and to generate singlet oxygen in their hypoxic centres.
Strikingly, after the treatment, the MCTSs were fully eradicated,
including in their large hypoxic centres. While the lead compound
of this study, 2 was found to be non-toxic in the dark, it is highly
phototoxic in MCTSs in the very low micromolar range upon 1-
Photon (595 nm) or 2-Photon (800 nm) irradiation. Worthy of note,
in comparison to the previously published 2,2´-bipyridine
analogous compounds, these complexes were found to be
photoactive upon even longer wavelengths by 1-Photon
irradiation. In addition, 2 was also able to eradicate an
adenocarcinomic human alveolar basal epithelial tumour inside a
mouse model upon clinically relevant 1-Photon (500 nm) or 2-
Photon (800 nm) excitation, demonstrating its high potential as a
PS. Overall, the compounds presented in this study can overcome
the limitations of currently applied PSs. We strongly believe that
these complexes, especially 2, has great potential for 1- and 2-
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Entry for the Table of Contents
The treatment of large cancer tumours with photodynamic therapy (PDT) remains a major medical challenge due to the problems of
the photosensitizers (PSs) to penetrate a 3D cellular structure and the low oxygen concentrations available in the tumor centre. Herein,
a series of Ru(II) polypyridine complexes is presented as PSs for 1- and 2-Photon PDT for the treatment of the hypoxic centre of
exceptionally large multicellular tumour spheroids.
Institute and/or researcher Twitter usernames:
Johannes Karges: @Johannes_Karges
Gilles Gasser: @GasserGroup
Universite PSL: @psl_univ
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