Rationally designed ruthenium complexes for 1- and 2-photon photodynamic therapy

DOI: 10.1038/s41467-020-16993-0

Source and publish data:

Nature Communications (2020)

Update date:2022-08-11

Topics:

Authors:

Blacque, Olivier

Chao, Hui

Ciofini, Ilaria

Gasser, Gilles

Karges, Johannes

Kuang, Shi

Maschietto, Federica

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Article abstract of DOI:10.1038/s41467-020-16993-0

The use of photodynamic therapy (PDT) against cancer has received increasing attention over recent years. However, the application of the currently approved photosensitizers (PSs) is limited by their poor aqueous solubility, aggregation, photobleaching an

Full text of DOI:10.1038/s41467-020-16993-0

https://doi.org/10.1038/s41467-020-16993-0

OPEN

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G a s s e r

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¹Chimie ParisTech, PSL University, CNRS, Institute of Chemistry for Life and Health Sciences, Laboratory for Inorganic Chemical Biology, 75005 Paris, France.

²MOE Key Laboratory of Bioinorganic and Synthetic Chemistry, School of Chemistry, Sun Yat-sen University, Guangzhou 510275, P.R. China. ³Chimie

ParisTech, PSL University, CNRS, Institute of Chemistry for Life and Health Sciences, Theoretical Chemistry and Modelling, 75005 Paris, France.

✉

⁴Department of Chemistry, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland. email: ceschh@mail.sysu.edu.cn; gilles.

gasser@chimieparistech.psl.eu

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ue to the increasing impact of cancer on the life quality photophysically and biologically evaluated in-depth. Strikingly,

and mortality of humans, increasing research efforts are while being able to overcome the limitations of clinically applied

devoted toward the development of anticancer drugs and PS, the compounds are phototoxic in various 2D monolayer cells,

strategies. Among the most commonly used techniques to ﬁght 3D multicellular tumor spheroids (MCTS) and able to eradicate a

this disease (i.e., surgery, chemotherapy, and radiotherapy), multiresistant tumor inside a mouse model upon clinically rele-

photodynamic therapy (PDT) is receiving increasing attention vant 1P and 2P excitation.

during the last decades. In PDT, a photosensitizer (PS) is acti-

vated upon light irradiation to generate reactive oxygen species

Results

(ROS)^1,2. As the majority of currently approved PSs are based on

Rational design. With the aim of enhancing the absorption

tetrapyrrolic structures (i.e., porphyrin, chlorin, and phthalocya-

properties of Ru(2,2′-bipyridine)₃without deterioration of its 1P

nine), these compounds share similar drawbacks (e.g., poor

absorption properties, the bipyridine ligand was functionalized

aqueous solubility, aggregation, photobleaching, slow clearance

with rigid π conjugated substituents acting as electron-donating

from the body, and hepatotoxicity)^3–5. To overcome these lim-

groups. This is expected to induce a 1P absorption red-shift

itations, there is a need for the development of new classes of PSs.

toward the biological spectral window by intercalation of donor-

Among others, the use of transition metal complexes^6–11and

centered orbitals in the Ru centered frontier orbitals manifold

especially, Ru(II) polypyridine complexes are gaining momentum

(allowing transitions of metal-to-ligand charge transfer (MLCT)

due to their attractive photophysical and chemical properties (i.e.,

and ligand-to-metal charge transfer (LMCT) character at low

strong luminescence, high singlet oxygen production, high che-

energy). As trans-stilbene are highly effective 2P dyes^39,40, it

mical, and photophysical stability)^12–22, with the compound

would be of high interest to extend the ligand scaffold with such

TLD-1433 having just entered phase II clinical trials for the

a moiety as well as to include terminal donor groups, namely

treatment of non-muscle invasive bladder cancer^23–25. Despite

(E,E′)-4,4′-bisstyryl-2,2′-bipyridine (L-H), (E,E′)-4,4′-bis[p-(N,N-

these remarkable properties, the vast majority of Ru(II) poly-

dimethylamino)styryl]-2,2′-bipyridine (L-NMe₂) and (E,E′)-4,4′-

pyridine complexes are excited using either blue or UV-A light.

bis[p-methoxystyryl]-2,2′-bipyridine (L-OMe). The predicted

As the light tissue penetration depth is rather poor at these

UV–vis spectra for the resulting complexes of L-H and the L-

wavelengths, the application of these compounds to treat deep-

OMe series (Supplementary Fig. 1) show the presence of two

seated or large tumors is limited^26–30. To circumvent this draw-

main bands in the 400–600 nm region, stemming from both

back, there is a need for the development of PSs with an

MLCT/LMCT type transitions but also from ligand-centered (LC)

absorption towards the biological spectral window (600–900 nm),

charge-transfer excitations. Detailed analysis of the 1P and of the

which can be achieved by a red-shifted one-photon (1P)

lowest lying 2P computed data (Supplementary Tables 1–4)

absorption or the use of a two-photon (2P) absorption process for

indicated that the most intense lowest lying 2P absorption pro-

2P PDT, a technique that is not employed yet in the clinic.

cesses are associated with LCCT transitions with an average CT

Worthy of note, the use of dihydrolipoic acid-coated gold

distance of 2.9–6.3 Å. Overall, the computed data suggests that Ru

nanoclusters was recently reported as an efﬁcient 2P PDT agent³¹.

(II) coordinated (E,E′)-4,4′-bisstyryl-2,2′-bipyridine complexes

However, the ability of Ru(II) polypyridine complexes to absorb

with donor substituents, have a red-shifted 1P and a strong 2P

2P simultaneously, expressed as the 2P cross-section, remains

absorption.

relatively weak (~40–250 Goeppert-Mayer (GM)), limiting their

applications in this ﬁeld of research^32–38

To tackle these drawbacks, herein, we report the design of Ru

(II) polypyridine complexes with a red-shifted 1P and excep-

tionally strong 2P absorption using an in silico optimization. The

resulting compounds were synthesized, characterized, and

Synthesis and characterization. Before the synthesis of the ﬁnal

compounds (Fig. 1), the experimental procedures to obtain the

corresponding ligands were optimized^41–44to a one-step high-

yielding synthesis using mild conditions (Supplementary Fig. 2).

R = NMe₂

OMe

R = NMe₂

OMe

R = NMe₂

OMe

Fig. 1 Chemical structures of complexes 1–7 investigated in this study.

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Having the ligands in hand, the desired complexes 1–7 were

synthesized. All compounds were analyzed by ¹H, ¹³C-NMR,

ESI-HRMS (Supplementary Figs. 3–25) and their purity con-

ﬁrmed by HPLC as well as elemental analysis. In addition, the

ligands L-H, L-NMe₂, and L-OMe as well as 3 were characterized

using single-crystal X-ray crystallography (Supplementary

Tables 5–6, Supplementary Figs. 26–29).

Photophysical evaluation. The photophysical properties of the

complexes (Table 1, Supplementary Fig. 30) were then experi-

mentally investigated to evaluate their potential as PDT PSs. In

agreement with theoretical ﬁndings, the compounds generally

show a red-shift of the ﬁrst 1P absorption (Fig. 2a) band max-

imum of about 50–70 nm for the symmetric Ru(II) complexes, in

comparison to Ru(2,2′-bipyridine)₃⁴⁵as well as an absorption tail

toward the near-infrared region. Strikingly and in agreement with

theoretical data, the reported compounds have an exceptionally

strong 2P absorption (Fig. 2b) with values up to ~6800 GM,

which is an order of magnitude higher than the ones reported

before for other Ru(II) polypyridine complexes^32–38. Interest-

ingly, the L-NMe₂coordinated complexes were found with larger

σ₂values than the L-OMe and L-H coordinated compounds.

Overall, the compounds have a 1P absorption tail towards, and a

strong 2P absorption, in the biological spectral window, poten-

tially allowing for the treatment of deep-seated or large tumors.

The luminescence quantum yields of the L-OMe coordinated

complexes (3: 1.1%, 5: 1.4%, 7: 2.8%) were found to be

signiﬁcantly higher than those of the L-H coordinated (1: 1.9%)

or L-NMe₂coordinated (2: >0.1%, 4: 0.4%, 6: 0.5%) compounds.

All complexes were found to have excited state lifetimes in the

nanosecond range (Supplementary Figs. 31–37) in degassed

(222–542 ns) and aerated saturated (36–96 ns) solutions. As the

lifetimes drastically decrease in the presence of air, it indicates

that the excited state can interact with a component in the air. For

identiﬁcation of the type of ROS produced upon light exposure,

electron spin resonance (ESR) spectroscopy was employed using

the singlet oxygen (¹O₂) scavenger 2,2,6,6–tetramethylpiperidine

and the ^•OOH or ^•OH radical scavenger 5,5-dimethyl-1-pyrroline

•

N-oxide. While no signals for the formation of OOH or OH

radicals were detected, the formation of O₂in acetonitrile and

PBS was conﬁrmed by observation of the characteristic ¹O₂-

induced triplet signal in the ESR spectrum (Supplementary

Figs. 38–44). The amount of generated ¹O₂was quantitatively

determined by two methods: directly by measurement of the

phosphorescence of O₂; indirectly by monitoring the change in

absorbance of a ¹O₂scavenger. The singlet oxygen quantum

yields were found to be between 16–77% in acetonitrile and

1–11% in an aqueous solution (Table 1). The comparison

between the values indicates that the L-OMe coordinated

complexes (3, 5, 7) are able to produce O₂more efﬁciently than

1 and the L-NMe₂coordinated Ru(II) complexes (2, 4, 6).

Overall, complex 7 was found with the highest singlet oxygen

quantum yield (acetonitrile: 68–77%, aqueous solution: 10–11%).

Stability. The stability of the compounds was investigated by

incubation in human plasma at 37 °C for 48 h. The comparison of

the HPLC chromatograms (Supplementary Figs. 45–51) showed

no change before and after incubation in human plasma for all

compounds, indicative of the stability of the complexes under

biological conditions. Additionally, the stability upon irradiation

was investigated (Supplementary Figs. 52–60) as the majority of

current clinically employed PSs suffer from this drawback.

Importantly, no signiﬁcant differences in the absorption spectra

for the L-OMe coordinated complexes (3, 5, 7) and 1 were

observed over time, indicating their photostability. On the

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Fig. 2 Absorption spectra of the complexes 1–7. a

contrary, small changes in the absorption spectra of the L-NMe₂

After an assessment of the uptake and the generation of O₂

coordinated complexes (2, 4, 6), especially of 2, were observed, upon light exposure in a cuvette, the generation of ROS inside of

suggesting that the complexes are slightly photobleaching. Wor- HeLa cells upon 1P-(488 nm, Supplementary Fig. 80) or 2P- (800

thy of note, under identical experimental conditions, the nm, Supplementary Fig. 81) excitation was conﬁrmed using the

absorption spectra of the known PS Protoporphyrin IX (PpIX) probe 2′, 7′ -dichloroﬂuorescein diacetate. To study their

was found to be drastically changed, indicating a signiﬁcantly efﬁciency as PDT PSs, their cytotoxicity in the dark as well as

stronger photobleaching. The study of the effect of the irradiation upon irradiation at 480 nm (10 min, 5.2 mW cm⁻², 3.1 J cm⁻²) or

on the molecular structure of 2 by NMR spectroscopy suggests 540 nm (40 min, 4.0 mW cm⁻², 9.5 J cm⁻²) toward noncancerous

the decomposition of the compound (Supplementary Fig. 61).

retinal pigment epithelium (RPE-1), HeLa, mouse colon carci-

noma (CT-26), and human glioblastoma astrocytoma (U373)

cells was investigated. Importantly, all complexes were found to

be nontoxic in the dark (IC_50,dark> 100 μM) in all cell lines

(Supplementary Tables 8–9). This is an important requirement

for a PDT agent. As desired, all compounds were found to be

phototoxic in the micromolar range (IC_{50,480 nm}= 0.7

0.4 – 53.6 3.2 μM, IC_{50,540 nm}= 0.9 0.3 – 83.1 6.7 μM) in

the different cell lines employed in this study. As the lead

compound of this study, complex 7 had an IC₅₀value in the

Biological evaluation on 2D monolayer cells. All compounds

were found to be lipophilic with high distribution coefﬁcients

between an organic octanol and an aqueous PBS phase (Supple-

mentary Table 7). The time-dependent cellular uptake of the

complexes was then investigated in human cervical carcinoma

(HeLa) cells by determining the amount of Ru inside the cells by

inductively coupled plasma mass spectrometry (ICP-MS) at each

time point. The results show that the asymptotic maximum

(Supplementary Figs. 62–68) of the uptake of the compounds was

reached within 8 h. As expected, the compounds with a higher

lipophilicity, with the exception of 2, were found to have the

highest uptake (Supplementary Fig. 69). The uptake mechanism

of the complexes was then investigated by blocking various

pathways by preincubation with a cationic transporter (tetra-

ethylammonium chloride), metabolic (2-deoxy-D-glucose and

oligomycin), and endocytotic inhibitors (ammonium chloride or

chloroquine) as well as at reduced temperature (4 °C). Since all

compounds were found with a similar proﬁle (Supplementary

Figs. 70–76), it suggests that these are internalized by the same

mechanism, namely an energy-dependent endocytosis pathway.

nanomolar range in CT-26 cells (IC_50,dark> 100 μM, IC_{50,480 nm}

0.7 0.4 μM, IC_{50,540 nm}= 0.9 0.3 μM) with a PI value > 143.

Under identical experimental conditions, the anticancer drug

cisplatin and the well-known PS PpIX display a magnitude lower

(photo-)toxicity. The cell death mechanism of the complexes was

then evaluated by measuring the cell viability upon preincubation

with autophagy (3-methyladenine), apoptosis (Z-VAD-FMK),

paraptosis (cycloheximide) and necrosis (necrostatin-1) inhibi-

tors (Supplementary Fig. 82). While apoptosis was found to be

the cell death mechanism for 1–5, 6–7 triggered cell death by a

combination of apoptosis and paraptosis pathways.

The localization of the complexes in HeLa cells was then inves- Biological evaluation on 3D multicellular tumor spheroids.

tigated by confocal laser scanning microscopy. The distribution After evaluation of the biological effects of the compounds on 2D

pattern of the luminescence of the compounds by 1P-(Supple- monolayer cells, their ability to act on 3D MCTS was investigated.

mentary Fig. 77) or 2P-(Supplementary Fig. 78) excitation was MCTS simulate the conditions found in clinically treated tumors

compared with the ones of commercial dyes for major cellular including hypoxia and proliferation gradients to the center⁴⁶.

organelles (i.e., nucleus, mitochondria, lysosomes, golgi appara- Consequently, the penetration of the compounds inside of HeLa

tus, and endoplasmic reticulum). As no signiﬁcant congruency MCTS with a diameter of 800 μm was investigated by 1P and 2P

was detected, it suggests that the complexes do not majorly z-stack confocal laser scanning microscopy. 4–7 completely

localize in these organelles. In addition, the cellular localization penetrated the MCTS within 12 h with a strong luminescence

was also investigated by separately extracting the cellular orga- signal at every section depth, whereas 1–3 were mostly found on

nelles (i.e., cytoplasm, mitochondria, lysosome, and nucleus) and the outer sphere (Supplementary Figs. 83–89). Upon an increased

determining the amount of Ru inside each organelle by ICP-MS. incubation time up to 60 h, the remaining compounds were also

The results (Supplementary Fig. 79) indicate that all complexes able to penetrate the MCTS, with the exception of 2, which

majorly localize in the cytoplasm with a small amount of unse- was still found mostly on the outer sphere (Supplementary

lective accumulation.

Figs. 90–94). Following this, the tumor growth inhibition effect of

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Time/day

Fig. 3 Tumor growth inhibition assay in HeLa MCTS.

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Fig. 4 Representative image of the cell viability assay in HeLa MCTS.

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1–7 (20 μM) in HeLa MCTS was investigated and compared with 1P or 2P irradiation (Fig. 3b, c, representative image of MCTS:

the well-known PS tetraphenylporphyrin (H₂TPP) (20 μM) and Supplementary Figs. 96–97) signiﬁcantly shrank, demonstrating

cisplatin (10 μM, 30 μM). The compounds were incubated for their strong tumor inhibition effect.

3 days in the dark as this time was shown to be required for

To further study the effect the complexes have on the tumor

complete MCTS penetration. The MCTS were then exposed to 1P survival, the treated MCTS were stained with a cellular living cell

(500 nm, 16.7 min, 10.0 mW cm⁻², and 10 J cm⁻²) or 2P irra- kit using Calcein AM (Fig. 4). The ﬂuorescence images conﬁrmed

diation (800 nm, 10 J cm⁻², and section interval of 5 μm) on day that the MCTS treated with 1–7 in the dark and with 1–3 upon a

3. During the whole time period, the shape and volume of the 1P irradiation (500 nm, 16.7 min, 10.0 mW cm⁻², and 10 J cm⁻²

)

MCTS was constantly monitored. As expected, the MCTS treated or 2P irradiation (800 nm, 10 J cm⁻², and section interval of 5

with 1–7 or H₂TPP in the dark (Fig. 3a, representative image of μm) are still intact. The low phototoxic effect of 1–3 is caused by

MCTS: Supplementary Fig. 95) were asymptotically growing in the poor MCTS penetration. Promisingly, the MCTS treated with

the same manner than the control group, indicating that the 4–7 and exposed to 1P or 2P light were completely eradicated.

compounds do not show any inhibitory effect, whereas cisplatin

The photodynamic effect using a 1P (500 nm, 16.7 min,

showed a weak effect on the tumor growth. On the contrary, the 10.0 mW cm⁻², and 10 J cm⁻²) and 2P irradiation (800 nm,

volume of the MCTS treated with complexes 1–7 and exposed to 10 J cm⁻², and section interval of 5 μm) was then quantiﬁed by

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determination of the ATP concentration (Supplementary to generate ¹O₂, causing a phototoxic effect by apoptosis and

Table 10). Importantly, no measurable cytotoxicity in the dark paraptosis pathways in various monolayer cells as well as MCTS.

could be observed in HeLa MCTS for all compounds and 1–3 In vivo studies conﬁrmed the impressive ability of 7 to act as a PS

upon light exposure in agreement with the previous investiga- upon treatment at clinically relevant 1P (500 nm) and 2P (800

tions. 4–6 were found to be phototoxic in the micromolar range nm) irradiation with an eradication of the tumor. Importantly,

(IC_{50,500 nm}= 6.8 0.2 – 78.3 5.1 μM, IC_{50,800 nm}= 1.4 0.2 – these compounds were found to be highly water soluble, stable in

87.3 6.8 μM). Strikingly, the lead compound of this study 7 human plasma, as well as upon constant irradiation and therefore

was found to be nontoxic in the dark at even higher are able to overcome the limitations of currently employed PSs.

concentrations (IC_50,dark> 300 μM), while being highly photo- We strongly believe that these complexes have great potential for

toxic in the low micromolar range (IC_{50,500 nm}> 6.8 0.2 μM, preclinical trials.

IC_{50,800 nm}> 1.4 0.2) with exceptionally high PI values (PI_{500 nm}

> 44, PI_{800 nm}> 250). Importantly, under identical experimental

conditions, treatment with H₂TPP did not show a cytotoxic effect Computational details. All calculations were performed at DFT (ground state)

(IC_{50, dark}= IC_{50,500 nm}= IC_{50,800 nm}> 100 μM), indicating that 7

is able to act at low drug and light doses compared to a clinically

Methods

and TD-DFT (excited state) level using the global hybrid B3LYP functional. The

Los Alamos (LANL2) effective core potential and the corresponding triple-zeta

basis set was applied to describe the Ruthenium atom, with all other atoms were

utilized tetrapyrrolic compound.

described by a Pople double-zeta basis set with a single set of polarization functions

on non-hydrogen atoms (6–31G(d)). All structures used correspond to ground

state minima on the ground state potential energy surfaces. Solvent effect (acet-

onitrile) were taken into account using the PCM model for structural optimizations

while single point calculations in the gas phase were performed to simulate 1P and

2P absorption spectra. All calculations were performed using the Gaussian suite of

programs except for the 2PA spectra which were simulated using the DALTON

software.

Biological evaluation inside a mouse model. Capitalizing on the

superior photophysical (i.e., highest O₂production) and biolo-

gical properties (i.e., highest phototoxicity in 2D monolayer cells

and 3D MCTS, high cell penetration), the properties of 7 to act as

a PDT PS were further investigated in a mouse model. To ensure

solubility and biocompatibility, the PS was converted to a chloride

salt using a counter ion exchange resin. We note at this stage that

a change of the counter ion can have a signiﬁcant effect on the

biological properties of a metal complex. It was previously shown

that a counter ion can cause an undesired cytotoxicity or the

formation of nanoaggregates or a colloid due to poor water

Materials. All chemicals were purchased from commercial sources and used

without further puriﬁcation. If necessary, solvents were dried over molecular sieves.

The Ru(II) precursors Ru(DMSO)₄Cl₂and Ru(2,2′-bipyridine)₂Cl₂were syn-

thesized according to previously reported procedures. The pooled human plasma

was obtained from Biowest. The cell culture media and reagents were purchased

from Fisher Scientiﬁc.

solubility⁴⁷. It was also reported that the cellular uptake of a Ru(II) Instrumentation and methods. ¹H and ¹³C-NMR spectra were measured on a

complex can dramatically change⁴⁸. A challenging to treat mul-

Bruker 400 MHz or 500 MHz NMR spectrometer. Chemical shifts (δ) are reported

in parts per million (ppm) referenced to tetramethylsilane (δ 0.00) ppm using the

residual proton solvent peaks as internal standards and coupling constants (J) in

tiresistant doxorubicin-selected P-gp-overexpressing human colon

cancer tumor model (SW620/AD300) was used to evaluate the PS

potential. The biodistribution of 7 (2 mg kg⁻¹) inside this mouse

model was then time-dependently (30 min, 1 h, 2 h) studied by

intravenous tail injection into nude mice. After each time point,

the mice were sacriﬁced and the major organs (i.e., blood, spleen,

intestine, stomach, liver, kidney, uterus, lung, heart, brain, and

tumor) were separated, ground and the Ru content determined by

ICP-MS. Interestingly, 7 was absorbed from the blood stream

within 1 h with a high accumulation in the intestine and some

accumulation in the tumor (Supplementary Fig. 98). Following

this, 1 h after an intravenous tail injection of 7 (2 mg kg⁻¹), in vivo

Hertz (Hz). The multiplicity of the peaks is abbreviated as follows: s (singlet), d

(doublet), dd (doublet of doublet), t (triplet), m (multiplet). ESI-MS experiments

were carried out using a LTQ-Orbitrap XL from Thermo Scientiﬁc and operated in

positive ionization mode with a spray voltage of 3.6 kV. No Sheath and auxiliary

gas was used. Applied voltages were 40 and 100 V for the ion transfer capillary and

the tube lens, respectively. The ion transfer capillary was held at 275 °C. Detection

was achieved in the Orbitrap with a resolution set to 100,000 (at m z⁻¹400) and

a m z⁻¹range between 150 and 2000 in proﬁle mode. Spectrum was analyzed using

the acquisition software XCalibur 2.1 (Thermo Fisher Scientiﬁc). The automatic

gain control allowed accumulation of up to 2 × 10⁵ions for FTMS scans, maximum

injection time was set to 300 ms and 1 µscan was acquired. Ten microliters was

injected using a Thermo Finnigan Surveyor HPLC system (Thermo Fisher Scien-

tiﬁc) with a continuous infusion of methanol at 100 µL min⁻¹. Elemental micro-

analyses were performed on a Thermo Flash 2000 elemental analyzer. For analytic

and preparative HPLC the following system has been used: 2 × Agilent G1361 1260

Prep Pump system with Agilent G7115A 1260 DAD WR Detector equipped with

an Agilent Pursuit XRs 5C18 (Analytic: 100 Å, C18 5 μm, 250 × 4.6 mm, Pre-

PDT experiments using a 1P (500 nm, 60 min, 10.0 mW cm⁻²

and 36 J cm⁻²) or 2P irradiation (800 nm, 50 mW, 1 kHz, pulse

width 35 fs, and 5 s mm⁻¹) were performed on mice with an

80 mm³tumor. Encouragingly, after only one PDT treatment, the parative: 100 Å, C18 5 μm 250 × 300 mm) column and an Agilent G1364B 1260-FC

fraction collector. The solvents (HPLC grade) were millipore water (0.1% tri-

ﬂuoroacetic acid, solvent A) and acetonitrile (0.1% triﬂuoroacetic acid, solvent B).

ICP-MS experiments were carried out on an iCAP RQ ICP-MS instrument

tumor drastically shrank until they were nearly eradicated whereas

the tumors treated with the light or 7 in the dark (Fig. 5a, c) kept

growing. Importantly, the animals treated with the compound

behaved normally, without signs of pain, stress or discomfort and

did not lose or gain weight (Fig. 5b). After the treatment, the mice

were sacriﬁced, the tumor and organs were separated and histo-

logically examined by an H&E stain. The tumor tissue treated with

7 and exposed to light displayed pathological alterations caused by

the PDT treatment (Supplementary Fig. 99), while all other organs

did not show any signiﬁcant effect (Supplementary Fig. 100).

Overall, this study demonstrates the enormous potential of 7 as a

PS for 1P and 2P PDT.

In summary, Ru(II) polypyridine complexes with (E,E′)-4,4′-

bisstyryl-2,2′-bipyridine ligands have been rationally designed

using DFT calculations and were found to have a remarkable red-

shifted 1P absorption as well as exceptionally high 2P cross

sections. The complexes were found to be taken up by cancerous

cells through an energy-dependent endocytosis pathway and to

(Thermo Fisher).

Synthesis. (E,E′)-4,4′-Bisstyryl-2,2′-bipyridine: The synthesis of (E,E′)-4,4′-Bis-

styryl-2,2′-bipyridine is already published but in this study another synthetic route

was employed. 4,4′-Dimethyl-2,2′-bipyridine (1000 mg, 5.43 mmol, 1.0 equiv.) was

dissolved in dry N,N-dimethylformamide under nitrogen atmosphere and ben-

zaldehyde (1.2 mL, 11.84 mmol, 2.2 equiv.) was added to the solution. Afterwards

potassium tert-butoxide (2436 mg, 21.72 mmol, 4.0 equiv.) was added slowly. The

color of the solution turned to green and the mixture was stirred for 24 h. After that

the mixture was poured into water (400 mL) and the suspension cooled down to

5 °C. The crude product which precipitated, was ﬁltered and washed with

methanol. The product was puriﬁed by recrystallization from boiling acetic acid.

The obtained solid was dissolved in dichloromethane and the mixture was washed

with a 5% aqueous lithium chloride solution, brine and water. The solvent was

removed and the product was isolated by recrystallization from boiling acetic acid.

1292 mg of (E,E′)-4,4′-Bisstyryl-2,2′-bipyridine (3.58 mmol, 66%) were yielded as a

beige solid. The experimental data obtained is in agreement with the previous

literature⁵¹

(E,E′)-4,4′-Bis[p-(N,N-dimethylamino)styryl]-2,2′-bipyridine: The synthesis of

accumulate in the cytoplasm. Upon irradiation, they were found (E,E′)-4,4′-Bis[p-(N,N-dimethylamino)styryl]-2,2′-bipyridine is already published but

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a ₂₀₀₀

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7 + 1P

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Fig. 5 Biological Evaluation of 7 inside a mouse model.

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)

(

)

. a

ﬁ

in this study another synthetic route was employed. 4,4′-Dimethyl-2,2′-bipyridine

(1000 mg, 5.43 mmol, 1.0 equiv.) was dissolved in dry N,N-dimethylformamide

(100 mL) under nitrogen atmosphere and potassium tert-butoxide (2437 mg, 21.72

added to the solution. Afterwards potassium tert-butoxide (1360 mg, 12.13 mmol,

4.2 equiv.) was added slowly. The color of the solution turned to green and the

mixture was stirred for 24 h. After that the mixture which turned bright was poured

into water (200 mL) and the suspension cooled down to 5 °C. The crude product

which precipitated, was ﬁltered, and washed with methanol. The product was

puriﬁed by recrystallization from boiling acetic acid. The obtained solid was

dissolved in dichloromethane and the mixture was washed with a 5% aqueous

lithium chloride solution, brine and water. The solvent was removed and the

product was isolated by recrystallization from boiling acetic acid. 925 mg of (E,E′)-

4,4′-Bis[p-methoxystyryl]-2,2′-bipyridine (2.20 mmol, 76%) were yielded as a beige

solid. The experimental data obtained is in agreement with the previous

mmol, 4.0 equiv.) was added slowly. After 1.5 h of stirring, 4-(dimethylamino)

benzaldehyde (1701 mg, 11.40 mmol, 2.1 equiv.) was added to the reaction mixture.

The color of the solution turned to yellow and the mixture was heated at 90 °C for

19 h. After that the mixture was poured into water (400 mL) and the suspension

cooled down to 5 °C. The crude product which precipitated, was ﬁltered and washed

with water and diethyl ether. The product was isolated by recrystallization from

dichloromethane/pentane. 1541 mg of (E,E′)-4,4′-Bis[p-(N,N-dimethylamino)styryl]-

2,2′-bipyridine (3.45 mmol, 64%) were yielded as a yellow solid. The experimental

data obtained is in agreement with the previous literature⁵²

literature⁵²

(E,E′)-4,4′-Bis[p-methoxystyryl]-2,2′-bipyridine: The synthesis of (E,E′)-4,4′-Bis

[p-methoxystyryl]-2,2′-bipyridine is already published but in this study another

synthetic route was employed. 4,4′-Dimethyl-2,2′-bipyridine (532 mg, 2.89 mmol,

1.0 equiv.) was dissolved in dry N,N-dimethylformamide (25 mL) under nitrogen

atmosphere and 4-methoxybenzaldehyde (0.88 mL, 7.22 mmol, 2.5 equiv.) was

[Ru(E,E′)-4,4′-Bisstyryl-2,2′-bipyridine)₃][PF₆]₂(1): (E,E′)-4,4′-Bisstyryl-2,2′-

bipyridine (400 mg, 1.11 mmol, 4.0 equiv.) and Ru(DMSO)₄Cl₂(134 mg, 0.28

mmol, 1.0 equiv.) were suspended in dry ethanol (150 mL) under nitrogen

atmosphere and the mixture was reﬂuxed for 24 h. Then the solution was cooled

down and undissolved residue was removed by ﬁltration. To the residual solution a

(

)

saturated aqueous solution of ammonium hexaﬂuorophosphate was added. The

crude product, which precipitated as a hexaﬂuorophosphate salt was collected by

0% A (100% B). The ﬂow rate was 20 mL min⁻¹and the chromatogram was

detected at 250, 350, and 450 nm. The collected product was dissolved in water and

a saturated aqueous solution of ammonium hexaﬂuorophosphate was added. The

product, which precipitated as a hexaﬂuorophosphate salt was collected by

ﬁltration and washed with water, diethyl ether and pentane. 89 mg of 4 (0.06 mmol,

24%) were yielded as a dark red solid. ¹H-NMR (400 MHz, CD₃CN): δ 8.61 (s, 4H),

8.50 (d, J = 8.2 Hz, 2H), 8.04 (td, J = 8.0, 1.5 Hz, 2H), 7.86 (ddd, J = 5.7, 1.4, 0.6 Hz,

2H), 7.66 (dd, J = 16.2, 1.9 Hz, 4H), 7.64 (d, J = 6.3 Hz, 2H), 7.56–7.50 (m, 10H),

7.43–7.34 (m, 6H), 7.02 (dd, J = 16.2 Hz, 4H), 6.81–6.77 (m, 8H), 3.02 (s, 12H),

3.01 (s, 12H); ¹³C-NMR (100 MHz, CD₃CN): δ 158.1, 158.0, 152.7, 152.6, 151.9,

151.9, 148.5, 138.3, 137.9, 130.0, 128.4, 125.1, 124.4, 120.7,119.6, 113.2, 40.4; HRMS

(m/z): [M]²⁺calcd. for C₇₀H₆₈N₁₀Ru, 575.2330; found, 575.2347; analysis (calcd.,

found for C₇₀H₆₈F₁₂N₁₀P₂Ru): C (58.37, 58.19), H (4.76, 4.62), N (9.72, 9.72).

[Ru(2,2′-bipyridine)((E,E′)-4,4′-Bis[p-methoxystyryl]-2,2′-bipyridine)₂][PF₆]₂(5):

(E,E′)-4,4′-Bis[p-methoxystyryl]-2,2′-bipyridine (490 mg, 1.17 mmol, 2.0 equiv.),

Ru(DMSO)₄Cl₂(282 mg, 0.58 mmol, 1.0 equiv.), and lithium chloride (2470 mg,

58.26 mmol, 100 equiv.) were suspended in dry N,N-dimethylformamide (75 mL)

under nitrogen atmosphere. The mixture was reﬂuxed for 6 h. The solution was

then cooled down and purged into water. The crude product, which precipitated

was collected by ﬁltration and washed with water and diethyl ether. The formation

of [Ru((E,E′)-4,4′-Bis[p-methoxystyryl]-2,2′-bipyridine)₂Cl₂] was analyzed via

HPLC. [Ru((E,E′)-4,4′-Bis[p-methoxystyryl]-2,2′-bipyridine)₂Cl₂] and 2,2′-

bipyridine (109 mg, 0.70 mmol, 1.2 equiv.) were suspended in dry ethanol (100 mL)

under nitrogen atmosphere. The mixture was reﬂuxed for 6 h. The solution was

then cooled down and undissolved solid was removed by ﬁltration. A saturated

aqueous solution of ammonium hexaﬂuorophosphate was added and the crude

product, which precipitated as a hexaﬂuorophosphate salt was collected by

centrifugation. The solid was washed with water and diethyl ether. The residue was

puriﬁed via preparative HPLC as a triﬂuoroacetic acid salt. The solvents were

millipore water with 0.1% triﬂuoroacetic acid (solvent A) and acetonitrile (solvent

B). The following HPLC gradient has been used: 0–3 min: isocratic 50% A (50% B);

3–17 min: linear gradient from 50% A (50% B) to 0% A (100% B); 17–23 min:

isocratic 0% A (100% B). The ﬂow rate was 20 mL min⁻¹and the chromatogram

was detected at 250, 350, and 450 nm. The collected product was dissolved in water

and a saturated aqueous solution of ammonium hexaﬂuorophosphate was added.

The product, which precipitated as a hexaﬂuorophosphate salt was collected by

ﬁltration and washed with water, diethyl ether and hexane. 248 mg of 5 (0.18

mmol, 31%) were yielded as a dark red solid. ¹H-NMR (400 MHz, CD₃CN): δ 8.72

(d, J = 1.6 Hz, 4H), 8.51 (d, J = 8.2 Hz, 2H), 8.06 (td, J = 8.0, 1.3 Hz, 2H), 7.85 (dd,

J = 5.6, 1.1 Hz, 2H), 7.75–7.68 (m, 6H), 7.67–7.61 (m, 8H), 7.60 (d, J = 5.9 Hz, 2H),

7.46–7.39 (m, 6H), 7.17 (dd, J = 16.4, 2.1 Hz, 4H), 7.05–6.99 (m, 8H), 3.85 (s, 6H),

3.84 (s, 6H); ¹³C-NMR (100 MHz, CD₃CN): δ 162.0, 158.1, 152.6, 152.2, 148.1,

138.6, 137.1, 132.8, 130.1, 129.5, 128.5, 125.2, 125.0, 122.7, 121.3, 115.5, 56.2;

HRMS (m/z): [M]²⁺calcd. for C₆₆H₅₆N₆O₄Ru, 549.1698; found, 549.1707; analysis

(calcd., found for C₆₆H₅₆F₁₂N₆O₄P₂Ru + C₆H₁₄): C (57.96, 58.33), H (4.44, 4.08),

N (5.87, 5.79).

centrifugation and washed with ethanol, water and diethyl ether. The product was

dissolved in dichloromethane and washed with a 5% aqueous lithium chloride

solution, brine and water. After drying, 323 mg of 1 (0.22 mmol, 79%) were yielded

as a red solid. ¹H-NMR (500 MHz, CD₃CN): δ 8.76 (d, J = 1.8 Hz, 6H), 7.78 (d, J =

16.4 Hz, 6H), 7.76 (d, J = 5.9 Hz, 6H), 7.71–7.69 (m, 12H), 7.51 (dd, J = 5.9, 1.8 Hz,

6H), 7.49–7.46 (m, 12H), 7.44–7.40 (m, 6H), 7.33 (d, J = 16.4 Hz, 6H); ¹³C-NMR

(125 MHz, CD₃CN): δ 158.2, 152.4, 147.6, 137.3, 136.8, 130.6, 130.1, 128.4, 125.4,

125.1, 121.7; HRMS (m/z): [M]²⁺calcd. for C₇₈H₆₀N₆Ru, 591.1956; found,

591.1978; analysis (calcd., found for C₇₈H₆₀F₁₂N₆P₂Ru + 4^.H₂O): C (60.66, 60.62),

H (4.44, 4.43), N (5.44, 5.76). [Ru(E,E′)-4,4′-Bisstyryl-2,2′-bipyridine)₃][Cl]₂: The

counter ion hexaﬂuorophosphate was exchanged to chloride by elution with

methanol from the ion exchange resin Amberlite IRA-410. Analysis (calcd., found

for C₇₈H₆₀Cl₂N₆Ru): C (74.75, 74.36), H (4.83, 4.51), N (6.71, 6.37).

[Ru((E,E′)-4,4′-Bis[p-(N,N-dimethylamino)styryl]-2,2′-bipyridine)₃][PF₆]₂(2):

(E,E′)-4,4′-Bis[p-(N,N-dimethylamino)styryl]-2,2′-bipyridine (338 mg, 0.76 mmol,

4.0 equiv.), Ru(DMSO)₄Cl₂(92 mg, 0.19 mmol, 1.0 equiv.) and lithium chloride

(401 mg, 9.46 mmol, 50.0 equiv.) were dissolved in dry N,N-dimethylformamide

(50 mL) under nitrogen atmosphere. The mixture was reﬂuxed for 48 h. The

solution was then cooled down and a saturated aqueous solution of ammonium

hexaﬂuorophosphate was added. The crude product, which precipitated as a

hexaﬂuorophosphate salt was collected by centrifugation and washed with ethanol,

water and diethyl ether. The residue was dissolved in dichloromethane and washed

with a 5% aqueous lithium chloride solution, brine and water. The solvent was

removed under reduced pressure and the crude product recrystallized from

dichloromethane/pentane. The product was isolated via fractionated precipitation

from acetonitrile by adding dropwise diethyl ether. 86 mg of 2 (0.05 mmol, 26%)

were yielded as a black solid. ¹H-NMR (500 MHz, CD₃CN): δ 8.62 (d, J = 1.9 Hz,

6H), 7.66 (d, J = 16.2 Hz, 6H), 7.65 (d, J = 6.1 Hz, 6H), 7.55–7.52 (m, 12H), 7.38

(dd, J = 6.1, 1.9 Hz, 6H), 7.03 (d, J = 16.2 Hz, 6H), 6.81–6.78 (m, 12H), 3.01 (s,

36H); ¹³C-NMR (125 MHz, CD₃CN): δ 159.4, 158.1, 152.6, 148.3, 137.7, 130.0,

124.5, 124.4, 120.6, 119.7, 113.2, 40.4; HRMS (m/z): [M]²⁺calcd. for C₉₀H₉₀N₁₂Ru,

720.3222; found, 720.3247; analysis (calcd., found for C₉₀H₉₀F₁₂N₁₂P₂Ru): C

(62.46, 62.54), H (5.24, 5.17), N (9.71, 9.79). [Ru((E,E′)-4,4′-Bis[p-(N,N-

dimethylamino)styryl]-2,2′-bipyridine)₃][Cl]₂: The counter ion

hexaﬂuorophosphate was exchanged to chloride by elution with methanol from the

ion exchange resin Amberlite IRA-410. Analysis (calcd., found for

C₉₀H₉₀Cl₂N₁₂Ru): C (71.51, 71.19), H (6.00, 5.93), N (11.12, 10.84).

[Ru((E,E′)-4,4′-Bis[p-methoxystyryl]-2,2′-bipyridine)₃][PF₆]₂(3): (E,E′)-4,4′-Bis

[p-methoxystyryl]-2,2′-bipyridine (286 mg, 0.68 mmol, 4.0 equiv.) and Ru

(DMSO)₄Cl₂(82 mg, 0.17 mmol, 1.0 equiv.) were suspended in dry ethanol (50

mL) under nitrogen atmosphere. The mixture was reﬂuxed for 15 h. The solution

was then cooled down and undissolved solid was removed by ﬁltration. A saturated

aqueous solution of ammonium hexaﬂuorophosphate was added and the crude

product, which precipitated as a hexaﬂuorophosphate salt was collected by

ﬁltration. The solid was washed with water and diethyl ether. The residue was

puriﬁed via fractionated precipitation from acetonitrile by adding dropwise diethyl

ether. The collected product was dissolved in dichloromethane and washed with a

5% aqueous lithium chloride solution, brine and water. After drying, 218 mg of 3

(0.13 mmol, 76%) were yielded as a black solid. ¹H-NMR (500 MHz, CD₃CN): δ

8.79 (d, J = 1.7 Hz, 6H), 7.79 (d, J = 16.4 Hz, 6H), 7.71 (d, J = 6.0 Hz, 6H), 7.64 (d,

J = 8.9 Hz, 12H), 7.44 (d, J = 6.0, 1.7 Hz, 6H), 7.17 (d, J = 16.4 Hz, 6H), 7.00 (d,

J = 8.9 Hz, 12H), 3.83 (s, 18H). ¹³C-NMR (125 MHz, CD₃CN): δ 162.0, 158.2,

152.2, 148.0, 137.0, 130.1, 129.5, 125.0, 122.7, 121.3, 115.5, 56.2; HRMS (m/z):

[M]²⁺calcd. for C₈₄H₇₂N₆O₆Ru, 681.2290; found, 681.2273; analysis (calcd.,

found for C₈₄H₇₂F₁₂N₆O₆P₂Ru): C (61.05, 61.17), H (4.39, 4.44), N (5.09, 5.21).

[Ru((E,E′)-4,4′-Bis[p-methoxystyryl]-2,2′-bipyridine)₃][Cl]₂: The counter ion

hexaﬂuorophosphate was exchanged to chloride by elution with methanol from the

ion exchange resin Amberlite IRA-410. Analysis (calcd., found for

[Ru(2,2′-bipyridine)₂((E,E′)-4,4′-Bis[p-(N,N-dimethylamino)styryl]-2,2′-

bipyridine)][PF₆]₂(6): Ru(2,2′-bipyridine)₂Cl₂(350 mg, 0.72 mmol, 1.0 equiv.) and

(E,E′)-4,4′-Bis[p-(N,N-dimethylamino)styryl]-2,2′-bipyridine (388 mg, 0.87 mmol,

1.2 equiv.) were suspended in dry ethanol (50 mL) under nitrogen atmosphere and

the mixture was reﬂuxed for 6 h. Then the solution was cooled down and a

saturated aqueous solution of ammonium hexaﬂuorophosphate was added. The

crude product, which precipitated as a hexaﬂuorophosphate salt was collected by

ﬁltration and washed with water and diethyl ether. The product was isolated via

fractionated precipitation from acetonitrile by adding dropwise diethyl ether. 449

mg of 6 (0.39 mmol, 54%) were yielded as a dark red solid. ¹H-NMR (500 MHz,

CD₃CN): δ 8.62 (d, J = 1.7 Hz, 2H), 8.50 (d, J = 8.2 Hz, 4H), 8.07–8.02 (m, 4H),

7.87–7.84 (m, 2H), 7.75–7.72 (m, 2H), 7.67 (d, J = 16.3 Hz, 2H), 7.57–7.52 (m, 4H),

7.52–7.49 (d, J = 6.0 Hz, 2H), 7.44–7.34 (m, 6H), 7.02 (d, J = 16.3 Hz, 2H),

6.81–6.76 (m, 4H), 3.01 (s, 12H); ¹³C-NMR (125 MHz, CD₃CN): δ 158.1, 158.0,

158.0, 152.7, 152.7, 152.6, 151.9, 148.8, 138.6, 138.0, 130.0, 128.5, 128.5, 125.2,

124.4, 124.3, 120.8, 119.5, 113.1, 40.4; HRMS (m/z): [M]²⁺calcd. for C₅₀H₄₆N₈Ru,

430.1439; found, 430.1441; analysis (calcd., found for C₅₀H₄₆F₁₂N₈P₂Ru): C (52.22,

51.97), H (4.03, 4.04), N (9.74, 9.71).

[Ru(2,2′-bipyridine)₂((E,E′)-4,4′-Bis[p-methoxystyryl]-2,2′-bipyridine)][PF₆]₂(7):

Ru(2,2′-bipyridine)₂Cl₂(432 mg, 0.89 mmol, 1.0 equiv.) and (E,E′)-4,4′-Bis[p-

methoxystyryl]-2,2′-bipyridine (450 mg, 1.07 mmol, 1.2 equiv.) were suspended in

dry ethanol (100 mL) under nitrogen atmosphere and the mixture was reﬂuxed for

6 h. Then the solution was cooled down and a saturated aqueous solution of

ammonium hexaﬂuorophosphate was added. The crude product, which

precipitated as a hexaﬂuorophosphate salt was collected by ﬁltration and washed

with water and diethyl ether. The product was isolated via fractionated

C₈₄H₇₂Cl₂N₆O₆Ru): C (70.38, 70.62), H (5.06, 5.28), N (5.86, 5.57).

[Ru(2,2′-bipyridine)((E,E′)-4,4′-Bis[p-(N,N-dimethylamino)styryl]-2,2′-

bipyridine)₂][PF₆]₂(4): (E,E′)-4,4′-Bis[p-(N,N-dimethylamino)styryl]-2,2′-

bipyridine (220 mg, 0.49 mmol, 2.0 equiv.), Ru(DMSO)₄Cl₂(119 mg, 0.25 mmol,

1.0 equiv.), and lithium chloride (1044 mg, 24.63 mmol, 100 equiv.) were

suspended in dry N,N-dimethylformamide (30 mL) under nitrogen atmosphere.

The mixture was reﬂuxed for 4 h. The solution was then cooled down and water

was added. The crude product, which precipitated was collected by ﬁltration and

washed with water and diethyl ether. The formation of [Ru((E,E′)-4,4′-Bis[p-(N,N-

dimethylamino)styryl]-2,2′-bipyridine)₂Cl₂] was analyzed via HPLC. [Ru((E,E′)-

4,4′-Bis[p-(N,N-dimethylamino)styryl]-2,2′-bipyridine)₂Cl₂] and 2,2′-bipyridine

(47 mg, 0.3 mmol, 1.2 equiv.) were suspended in dry ethanol (50 mL) under

nitrogen atmosphere. The mixture was reﬂuxed for 7 h. The solution was then

cooled down and undissolved solid was removed by ﬁltration. A saturated aqueous

solution of ammonium hexaﬂuorophosphate was added and the crude product,

which precipitated as a hexaﬂuorophosphate salt was collected by ﬁltration. The

solid was washed with water and diethyl ether. The residue was puriﬁed via

preparative HPLC as a triﬂuoroacetic acid salt. The solvents were millipore water

with 0.1% triﬂuoroacetic acid (solvent A) and acetonitrile (solvent B). The

following HPLC gradient has been used: 0–3 min: isocratic 50% A (50% B); 3–17

min: linear gradient from 50% A (50% B) to 0% A (100% B); 17–23 min: isocratic

precipitation from acetonitrile by adding dropwise diethyl ether. 358 mg of 7 (0.32

mmol, 36%) were yielded as a dark red solid. ¹H-NMR (500 MHz, CD₃CN): δ 8.71

(d, J = 1.4 Hz, 2H), 8.51 (dd, J = 8.2, 0.7 Hz, 4H), 8.06 (td, J = 8.0, 1.5 Hz, 4H),

7.86–7.84 (m, 2H), 7.76–7.73 (m, 2H), 7.72 (d, J = 16.4 Hz, 2H), 7.66–7.62 (m, 4H),

7.59 (d, J = 6.0 Hz, 2H), 7.45–7.38 (m, 6H), 7.16 (d, J = 16.4 Hz, 2H), 7.03–6.98

(m, 4H), 3.83 (s, 6H). ¹³C-NMR (125 MHz, CD₃CN,): δ 162.0, 158.1, 158.0, 152.7,

152.6, 152.2, 148.2, 138.7, 138.7, 137.1, 130.1, 129.5, 128.6, 128.5, 125.2, 125.0,

122.6, 121.4, 115.5, 56.2; HRMS (m/z): [M]²⁺calcd. for C₄₈H₄₀N₆O₂Ru, 417.1123;

found, 417.1126; analysis (calcd., found for C₄₈H₄₀F₁₂N₆O₂P₂Ru): C (51.30, 51.23),

(

)

H (3.59, 3.48), N (7.48, 7.61). [Ru(2,2′-bipyridine)₂((E,E′)-4,4′-Bis[p-(N,N-

in acetonitrile (Φ_em= 5.9%)⁵⁸applying the following formula:

ꢀ

ꢁ

methoxy)styryl]-2,2′-bipyridine)][Cl]₂: The counter ion hexaﬂuorophosphate was

exchanged to chloride by elution with methanol from the ion exchange resin

Amberlite IRA-410. Analysis (calcd., found for C₄₈H₄₀Cl₂N₆O₂Ru): C (63.70,

63.51), H (4.46, 4.30), N (9.29, 9.11).

Φ_em;sample¼ Φ_em;reference

_reference=F_sample

ð2Þ

ð3Þ

ꢀ

ꢁ

ꢀ

ꢁ

_sample=I_reference

_sample=n_reference

;

F ¼ 1 À 10^ÀA

X-ray crystallography. Single-crystal X-ray diffraction data were collected at 160

(1) K for compounds L-H, L-NMe₂, L-OMe, and 3 on a Rigaku OD XtaLAB

Synergy Dualﬂex (Pilatus 200K detector) diffractometer associated with an Oxford

liquid-nitrogen Cryostream cooler. The wavelength used for all experiments is

1.54184 Å and corresponds to the Cu K_αradiation (from a micro-focus sealed X-

ray tube). The single crystals were selected and cut (if needed) in polybutene oil,

mounted on a ﬂexible loop, and transferred to the diffractometer on the goni-

ometer head. The program suite CrysAlisPro (version 1.171.39.13a) was used for

the pre-experiments, data collections, data reductions, and also for the analytical

Φ_em= luminescence quantum yield, F = fraction of light absorbed, I = integrated

emission intensities, n = refractive index, A = absorbance of the sample at irra-

diation wavelength.

Lifetime. The sample was prepared in an air saturated as well as a degassed

acetonitrile solution with an absorption of 0.2 at 355 nm. The sample was irra-

diated at 355 nm with a NT342B Nd-YAG pumped optical parametric oscillator

(Ekspla). The emission was focused at right angle to the excitation pathway and

directed to a Princeton Instruments Acton SP-2300i monochromator. The signal

was detected with a R928 photomultiplier tube (Hamamatsu).

absorption corrections⁵³based on the indexed faces. Using the Olex2 software⁵⁴

the crystal structures were solved with the SHELXT⁵⁵small molecule structure

solution program and reﬁned with the SHELXL program package⁵⁶by full-matrix

least-squares minimization on F². Molecular graphics were generated using Mer-

cury 4.0⁵⁷. The crystal data collections and structure reﬁnement parameters are

summarized in Tables S1 and S2. CCDC 1951466 (for L-H), CCDC 1951467 (for

3), CCDC 1951468 (for L-NMe₂), and CCDC 1951469 (for L-OMe) contain the

supplementary crystallographic data for these compounds, and can be obtained

free of charge from the Cambridge Crystallographic Data Center via www.ccdc.

cam.ac.uk/data_request/cif.

Electron spin resonance (ESR). The sample (10 μM) was dissolved in acetonitrile

or PBS containing 20 mM TEMP (2,2,6,6–tetramethylpiperidine) as a ¹O₂sca-

•

venger or 20 mM DMPO (5,5-dimethyl-1-pyrroline N-oxide) as a OOH or OH

radical scavenger. Capillary tubes were ﬁlled with the solution and sintered by ﬁre.

EPR spectra were recorded on a Bruker A300 spectrometer with 1 G ﬁeld mod-

ulation, 100 G scan range, and 20 mW microwave power. The samples were

measured in exclusion from light and after irradiation (450 10 nm, 60 s, and

21.8 mW cm⁻²).

In the crystal structure of L-H, the substituted bipyridine is located on a center

of inversion, only one half of the molecule had to be reﬁned, the second part being

reproduced by a symmetry operation. The main species cocystallized with solvent

molecules of acetic acid in a ratio 1:2, respectively.

Singlet oxygen. Direct evaluation: the sample was prepared in an air-saturated

acetonitrile or deuterated water solution with an absorption of 0.2 at 450 nm. The

sample was irradiated at 450 nm with a mounted M450LP1 LED (Thorlabs), whose

light was focused with aspheric condenser lenses. Using a T-Cube LED Driver

(Thorlabs), the intensity of the irradiation was varied and monitored with an

optical power and energy meter. The emission was focused at right angle to the

excitation pathway and directed to a Princeton Instruments Acton SP-2300i

monochromator. To cut off light at wavelengths shorter than 850 nm, a longpass

glass ﬁlter was placed in front of the monochromator entrance slit. The signal was

detected with an EO-817L IR-sensitive liquid-nitrogen cooled germanium diode

detector (North Coast Scientiﬁc Corp.). The luminescence signal, centered at

1270 nm, was measured from 1100 to 1400 nm. The obtained data were analyzed

upon plotting the integrated luminescence peaks against the percentage of the

irradiation intensity. The slope of the linear regression was calculated and com-

pared with the reference Rose Bengal (Φ = 76%)⁵⁹. The absorption of the sample

was corrected with an absorption correction factor. The singlet oxygen quantum

yields were calculated using the following formula:

In the crystal structure of L-OMe, there are two crystallographically

independent bipyridine molecules in the asymmetric unit: one complete molecule

and two half molecules on special positions for which the second half is reproduced

by a symmetry operation. Solvent molecules of acetic acid cocrystallized with the

main species in a ratio 2:1.

In the crystal structure of 3, the asymmetric unit contains two dicationic Ru

species, four anionic hexaﬂuorophosphate counterions and one solvent molecule of

tetrahydropyran (disordered over three positions). The model was difﬁcult to reﬁne

because of the large number of non-hydrogen atoms in the asymmetric unit, and of

the disorders observed for the ﬂexible terminal C=C(H)-C₆H₄OCH₃groups. The

crystal and the data collection were of a rather good quality but the ﬁnal reﬁnement

parameters remained relatively high (R₁= 12% and wR₂= 40%) or poor (Goof =

1.5). The solvent molecules could be identiﬁed in residual density peaks but

reﬁnement was complicated and ﬁnally the real geometry of the tetrahydropyran

molecule was substituted by a regular planar six-membered ring. Many restraints

and constraints were used to get stable reﬁnement cycles but the result seems to be

reliable, especially there is no ambiguity about the main charged species.

ꢀ

ꢁ

ꢀ

ꢁ

Φ_sample¼ Φ_reference

_sample=S_reference

_reference=I_sample

;

ð4Þ

Spectroscopic measurements. The absorption spectra of the sample was mea-

sured with a SpectraMax M2 Spectrometer (Molecular Devices). For the mea-

surements of the emission, the sample was irradiated at 355 nm with a NT342B

Nd-YAG pumped optical parametric oscillator (Ekspla). The emission was focused

at right angle to the excitation pathway and directed to a Princeton Instruments

Acton SP-2300i monochromator. The signal was detected with a XPI-Max 4 CCD

camera (Princeton Instruments).

I ¼ I₀

1 À 10^ÀA

ð5Þ

Φ = singlet oxygen quantum yield, S = slope of the linear regression of the plot

of the areas of the singlet oxygen luminescence peaks against the irradiation

intensity, I = absorption correction factor, I₀= light intensity of the irradiation

source, and A = absorption of the sample at irradiation wavelength.

Indirect evaluation: measurement in acetonitrile: the sample was prepared in an

air-saturated acetonitrile solution with an absorption of 0.2 at the irradiation

wavelength, N,N-dimethyl-4-nitrosoaniline aniline (RNO, 24 µM) and imidazole

(12 mM). Measurement in PBS buffer: the sample was prepared in an air-saturated

PBS solution containing the complex with an absorption of 0.2 at the irradiation

wavelength, N,N-dimethyl-4-nitrosoaniline aniline (RNO, 20 µM) and histidine

(10 mM). The samples were irradiated for various time points with an Atlas

Photonics LUMOS BIO irradiator. The absorption of the samples was constantly

monitored with a SpectraMax M2 Microplate Reader (Molecular Devices). The

difference in absorption (A₀− A) at 420 nm for the measurement in acetonitrile or

at 440 nm for the measurement in PBS was determined. The difference in

absorption was then plotted against the irradiation times and the slope of the linear

regression calculated. The absorption of the sample was corrected with an

absorption correction factor. The singlet oxygen quantum yields were calculated

using the same formulas as used for the direct evaluation.

Two-photon absorption cross section. The two-photon absorption cross section

spectra of the sample was determined upon direction comparison to Rhodamine B.

The sample was irradiated with an OpoletteTM 355II (pulse width ≤ 100 fs, 80

MHz repetition rate, Spectra Physics Inc.) laser. The experimental excitation and

detection conditions were conducted with negligible reabsorption processes, which

could affect two-photon absorption measurements. The quadratic dependence of

the two-photon induced luminescence intensity on the excitation power was ver-

iﬁed at an excitation wavelength of 800 nm. The two-photon absorption cross

section was calculated using the following equation:

ꢀ

ꢁ

σ_2;sample¼ σ_2;reference

Φ_reference´ c_reference´ I_sample´ n_sample

ꢀ

ꢁ

;

ð1Þ

Φ_sample´ c_sample´ I_reference´ n_reference

σ₂= two-photon cross section, Φ = luminescence quantum yield, c =

concentration, I = integrated luminescence intensity, n = refractive index

Stability in human plasma. The stability of the sample was evaluated upon

incubation in pooled human plasma with caffeine as an internal standard, which

previously demonstrated to be stable under these conditions⁶⁰. The sample (20 μM)

and caffeine (40 μM) were prepared in dimethyl sulfoxide stock solutions. One

aliquot of both solutions was added to 975 μL of human plasma achieving a total

volume of 1000 μL. The resulting solutions were incubated upon continuous gentle

shaking (ca. 300 rpm) for 48 h at 37 °C. After this time, the incubation was ended

by addition of 3 mL of methanol. The mixture was centrifuged for 60 min at

3000 rpm/958 gand 4 °C. The solution was ﬁltered through a 0.2 μm membrane

ﬁlter. The solvent was evaporated and the residue was redissolved in 1:1 (v/v)

Luminescence quantum yield. The sample was prepared in an acetonitrile solu-

tion with an absorption of 0.1 at 355 nm. The sample was irradiated at 355 nm with

a NT342B Nd-YAG pumped optical parametric oscillator (Ekspla). The emission

was focused at right angle to the excitation pathway and directed to a Princeton

Instruments Acton SP-2300i monochromator. The signal was detected with a

XPI-Max 4 CCD camera (Princeton Instruments). The luminescence quantum

yields were determined by comparison with the reference [Ru(2,2′-bipyridine)₃]Cl₂

(

)

acetonitrile/water with 0.1% triﬂuoroacetic acid. The resulting solution was then

compared with the Ru references. The Ru content was then associated with the

number of cells.

Endocytic inhibition: the cells were preincubated with ammonium chloride

ﬁltered through a 0.2 μm membrane ﬁlter and analyzed with a HPLC System. The

solvents (HPLC grade) were millipore water with 0.1% triﬂuoroacetic acid (solvent

A) and acetonitrile (solvent B). Method M1: 0–3 min: isocratic 50% A (50% B);

3–17 min: linear gradient from 50% A (50% B) to 0% A (100% B); Method M2:

0–3 min: isocratic 95% A (5% B); 3–17 min: linear gradient from 95% A (5% B)

to 0% A (100% B); 17–23 min: isocratic 0% A (100% B). The ﬂow rate was

1 mL min⁻¹and the chromatogram was detected at 250 nm.

(50 mM) or chloroquine (100 μM) for 1 h. After this time, the cells were incubated

with the sample (10 μM, 2% DMSO, v%) for 1 h at 37 °C and then washed with

PBS. The cells were detached with trypsin, harvested, centrifuged, and resuspended.

The number of cells on the dish was counted. The sample was digested using a 60%

HNO₃solution for 3 days and following this diluted to a 2% HNO₃solution in

water. The Ru content was determined with an ICP-MS apparatus and the results

compared with the Ru references. The Ru content was then associated with the

number of cells.

Photostability. The sample was prepared in an air-saturated acetonitrile solution.

Using a Atlas Photonics LUMOS BIO irradiator, the sample was constantly irra-

diated at 450 nm in time intervals from 0 to 10 min (13.2 J cm⁻²). The absorption

spectra from 350 to 700 nm was monitored with a SpectraMax M2 Microplate

Reader (Molecular Devices) at each time interval. As positive and negative controls,

respectively, [Ru(2,2′-bipyridine)₃]Cl₂and Protoporphyrin IX were used.

Cation transporter inhibition: the cells were preincubated with

tetraethylammonium chloride (1 mM) for 1 h. After this time, the cells were

incubated with the sample (10 μM, 2% DMSO, v%) for 1 h at 37 °C and then

washed with PBS. The cells were detached with trypsin, harvested, centrifuged, and

resuspended. The number of cells on the dish was counted. The sample was

digested using a 60% HNO₃solution for 3 days and following this diluted to a 2%

HNO₃solution in water. The Ru content was determined with an ICP-MS

apparatus and the results compared with the Ru references. The Ru content was

then associated with the number of cells.

Photobleaching. The sample was dissolved in air saturated 0.7 mL CD₃CN and

stored in a NMR tube in the dark. A ¹H-NMR spectrum was measured directly

after preparation to ensure the identity of the sample. The sample was then

irradiated at 450 nm with an Atlas Photonics LUMOS BIO irradiator (10 min,

13.2 J cm⁻²). Following this treatment, a ¹H-NMR spectrum was again measured.

Intracellular distribution by confocal luminescence imaging. A total of 1 × 10⁴

cells were seeded on confocal dishes and allowed to adhere overnight. The cells

were incubated with the sample (10 μM, 2% DMSO, v%) for 8 h at 37 °C in the

dark. After this time, the cells were washed with PBS. The cells were further each

incubated with LysoTracker Green, MitoTracker Deep Red, Cell-light Golgi-GFP,

ER tracker Green and Hoechst 33342 for 30 min at 37 °C in the dark. The cells were

washed three times with PBS. Confocal images were taken with a LSM 880 (Carl

Zeiss) laser scanning confocal microscope. The organelle trackers were excited and

detected as recommended by the supplier. The samples were detected under usage

of their 1-Photon (λ_ex= 458 nm, λ_em= 600–750 nm) or 2-Photon (λ_ex= 800 nm,

λ_em= 600–750 nm) luminescence properties.

Distribution coefﬁcient. Using the “shake-ﬂask” method, the distribution coefﬁ-

cient between the PBS and octanol phase of the sample was determined. The PBS

and octanol phases were previously saturated in each other. The sample was dis-

solved in the phase, which is dissolving best the compound. This solution was

added to an equal volume of the other phase and mixed at 190 rpm overnight with

a sample mixer. Following this, the solutions were equilibrated for 4 h. The phases

were carefully separated from each other. The amount of the sample in each phase

was determined by ICP-MS. The evaluation was repeated three times and the ratio

between the organic and aqueous phase determined.

Intracellular distribution by ICP-MS. A total of 10 × 10⁶cells were incubated with

the sample (10 μM, 2% DMSO, v%) for 8 h at 37 °C in the dark. After this time, the

cells were detached with trypsin and harvested. The number of cells was counted

and equally divided into four portions. In the ﬁrst portion, the nucleus was

extracted using a nucleus extraction kit (Sangon Biotech); in the second portion,

the mitochondria was extracted using a mitochondria extraction kit (Sangon

Biotech), and in the third portion, the lysosome was extracted using a lysosome

extraction kit (GenMed Scientiﬁc). Using the fourth portion, the cytoplasm was

extracted. The cells were detached with trypsin, harvested, and centrifuged. The cell

pellet was resuspended in lysis buffer and the cells lysed. The cytoplasm was

separated by centrifugation(Optima MAX-XP ultracentrifuge, Beckman Coulter)

for 150 min at 200,000 g at 4 °C. The supernatant solution was separated. The

sample was digested using a 60% HNO₃solution for 3 days and following this

diluted to a 2% HNO₃solution in water. The Ru content was determined with an

ICP-MS apparatus and the results compared with the Ru references. The Ru

content was then associated with the number of cells.

Cell culture. Human glioblastoma astrocytoma (U373) cells were cultured in MEM

medium supplemented with 10% FBS, 1% NEAA (nonessential amino acids), and

1% penicillin/streptomycin. Human cervical carcinoma (HeLa), doxorubicin-

resistant human colon adenocarcinoma (SW620/AD300), and the mouse colon

carcinoma (CT-26) cells were cultured in DMEM medium supplemented with 10%

FBS and 1% penicillin/streptomycin. Human retinal epithelial (RPE-1) cells were

cultured in DMEM-F12 medium supplemented with 10% FBS and 1% penicillin/

streptomycin. All cell lines were obtained from the American Type Culture Col-

lection and cultured at 37 °C and 5% CO_2.Before an experiment, the cells were

passaged three times.

Time-dependent cellular uptake. A total of 6 × 10⁶cells were incubated with

the sample (10 μM, 2% DMSO, v%) for varying time points (0.5, 1, 2, 4, 8, 12 h) at

37 °C. After this time, the media was removed and the cells washed with cell media.

The cells were trypsinised, harvested, centrifuged, and resuspended. The number of

cells on the dish was counted. The sample was digested using a 60% HNO₃solution

for 3 days and, following this, diluted to a 2% HNO₃solution in water. The Ru

content was determined with an ICP-MS apparatus and the results compared with

the Ru references. The Ru content was then associated with the number of cells.

One-photon induced cellular singlet oxygen generation. A total of 1 × 10⁴cells

were seeded on confocal dishes and allowed to adhere overnight. The cells were

incubated with the sample (10 μM, 2% DMSO, v%) for 4 h at 37 °C in the dark.

After this time, the cells were washed with PBS. The cells were further incubated

with 2′,7′-dichloroﬂuorescein diacetate for 30 min. The cells were washed three

Cellular uptake mechanism. The cellular uptake mechanism was investigated

upon pretreatment of 1 × 10⁶cells with pathways inhibitors and determination of

the amount of Ru inside the cells with an ICP-MS apparatus.

times with PBS. The sample was irradiated upon 1-Photon excitation (λ_ex

488 nm). Confocal images (λ_ex= 488 nm, λ_em= 510–550 nm) were taken with a

LSM 880 (Carl Zeiss) laser scanning confocal microscope.

Control: the cells were incubated with the sample (10 μM, 2% DMSO, v%) for

1 h at 37 °C and then washed with PBS. The cells were detached with trypsin,

harvested, centrifuged, and resuspended. The number of cells on the dish was

counted. The sample was digested using a 60% HNO₃solution for 3 days and

following this diluted to a 2% HNO₃solution in water. The Ru content was

determined with an ICP-MS apparatus and the results compared with the Ru

references. The Ru content was then associated with the number of cells.

Low temperature: the cells were preincubated at 4 °C for 1 h. After this time, the

cells were incubated with the sample (10 μM, 2% DMSO, v%) for 1 h at 37 °C and

then washed with PBS. The cells were detached with trypsin, harvested,

centrifuged, and resuspended. The number of cells on the dish was counted. The

sample was digested using a 60% HNO₃solution for 3 days and following this

diluted to a 2% HNO₃solution in water. The Ru content was determined with an

ICP-MS apparatus and the results compared with the Ru references. The Ru

content was then associated with the number of cells.

Metabolic inhibition: the cells were preincubated with 2-deoxy-D-glucose (50

mM) and oligomycin (5 μM) for 1 h. After this time, the cells were incubated with

the sample (10 μM, 2% DMSO, v%) for 1 h at 37 °C and then washed with PBS. The

cells were detached with trypsin, harvested, centrifuged, and resuspended. The

number of cells on the dish was counted. The sample was digested using a 60%

HNO₃solution for 3 days and following this diluted to a 2% HNO₃solution in

water. The Ru content was determined with an ICP-MS apparatus and the results

Two-photon induced cellular singlet oxygen generation. A total of 1 × 10⁴cells

were seeded on confocal dishes and allowed to adhere overnight. The cells were

incubated with the sample (10 μM, 2% DMSO, v%) for 4 h at 37 °C in the dark.

After this time, the cells were washed with PBS. The cells were further incubated

with 2′,7′-dichloroﬂuorescein diacetate for 30 min. The cells were washed three

times with PBS. The sample was irradiated upon 2-Photon excitation (λ_ex= 800

nm). Confocal images (λ_ex= 488 nm, λ_em= 510–550 nm) were taken with a LSM

880 (Carl Zeiss) laser scanning confocal microscope.

(Photo-)cytotoxicity on 2D cell monolayers. A total of 4 × 10³cells were seeded

on 96-well plates and allowed to adhere overnight. The cells were treated with

increasing concentrations of the sample diluted in cell media achieving a total

volume of 200 μL. The cells were incubated with the sample for 8 h and, after this

time, the medium refreshed. To study the phototoxic effect of the sample, the cells

were exposed to 480 nm (spectral half-width: 20 nm, 10 min, 5.2 mW cm⁻², 3.1 J

cm⁻²) or 540 nm (spectral half-width: 30 nm, 40 min, 4.0 mW cm⁻², 9.5 J cm⁻²

light using an Atlas Photonics LUMOS BIO irradiator. To study the dark cyto-

)

toxicity of the sample, the cells were not irradiated and the medium exchanged.

The cells were grown for an additional 40 h at 37 °C. After this time, the medium

was replaced with fresh medium containing resazurin with a ﬁnal concentration of

(2 0 2 0 ) 1 1 : 3 2 6 2 | h t t p s : / / d o i . o r g / 1 0 . 1 0 3 8 / s 4 1 4 6 7 - 0 2 0 - 1 6 9 9 3 - 0 | w ww . n a t u r e . c o m / n a t u re c o m m u n i c a t i o n s

0.2 mg mL⁻¹. The cells were incubated for 4 h and the amount of the ﬂuorescent

product resoruﬁn determined (λ_ex= 540 nm, λ_em= 590 nm) with a SpectraMax

M2 Microplate Reader (Molecular Devices). The obtained data was analyzed with

the GraphPad Prism software.

In vivo experiment. Six weeks old nu/nu female mice were purchased from

Charles River. For the tumor xenograft, 6 × 10⁶SW620/AD300 cells were sub-

cutaneously (s.c.) injected with a 150 μL Matrigel and saline (1:1, v/v) suspension

into the mice. After a week, the tumor volumes of the mice reached ~80 mm³. All

experiments were conducted in compliance with the ethical regulations for animal

testing and received approval of the experimental animal managing committee of

Sun Yat Sen University.

Cell death mechanism. The cell death mechanism was investigated by measuring

the cell viability upon preincubation with various cell death pathway inhibitors. 3-

Methyladenine (100 μM), Z-VAD-FMK (20 μM), cycloheximide (0.1 μM), and

necrostatin-1 (60 μM) were preincubated with the cells for 40 min. Following this,

the sample (5 μM) was added and incubated for 8 h. The cells were washed, irra-

diated with a LED (500 nm, 10 min, 10.0 mW cm⁻², 6 J cm⁻²) light source, and

incubated for 12 h. After this time, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-

trazolium bromide was added and the cells were incubated for additional 4 h. The

liquid was disposed and 150 μL DMSO was added. The plates were shaken for

2 min and the absorbance at 595 nm was measured using a microplate reader

(TecanInﬁniteF200, Bio-rad).

In vivo biodistribution. Three nude mice were tail-intravenous (i.v.) injected with

2 mg kg⁻¹of the sample. One hour later, the mice were sacriﬁced and the major

organs (including the heart, liver, spleen, lung, kidney, brain, intestine, and blood)

were separated, weighted and ground. The grinded organ as well as the blood were

digested using a 60% HNO₃solution for 3 days, and following this, diluted to a 2%

HNO₃solution in water. The Ru content was determined with an ICP-MS appa-

ratus and the results compared with the Ru references.

In vivo time-dependent biodistribution. Six SW620/AD300 nude tumor-bearing

nude mice were separated into three groups, and i.v. injected with 2 mg kg⁻¹of the

sample. 30 min, 1 h, 2 h later, the mice were euthanized and the distribution of the

sample determined using the previous described method.

Generation and analysis of 3D multicellular tumor spheroids (MCTS). A

suspension of 0.75% agarose in PBS was heated inside a high-pressure autoclave.

The hot emulsion was transferred into a 96-well plate (50 μL per well). The plates

were exposed for 3 h to UV irradiation and allowed to cool down. After this time, a

cell suspension of 3 × 10³cells was seeded on top the agarose ground layer. Within

2–3 days, MCTS were formed from the cell suspension. The MCTS were cultivated

and maintained at 37 °C in a cell culture incubator at 37 °C with 5% CO₂atmo-

sphere. The culture medium was replaced every 2 days. The formation, integrity,

diameter, and volume of the MCTS was monitored by an Axio Observer Z1 (Carl

Zeiss) phase contrast microscope. The volume was calculated using the following

equation: V = 4/3πr³. The luminescence images along the z-axis were captured by

In vivo (Photo-)cytotoxicity. 30 SW620/AD300 nude tumor-bearing nude mice

were randomly separated into six groups, resulting in ﬁve mice for each group.

After been anaesthetized, the mice was ﬁxed in a warm three-axes holder. Before

irradiation, the tumor site was disinfected with ethanol. The tumor was exposed to

1P or 2P light. Following the irradiation, the operative area was disinfection by

iodophor.

Group 1: injected 7 (2 mg kg⁻¹, 50 μL) intravenously and irradiate under

800 nm laser (50 mW, 1 kHz, pulse width 35 fs, 5 s mm⁻¹) 1 h after injection;

Group 2: injected 7 (2 mg kg⁻¹, 50 μL) intravenously and irradiate under

500 nm light (60 min, 10.0 mW cm⁻², 36 J cm⁻²) 1 h after injection;

Group 3: injected by physiological saline (50 μL) and treated with 800 nm laser

(50 mW, 1 kHz, pulse width 35 fs, 5 s mm⁻¹) 1 h after injection;

Group 4: injected by physiological saline (50 μL) and treated with 500 nm light

(60 min, 10.0 mW cm⁻², 36 J cm⁻²) 1 h after injection;

1-Photon (λ_ex= 458 nm, λ_em= 600–750 nm) or 2-Photon (λ_ex= 800 nm, λ_em

600–750 nm) excitation in the z-stack mode with a an LSM 880 (Carl Zeiss) laser

scanning confocal microscope equipped with Argon or Coherent Chameleon

2-Photon laser and a GaAsP detector.

3D multicellular tumor spheroids (MCTS) growth inhibition assay. MCTS were

treated with the sample (20 μM 1–7, 20 μM tetraphenylporphyrin H₂TPP, 10 μM

cisplatin, 30 μM cisplatin, 2% DMSO, v%) by replacing 50% of the media with drug

supplemented media in the dark for 3 days. After this time, the MCTS were

exposed to a 1-Photon (500 nm, 16.7 min, 10.0 mW cm⁻², and 10 J cm⁻²) irra-

diation using a LED light source or 2-Photon (800 nm, 10 J cm⁻²with a section

interval of 5 μm) irradiation using a LSM 880 (Carl Zeiss) laser scanning confocal

microscope equipped with a Coherent Chameleon 2-Photon laser. The cell culture

media was replaced every 2 days. The integrity and diameter of the MCTs was

monitored with an Axio Observer Z1 (Carl Zeiss) phase contrast microscope

every 24 h.

Group 5: intravenous injected 7 (2 mg kg⁻¹, 50 μL);

Group 6: injected 50 μL physiological saline.

The mice were anesthetized by injection of a 4% chloral hydrate aqueous

solution (0.2 mL, 20 g⁻¹) before the treatment. The tumor volume and body weight

were measured and recorded every 2 days. Tumor volume was calculated by the

following formula:

Volume ¼ Length ´ Width²=2:

ð6Þ

Histological examination. The tumor and all major organs (including the heart,

liver, spleen, lung, kidney, brain, intestine, and blood) were collected and a slice of

each one was ﬁxated with 4% paraformaldehyde. The obtained slices were stained

with hematoxylin and eosin. The histological images were taken with a Carl Zeiss

Axio Imager Z2 microscope.

3D multicellular tumor spheroids (MCTS) viability assay. MCTS were treated

with the sample (20 μM, 2% DMSO, v%) by replacing 50% of the media with drug

supplemented media in the dark for 3 days. After this time, the MCTS were

exposed to a 1-Photon (500 nm, 16.7 min, 10.0 mW cm⁻², and 10 J cm⁻²) irra-

diation using a LED light source or 2-Photon (800 nm, 10 J cm⁻²with a section

interval of 5 μm) irradiation using a LSM 880 (Carl Zeiss) laser scanning confocal

microscope equipped with a Coherent Chameleon 2-Photon laser. The cell culture

media was replaced every 2 days. Two days after the irradiation the MCTS viability

was tested using a Viability/Cytotoxicity Kit for mammalian cells (Invitrogen).

Living cells can be identiﬁed from dead cells through the presence of ubiquitous

intracellular esterase activity which can be monitored by the enzymatic conversion

Reporting summary. Further information on research design is available in

the Nature Research Reporting Summary linked to this article.

Data availability

The authors declare that all data supporting the ﬁndings of this study are available within

the paper and its Supplementary Information ﬁles. Correspondence and requests for

materials should be addressed to H.C. or G.G.

of the non-ﬂuorescent calcein AM to the intensely ﬂuorescent calcein (λ_ex

495 nm, λ_em= 515 nm). As the spectroscopic properties of the dead cell probe

EthD-1 overlaps with the one of the investigated compounds, this probe was not

used and only calcein AM as a probe for living cells was used. The MCTS were

incubated with calcein AM (2 μM) for 30 min and images of the MCTS taken with

an Axio Observer Z1 (Carl Zeiss, Germany) inverted ﬂuorescence microscope.

Received: 4 February 2020; Accepted: 5 June 2020;

(Photo-)cytotoxicity on 3D multicellular tumor spheroids (MCTS). The cyto-

toxicity of the compounds in 3D MCTS was assessed by measurement of the ATP

concentration. MCTS were treated with increasing concentrations of the com-

pound (2% DMSO, v%) by replacing 50% of the media with drug supplemented

media and incubation for 12 h. After this time, the MCTS were divided in three

identical groups. The ﬁrst group was strictly kept in the dark. The second group

was exposed to a 1-Photon irradiation (500 nm, 16.7 min, 10.0 mW cm⁻², and

10 J cm⁻²) using a LED light source and the third group was exposed to a 2-Photon

irradiation (800 nm, 10 J cm⁻²with a section interval of 5 μm) using a LSM 880

(Carl Zeiss, Germany) laser scanning confocal microscope equipped with a

Coherent Chameleon 2-Photon laser. After the irradiation, all groups were incu-

bated additional 48 h. The ATP concertation was measured using a CellTiter-Glo

3D Cell Viability kit (Promega) by measuring the generated chemiluminescence

with an inﬁnite M200 PRO (Tecan) plate reader. The obtained data was analyzed

with the GraphPad Prism software.

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Supplementary information is available for this paper at https://doi.org/10.1038/s41467-

chromatography/mass spectrometry. Anal. Chem. 81, 3285–3296 (2009).

020-16993-0.

Correspondence and requests for materials should be addressed to H.C. or G.G.

Acknowledgements

Peer review information Nature Communications thanks the anonymous reviewer(s) for

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.). I.C and F.M

were ﬁnancially supported from the European Research Council (ERC) under the Eur-

opean Union’s Horizon 2020 research and innovation program (grant agreement No

648558, STRIGES CoG grant).

their contribution to the peer review of this work.

Reprints and permission information is available at http://www.nature.com/reprints

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licenses/by/4.0/.

Author contributions

All authors were involved with the design and interpretation of experiments and with the

writing of the paper. DFT calculations were performed by J.K. and F.M. Chemical,

photophysical, and biological experiments were carried out by J.K. X-ray crystallography

was carried out by J.K. and O.B. Animal testing was carried out by J.K. and S.K. The work

was supervised by I.C., H.C., and G.G. All authors have given approval to the ﬁnal

version of the paper.

Competing interests

The authors declare no competing interests.

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