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Cyclometalated Iridium(III) Complexes as High-Sensitivity Two-Photon Excited Mitochondria Dyes and Near-Infrared Photodynamic Therapy Agents.
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Article
Cyclometalated Iridium(III) Complexes as High-Sensitivity TwoPhoton Excited Mitochondria Dyes and Near-Infrared Photodynamic
Therapy Agents
Xu-Dan Bi, Rong Yang, Yue-Chen Zhou, Daomei Chen, Guo-Kui Li, Yuan-Xiao Guo, Meng-Fan Wang,
Dandan Liu,* and Feng Gao*
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ABSTRACT: Photodynamic therapy (PDT) using two-photon near-infrared light excitation is a very effective way to avoid the use of shortwavelength ultraviolet or visible light which cannot efficiently penetrate into
the biological tissues and is harmful to the healthy cells. Herein, a series of
cyclometalated Ir(III) complexes with a structurally simple diimine ligand
were designed and the synthetic route and preparation procedure were
optimized, so that the complexes could be obtained in apparently higher
yield, productivity, and efficiency in comparison to the traditional methods.
Their ground state and excited singlet and triplet state properties were
studied by spectroscopy and quantum chemistry theoretical calculations to
investigate the effect of substituent groups on the photophysical properties of
the complexes. The Ir(III) complexes, especially Ir1 and Ir3, showed very
low dark toxicities and high phototoxicities under both one-photon and twophoton excitation, indicating their great potential as PDT agents. They were also found to be highly sensitive two-photon
mitochondria dyes.
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INTRODUCTION
Photodynamic therapy (PDT) is a minimally invasive
treatment approved for the treatment of cancerous tumors
and of noncancerous diseases, involving the interaction of the
photosensitizer (PS), light component, and molecular oxygen.1−3 The vast majority of current PSs clinically approved for
PDT are activated by short-wavelength ultraviolet (UV) or
visible (Vis) light, which cannot efficiently penetrate into the
biological tissues, and PDT modality is restricted to the
treatment for superficial lesions.4 Notably, near-infrared (NIR)
light in the range of around 700−1000 nm, known as the
“biological transparency window”, displays minimum photodamage to healthy cells, reduced scattering from tissue
components, and better tissue penetration (e.g., <10 cm)
than UV or Vis light (e.g., <1 cm).5 Therefore, more attention
has recently focused on shifting the excitation wavelength to
the NIR region for deep PDT treatment.
One approach to reach the NIR region relies on organic PSs,
such as derivatives of boron dipyrromethene (BODIPY),
porphyrin, and phthalocyanine that absorb in the 650−750 nm
region.6 Another choice is upconverting nanoparticles
(UCNPs), a class of nanoparticles that emit UV or Vis light
under NIR light excitation.7−9 More recently, PSs with twophoton (TP) absorption properties have attracted a great deal
of attention in PDT.10−12 The TP-PDT agent molecule is
promoted to an excited state, the same PS-active excited state
© XXXX American Chemical Society
as for one higher-energy photon excitation, by the simultaneous absorption of two lower-energy NIR photons, each of
which contributes half of the total energy required to induce
emission. In comparison with traditional one-photon (OP)
PDT agents, TP-PDT agents require the application of a lowenergy NIR laser as a light source, which gives a possibility of
higher light dose administration and minimizes the side effects
thanks to the reduced interaction between the NIR light and
the tissue.13
Metal-containing PSs have emerged as promising PDT
systems because of their rich photochemical and photophysical
properties derived from various excited-state electronic
configurations.12,14−17 On one hand, their energy- and
electron-transfer processes can yield highly potent oxygendependent and/or oxygen-independent photobiological activity.18 On the other hand, the large quantum yields for tripletstate formation and the characteristic reactivities of the
different excited-state configurations offer the opportunity to
Received: May 23, 2020
A
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oxygen (1O2) and/or other reactive oxygen species (ROS), and
their large Stokes shift can minimize the possible selfquenching effect even at high concentration.26−29 In addition
to d-block metals, f-block-element complexes, particularly
Gd(III) and Lu(III), have also been reported for their TPPDT activity.30,31
It is encouraging that, after years of efforts, TLD1433
became the first Ru(II)-based PS that entered a human clinical
trial (ClinicalTrials.gov, identifier NCT03053635) for nonmuscle invasive bladder cancer in 2018. As summarized in the
recent review by McFarland,32 the developer of TLD1433, an
ideal PDT agent should meet the following standards: (1)
effective generation of cytotoxic 1O2 and/or other ROS, (2)
large molar extinction coefficient in the PDT window, (3)
preferential tumor accumulation and rapid systemic clearance,
(4) amphiphilic structure, (5) no dark toxicity, (6) chemical
stability, (7) solubility in injectable formulations, and (8)
chemically pure and easy to obtain via high-yielding
reactions.33 This last standard concerning preparation has
been placed in an unimportant position in the pursuit of high
activity. Almost all reported Ir(III)-based PDT agents have
complex ligand structures that are difficult to synthesize, and
the amount of preparation is usually in milligrams. This is
undoubtedly an obstacle for further in vivo experiments and
clinical research.
Overall, compounds with a high 1O2 yield upon NIR
irradiation and a simple chemical structure will be the ideal
choice for PDT. Therefore, the focus of this study is to
introduce structurally simple functional groups which are
beneficial to the production of 1O2 into Ir(III) complexes
which have been proven to have good TP-PDT properties.
Cinnamaldehyde, as an active compound isolated from the
traditional medicinal herb Cinnamomum cassia, has been
reported to induce a ROS-mediated mitochondrial permeability transition and resultant cytochrome c release.34
Recently, a tumor-specific enhanced oxidative stress polymer
conjugate (TSEOP) has been designed to boost tumor-specific
antitumor immunity, by the release of cinnamaldehyde, which
was proven to amplify ROS.35
In this paper, a series of cyclometalated Ir(III) complexes
(Ir1−Ir4) with the structurally simple ligand (E)-2-styryl-1Himidazo[4,5-f ][1,10]phenanthroline (sip) was designed by the
combination of a cinnamaldehyde group which can amplify
ROS and an Ir(III) complex with good TP-PDT properties.
The synthesis route and preparation procedure were
optimized, so that the complexes could be obtained in
apparently higher yield, productivity, and efficiency in
comparison to the traditional methods. Their ground state
and excited singlet and triplet state properties have been
studied by spectroscopy, and density functional theory (DFT)
and time-dependent DFT (TDDFT) calculations to discuss
the effect of substituent groups on the photophysical properties
of complexes. In-depth biological explorations of these Ir(III)
complexes as highly sensitive TP mitochondria dyes and highly
effective OP and TP excited PDT agents are presented. With
both promising 1O2 quantum yields and low dark toxicities,
Ir1−Ir4 showed remarkable phototherapeutic indexes (PIs)
which are quite comparable to those of the most potent
phototherapeutic Ir(III) complexes reported with structurally
complicated ligands.
rationally design transition-metal complexes with desirable
photobiological mechanisms that are simply not possible with
organic PSs.
In 2014, You and Nam synthesized the first Ir(III)-based
molecular dyad (Irbtp−RhB) capable of lysosomal staining,
1
O2 sensitization, and TP-PDT (Figure 1).19 Lemercier and
Figure 1. Representive metal complexes for PDT and complexes Ir1−
Ir4 designed in this study.
Natrajan also developed a cyclometalated Ir(III) complex
(Figure 1) as a PS for TP-PDT using the ligand of RuL1TEG,
the first Ru(II) complex as a PS for TP-PDT.20,21 Since then,
luminescent cyclometalated Ir(III) complexes with TP
absorption behavior have been widely investigated, and their
targets in tumor cells have been reported to be DNA,
intracellular nucleus, endoplasmic reticulum, lysosome, mitochondria, cytoplasm, peptide, and protein.12,22−24 In comparison to Ru(II) complexes, Ir(III) complexes have higher ligand
field strength and stronger spin−orbital coupling provided by
the third-row transition metal. Furthermore, heteroleptic
Ir(III) complexes with bipyridyl (N^N) and cyclometalating
(C^N) ligands have shown high photostability due to highlying metal-centered states and easily tunable highest occupied
molecular orbital (HOMO) and lowest unoccupied molecular
orbital (LUMO) energy levels. Their high photostability allows
cyclometalated Ir(III) complexes to be used for continuous
irradiation and real-time monitoring of intracellular trafficking.25 Their long-lived triplet states result in long lifetimes and
facilitate possible reactions with oxygen to generate singlet
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(d, J = 5.0 Hz, 3H), 7.79 (d, J = 6.7 Hz, 2H), 7.61 (q, J = 5.6 Hz, 4H),
7.49 (q, J = 6.4 Hz, 3H), 7.43 (t, J = 3.4 Hz, 1H), 7.35 (t, J = 6.5 Hz,
2H). 13C{1H} NMR (CDCl3, 150 MHz): δ 168.2, 162.6, 148.4, 138.0,
136.9, 135.7, 131.9, 131.1, 129.1, 128.9, 127.6, 124.9, 123.3, 123.1,
122.8, 119.7, 115.5. FTIR (KBr) νmax (cm−1): 3334 (NH), 3086 (CH,
aryl), 2962 (CH, aryl), 1606 (CC, styryl), 1478 (CC, aryl), 1387
(CN, pyridyl), 1110 (CN, imidazolyl), 848 (PF), 758 (CC, aryl),
552 (PF). ESI-MS (MeOH, m/z): calcd for C43H30IrN8 ([M −
PF6]+), 823.22; found, 823.27.
[Ir(ppy)2(nsip)](PF6) (Ir2). This compound was synthesized
according to the same procedure as for Ir1, using 0.442 g (2.50
mmol) of (E)-4-nitrocinnamaldehyde instead of (E)-cinnamaldehyde.
Yield: 2.82 g, 93%. Anal. Calcd for C43H29F6IrN7O2P·2H2O: C, 49.24;
H, 3.17; N, 9.35. Found: C, 48.98; H, 3.43; N, 9.07. 1H NMR
((CD3)2SO, 300 MHz): δ 9.13 (d, 2H, J = 6.12 Hz), 8.28 (m, 4H),
8.16 (d, 2H, J = 3.21 Hz), 8,09 (m, 2H), 8.05 (m, 2H), 7.96 (d, 3H, J
= 5.55 Hz), 7.89 (m, 3H), 7.68 (d, 1H, J = 12.36 Hz), 7.52 (d, 2H, J =
4.23 Hz), 7.07 (m, 2H), 6.98 (m, 4H), 6.30 (d, 2H, J = 5.58 Hz).
13
C{1H} NMR ((CD3)2SO, 100 MHz): δ 167.4, 151.9, 150.8, 149.7,
149.0, 147.6, 144.8, 144.5, 142.6, 139.2, 133.0, 132.7, 131.7, 130.7,
128.7, 127.6, 125.5, 124.6, 124.3, 122.8, 121.7, 120.4. FTIR (KBr)
νmax (cm−1): 3398 (NH), 3078 (CH, aryl), 2943 (CH, aryl), 1605
(CC, styryl), 1523 (NO), 1415 (CC, aryl), 1353 (CN, pyridyl),
1110 (CN, imidazolyl), 844 (PF), 749 (CC, aryl), 566 (PF). ESIMS (MeOH, m/z): calcd for C43H29IrN7O2 ([M − PF6]+), 868.20;
found, 868.23.
[Ir(ppy)2(osip)](PF6) (Ir3). This compound was synthesized
according to the same procedure as for Ir1, using 0.405 g (2.50
mmol) of (E)-4-methoxycinnamaldehyde instead of (E)-cinnamaldehyde. Yield: 2.22 g, 89%. Anal. Calcd for C44H32F6IrN6OP·2H2O: C,
51.11; H, 3.51; N, 8.13. Found: C, 50.89; H, 3.76; N, 7.87. 1H NMR
((CD3)2SO, 300 MHz): δ 12.13 (s, 1H), 9.31 (d, 1H, J = 5.91 Hz),
9.10 (d, 1H, J = 6.09 Hz), 8.17 (d, 1H, J = 3.45 Hz), 8.14 (d, 1H, J =
3.27 Hz), 7.94 (d, 2H, J = 6.12 Hz), 7.79 (m, 2H), 7.73 (m, 5H), 7.53
(d, 2H, J = 6.42 Hz), 7.42 (d, 1H, J = 3.9 Hz), 7.33 (d, 1H, J = 3.99
Hz), 7.12 (m, 3H), 6.99 (m, 2H), 6.92 (m, 1H), 6.81 (m, 3H), 6.42
(t, 2H, J = 6.39 Hz), 3.81 (s, 3H). 13C{1H} NMR (DMSO-d6, 150
MHz): δ 168.1, 150.4, 148.8, 148.6, 148.0, 144.0, 138.0, 131.9, 130.9,
129.0, 128.8, 126.0, 124.8, 123.2, 122.7, 119.6, 112.0, 46.2. FTIR
(KBr) νmax (cm−1): 3398 (NH), 3072 (CH, aryl), 2963 (CH, aryl),
1605 (CC, styryl), 1462 (CC, aryl), 1381 (CN, pyridyl), 1251
(CO), 1103 (CN, imidazolyl), 1028 (CO), 845 (PF), 763 (CC,
aryl), 593 (PF). ESI-MS (MeOH, m/z): calcd for C44H32IrN6O ([M
− PF6]+), 853.23; found, 853.28.
[Ir(ppy)2(dsip)](PF6) (Ir4). This compound was synthesized
according to the same procedure as Ir1, using 0.438 g (2.50 mmol)
of (E)-4-dimethylaminocinnamaldehyde instead of (E)-cinnamaldehyde. Yield: 2.15 g, 85%. Anal. Calcd for C45H35F6IrN7P·2H2O: C,
51.62; H, 3.75; N, 9.36. Found: C, 51.38; H, 3.93; N, 9.03. 1H NMR
((CD3)2SO, 300 MHz): δ 9.19 (d, 2H, J = 6.03 Hz), 8.27 (d, 2H, J =
5.58 Hz), 8.12 (s, 2H), 8.04 (m, 2H), 7.96 (d, 2H, J = 5.49 Hz), 7.88
(m, 2H), 7.53 (m, 4H), 7.07 (m, 4H), 6.97 (m, 4H), 6.76 (d, 2H, J =
6.18 Hz), 6.58 (d, 1H, J = 3.81 Hz), 6.30 (d, 2H, J = 5.43 Hz), 2.98
(d, 6H, J = 12.36 Hz). 13C{1H} NMR (DMSO-d6, 150 MHz): δ
168.1, 160.4, 154.1, 148.7, 148.2, 143.7, 138.0, 136.4, 131.9, 130.9,
129.0, 128.6, 124.8, 122.8, 123.3, 123.1, 119.6, 114.1, 113.3, 55.3.
FTIR (KBr) νmax (cm−1): 3397 (NH), 3058 (CH, aryl), 2904 (CH,
aryl), 1605 (CC, styryl), 1531 (CC, aryl), 1476 (CN, pyridyl),
1361 (CN, −NMe2), 1123 (CN, imidazolyl), 838 (PF), 763 (CC,
aryl), 546 (PF). ESI-MS (MeOH, m/z): calcd for C45H35IrN7 ([M −
PF6]+), 866.32; found, 866.26.
Singlet Oxygen Quantum Yields. ΦΔ for 1O2 generation in airsaturated MeOH was determined by monitoring the photo-oxidation
of 1,3-diphenylisobenzofuran (DPBF) promoted by the Ir(III)
complex. The absorbance of DPBF was adjusted to 1.0 at 409 nm
in air-saturated MeOH, and the absorbance of the Ir(III) complex was
adjusted to 0.1 at the irradiation wavelength. The photo-oxidation of
DPBF was monitored at the interval of 10 s. The ΦΔ value for 1O2
was calculated by a relative method using RB (ΦΔstd = 0.76 in airsaturated MeOH) as the reference.40 Similarly, the 1O2 generation
EXPERIMENTAL SECTION
1
Instruments. H NMR spectra were recorded on a Bruker Avance
300 or 400 spectrometer at 300 or 400 MHz. 13C{1H} NMR spectra
were recorded on a Bruker Avance 600 spectrometer at 600 MHz. All
chemical shifts are given relative to tetramethylsilane (TMS).
Microanalysis (C, H, and N) was carried out with a PerkinElmer
240Q elemental analyzer. Electron spray ionization mass spectra (ESIMS) were recorded on an AB QSTAR Pulsar mass spectrometer or an
Agilent LC/MSD TOF mass spectrometer. IR spectra were recorded
on an FT-IR Thermo Nicolet Avatar 360 spectrometer using a KBr
pellet. UV−vis spectra were recorded on a Shimadzu UV-2700
spectrophotometer equipped with a temperature controller accessory
and circulating water system. Emission spectra were recorded on an
Agilent Cary Eclipse fluorescence spectrophotometer using [Ru(bpy)3]2+ (Φr = 0.028 in air-equilibrated water solution36) as the
reference for calculating the quantum yield (Φ). The emission
lifetimes of the Ir(III) complexes were recorded on an Edinburgh
LFS-920 spectrometer with a hydrogen-filled excitation source. Cyclic
voltammetry (CV) measurements were performed with a CH
Instruments 840D electrochemical workstation. Samples were
dissolved in acetonitrile (water ≤30 ppm by Karl Fischer) with 0.1
M tetrabutylammonium hexafluorophosphate (TBAPF6) as a
supporting electrolyte and the ferrocene/ferrocenium couple (Fc/
Fc+) used as an internal standard. A 3 mm diameter platinum-plate
working electrode, a Ag+/AgCl (0.1 M AgNO3 in acetonitrile)
reference electrode, and a platinum-wire counter electrode were used.
All potentials are reported relative to the ferrocene/ferrocenium
couple (Fc/Fc+). All experiments using one-photon excitation were
performed with a blue light-emitting diode (LED) lamp (450 nm, 12 J
cm−2) as the light source. Two-photon photoluminescence spectra
were recorded on a SpectroPro300i instrument, and the pump laser
beam came from a Ti:sapphire laser system (pulse duration, 200 fs;
repetition rate, 76 MHz; Coherent Mira900-D). TPA cross sections
were measured by using the two-photon-induced fluorescence.37
Materials. The compounds 1,10-phenanthroline-5,6-dione38 and
[Ir2(ppy)4(μ-Cl)2]39 (ppy = 2-phenylpyridine) were synthesized
according to the literature methods. (E)-Cinnamaldehyde, (E)-4nitrocinnamaldehyde, (E)-4-dimethylaminocinnamaldehyde, and ammonium hexafluorophosphate (NH4PF6) were purchased from
Adamas-beta. (E)-4-Methoxycinnamaldehyde and silver triflate were
purchased from Energy Chemical. Ammonium acetate, propanoic
acid, aqueous ammonia, silica gel (200−300 mesh), N,N-dimethylformamide (DMF), acetonitrile (ACN), dichloromethane (DCM),
ethanol (EtOH), and methanol (MeOH) were purchased from
Greagent.
Synthesis and Characterization of Ir(III) Complexes. [Ir(ppy)2(sip)](PF6) (Ir1). A suspension of [Ir2(ppy)4(μ-Cl)2] (1.340 g,
1.25 mmol), 1,10-phenanthroline-5,6-dione (phendione, 0.788 g, 3.75
mmol), and silver triflate (0.642 g, 2.5 mmol) in 100 mL of 1/1 (v/v)
DCM/EtOH was stirred for 3 h under argon in a sealed reaction tube.
The volume of the DCM/EtOH solution was reduced to 20 mL by
reduced-pressure distillation and filtered. NH4PF6 (0.815 g, 5.0 mmol,
in 2 mL of H2O) was added, and [Ir(ppy)2(phendione)]PF6 (Ir0)
precipitated as a yellow solid (2.01 g, 94% yield) upon standing at 0
°C. Ir0 was dissolved in 100 mL of propanoic acid, and refluxed with
(E)-cinnamaldehyde (0.330 g, 2.50 mmol) and ammonium acetate
(3.88 g, 50.3 mmol) for 2 h. After it was cooled, the reaction mixture
was poured into a 200 mL aqueous solution of NH4PF6 (2.00 g, 12.4
mmol) and neutralized with concentrated aqueous ammonia. The
complex Ir1 precipitated as an orange solid, which was isolated by
suction filtration and washed with a small amount of cold methanol.
The crude product was purified by flash column chromatography (4
cm in diameter and 6 cm in length) on silica gel (200−300 mesh)
with DCM as eluent. Yield: 2.226 g, 92% based on [Ir2(ppy)4(μ-Cl)2].
Anal. Calcd for C43H30F6IrN8P·2H2O: C, 51.44; H, 3.41; N, 8.37.
Found: C, 51.21; H, 3.68; N, 8.09. 1H NMR ((CD3)2SO, 300 MHz):
δ 9.01 (d, J = 8.0 Hz, 2H), 8.87 (d, J = 8.5 Hz, 2H), 8.84 (d, J = 8.6
Hz, 2H), 8.23 (t, J = 7.8 Hz, 2H), 8.12 (d, J = 7.6 Hz, 2H), 8.07 (t, J =
4.7 Hz, 3H), 7.94 (d, J = 5.5 Hz, 1H), 7.91 (d, J = 5.4 Hz, 1H), 7.85
C
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Scheme 1. Synthetic Route of Cyclometalated Ir(III) Complexes in This Study
quantum yields in air-saturated aqueous solution ΦΔ(aq) were also
determined, using N,N′-bis(2,3-dihydroxypropyl)-9,10-anthracenedipropanamide (DHPA) instead of DPBF.
Quantum Chemistry Calculations. The DFT calculations were
carried out with the Gaussian 09 program package41 using the M06
(hybrid meta exchange-correlation functionals, which were parametrized including both transition metals and nonmetals and
containing dispersion correction) method and LanL2DZ basis set
(a double-ζ basis set containing effective core potential).42 Full
geometry optimization computations were carried out. The stability of
the optimized conformation of the complexes was confirmed by a
frequency analysis, which showed no imaginary frequency for each
energy minimum. Both geometry optimization and optical property
calculations were performed in water using the conductor polarized
continuum model (CPCM).43 For absorption spectral calculations,
the 80 lowest singlet excited states were calculated to reproduce the
experimental spectra in the 250−600 nm range. The discrete optical
transitions with their corresponding oscillator strengths were
broadened by a Gaussian function with a full width at half-maximum
(fwhm) of 3000 cm−1 to represent an experimental inhomogeneous
spectral broadening. To obtain the triplet (phosphorescence) and
singlet (fluorescence) emission energies, the molecular structures of
the lowest singlet excited state (S1) and lowest triplet excited state
(T1) were optimized using analytical TDDFT gradients. A compact
representation of an excited state via a photoexcited electron−hole
pair, which fits for chemical intuition, can be obtained using natural
transition orbitals (NTOs).44 When NTO calculations were
performed, an electron−hole pair transition from a ground state to
an excited state could be realized through unitary transformation of
the transition density matrix of a specific excited state.44 NTO pairs
contributing to the most important optical transitions were visualized
for plotting excited charge densities by setting the isovalue as 0.02.
For phosphorescence, we also used an additional method based on
the ΔSCF approach,45 which is computationally less expensive than
the optimization of the excited state and provides relatively accurate
results because of the inclusions of orbital relaxations. It involves a
geometry optimization on the ground triplet state using unrestricted
DFT and then a TDDFT calculation of a few lowest triplet excited
states using that ground-state triplet geometry.46
DNA Binding Experiments. Calf thymus DNA (CT-DNA) was
obtained from the Sigma Co. The preparation of the DNA stock
solution, determination of the DNA concentration, and DNA-binding
experiments of the Ir(III) compounds, including absorption spectral
titration, viscosity measurements, and thermal denaturation studies,
were performed as described previously.47
Two-Photon Confocal Laser Scanning Microscopy. The stock
solutions (100 μM) of Ir(III) complexes for cell experiments were
prepared in PBS containing 1% (v/v) DMSO (Sangon). Human lung
cancer cell lines A549 were grown on a 6 Chamber Glass Slide
(Thermo Fisher Scientific, USA) at a density of 6 × 104 cells/mL and
incubated for 1 h with the Ir(III) compounds at 10 μM in PBS
containing 0.1% (v/v) DMSO. The cells were washed twice with PBS
for 5 min. Nuclei and mitochondria were counterstained with 1.0 μg/
mL of DAPI (4′,6-diamidino-2-phenylindole, Solarbio) and 0.2 μM
MTG (MitoTracker Green, Invitrogen) for 30 min and washed with
PBS twice for 5 min. The two-photon laser microscopy system
consisted of a Nikon A1plus confocal microscope (Apo LWD 25x/
1.10 DIC N2 Objective, Galvano Scanner, NDD Reflect Detector, IR-
DM First Dichroic Mirror) and a COHERENT Chameleon Vision II
widely tunable laser (800.0 nm, 140 fs, 3.5 W, 80 MHz). Fluorescence
images were collected on three detection channels (blue, 420 nm;
green, 515 nm; red, 590 nm) and processed by NIS Elements
(Nikon) software. Z-stack images (12 loops) were collected with
800.0 nm excitation wavelength and 575.0 nm emission wavelength.
The Pearson correlation coefficients (R) were calculated by ImageJ
(1.53a) software48 for the colocalization of MTG and Ir(III)
complexes.
Photocytotoxicity. The photocytotoxicity of Ir1−Ir4 toward
three human cancer cell lines (lung A549, breast MCF7, and prostate
22Rv1) were tested by both one- and two-photon excitation
techniques. Exponentially grown cancer cells were seeded in triplicate
into 384-well plates at 1 × 104 cells per well. After incubation for 24 h,
the cells were treated with increasing concentrations of the tested
complexes. The culture medium alone was used as the blank. The
plates were incubated in the dark for 24 h. Then, all of the culture
media were refreshed. For the cell cultures exposed to light, all wells
were irradiated by a one- or two-photon light source for 10 min and
incubated for an additional 48 h. Finally, IC50 values of the
compounds in the two groups were measured by plotting the
percentage of viability that was tested by a CCK-8 kit (Sigma) versus
concentration on a logarithmic graph. The cell survival rate in the
control wells of the dark group was considered to represent 100% cell
survival. Phototherapeutic indexes (PIs) were calculated from the
ratio of dark to light IC50 values.
■
RESULTS AND DISCUSSION
Synthesis. Most current synthesis routes of [Ir(ppy)2L]+
type compounds have involved the reaction between
[Ir2(ppy)4(μ-Cl)2] and L, which usually required at least a 3fold excess of L to complete the conversion of [Ir2(ppy)4(μCl)2]. As more and more complicated structures of L were
designed for diverse functions of interest and synthesized with
more and more steps, this synthetic route has become less and
less efficient. Here we present an optimized route for the
preparation of [Ir(ppy)2L]+ type complexes (Scheme 1).
[Ir(ppy)2(phendione)]PF6 (Ir0) was first prepared with the
relatively economical 1,10-phenthroline-5,6-dione as L. The
obtained Ir0 was sufficiently pure to proceed to the next
reaction without column chromatography. Ir1−Ir4 were then
synthesized by the condensation of Ir0 and cinnamaldehydes.
The overall yields based on [Ir2(ppy)4(μ-Cl)2] and aldehydes
were 85−93%, which greatly improved the economy of both
the precious metal iridium and the material for ligand
synthesis. Although the cinnamaldehydes used in this paper
are inexpensive, this is a very economical synthetic route for
other precious aldehyde materials. These four Ir(III)
complexes could be prepared by a simple two-step reaction
with apparently higher yield, productivity, and efficiency in
comparison to the traditional reactions, facilitating its further
application in animal experiments and pharmaceutical
preparations. The prepared Ir(III) complexes have been
characterized by 1H NMR, 13C NMR, IR, ESI-MS, and
D
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ppy ligand and the d orbital of the Ir(III) ion and the LUMOs
are exclusively on the ligand, similar to the case for
[Ir(ppy)2(bpy)]+ (bpy = 2,2′-bipyridine) and some other
[Ir(ppy)2L]+ type complexes.40 For Ir4, however, the HOMO
is delocalized on the styryl part of the N^N ligand (location of
HOMO-1 energy levels for Ir1−Ir3). The MO which is
delocalized on the ppy ligand and the d orbital of the Ir(III)
ion (HOMOs for Ir1−Ir3) for Ir4 lowers to HOMO-1. This
difference on the MO population is thought to be the result of
a conjugation function between the lone pair on the N atom
and phenyl ring for Ir4, as the optimized structure showed the
two −CH3 groups are located nearly coplanar with the phenyl
group of sip.
These different frontier MO populations give rise to
different ground state energy levels and excitation properties.
The ground state energy diagram for Ir1−Ir4 is shown in
Figure 3, and the calculated electronic absorption spectral data
elemental analysis. Ir1−Ir4 have good solubility in water. With
5% (v/v) DMSO, the concentration of the stock solution could
reach 400 μM in water or phosphate-buffered saline (PBS).
Electronic Absorption. The UV−vis absorption spectra of
complexes Ir1−Ir4 were recorded in MeOH at room
temperature (Figure 2), and the absorption band maxima
Figure 2. Absorption (solid) and normalized emission spectra
(dashed) of Ir1−Ir4 in air-saturated MeOH solution. The excitation
wavelengths are 404, 418, 416, and 392 nm for Ir1−Ir4, respectively.
and molar extinction coefficients (ε) are summarized in Table
1. The absorption follows the Beer−Lambert law in the
concentration range of 1−100 μM, suggesting the absence of
ground state aggregation in this tested concentration range.
The absorption spectra for Ir(III) complexes are comprised of
two main absorption bands (an intense band below 400 nm
and a weaker band extended to 404−473 nm), in line with the
reported Ir(III) complexes with extended π-conjugated
diamine (N^N) ligands.46,49,50
To better understand the effect on the absorption spectra of
the substituent groups on the N^N ligand, DFT calculations
were performed for Ir1−Ir4. As we have focused on the
biological applications of the Ir(III) complexes in this paper,
the solvent effect was implicitly included by CPCM for water
in the theoretical study and discussed with the experimental
data in aqueous solution as well. The optimized structures
showed that all complexes adopted a similar octahedral
geometry around the Ir(III) center and the dihedral angles
within the N^N ligand are very close to 0 or 180° (Figure S1).
The calculated electron density distributions of the HOMO
and LUMO are different for the four complexes (see the
representative frontier molecular orbital plots for Ir1−Ir4 in
Figure S1). For Ir1−Ir3, the HOMOs are delocalized on the
Figure 3. Ground state energy diagram for Ir1−Ir4 (M06/Lanl2dz).
The solvent water was included by the conductor polarized
continuum model (CPCM).
(wavelengths, oscillator strengths, excitation energies, assignments of contributions to excited states) and their comparisons
with the corresponding experimental data in aqueous solution
are summarized in Table S1. The simulated absorption spectra
are in good accordance with the experimental spectra (Figure
S2). For Ir1−Ir3, there are two theoretical metal to ligand
charge transfer (MLCT) transitions. The first MLCTs
(HOMO → LUMO) are mixed with ligand to ligand charge
transfer (LLCT) transitions (πppy → π*sip), and the calculated
Table 1. Photophysicala and Electrochemistryb Data for Ir(III) Complexes
emission/nm
(Φ)
ΦΔ
ΦΔ(aq)d
τ/ns
δe/GM
468 (sh, 3.8), 404 (sh, 2.2), 365 (51.2),
342 (sh, 77.0)
473 (sh, 10.2), 418 (sh, 46.7), 373 (76.2)
572 (0.12)
0.85
0.24
875
162
+0.80
590 (0.04)
0.37
0.14
836
113
+0.84
467 (sh, 4.1), 416 (sh, 18.5), 357 (sh, 77.6),
344 (80.5)
473 (sh, 13.0), 392 (70.3)
572 (0.18)
0.95
0.31
942
168
+0.78
577 (0.03)
0.57
0.17
851
152
+0.76, +0.50
complex
absorption/nmc (ε/103 Μ−1 cm−1)
Ir1
Ir2
Ir3
Ir4
Eox/V
Ered/V
Egap/V
ΔE/eV
−1.28, −1.62,
−2.14
−1.12, −1.52,
−2.08
−1.36, −1.54,
−2.04
−1.42, −1.58,
−2.12
2.08
3.48
1.96
2.54
2.14
3.49
1.92
3.01
In air-saturated MeOH solution (2 × 10−5 M), except for ΦΔ(aq). bCyclic voltammograms performed at a scan rate of 200 mV s−1 versus Fc/Fc+
using 0.1 M TBAPF6 as a supporting electrolyte in degassed MeCN. cShoulder peaks are marked as sh. dIn aqueous solution (2 × 10−5 M). eTwophoton absorption cross sections (δ) upon excitation at λex = 800 nm (1 GM = 10−50 cm4 s photon−1).
a
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Table 2. Natural Transition Orbitals (NTOs) for Low-Energy Transitions of Ir1−Ir4
a
Experimental emission wavelengths in aqueous solution. bTriplet emission wavelengths calculated by TDDFT optimized excited triplet state
geometries. Values in parentheses represent the emission wavelengths calculated by the ΔSCF approach.
oscillator strengths (f) are very low (from 0 to 0.0004).
Therefore, the second MLCTs H-2 → LUMO (f = 0.1244) for
Ir1, H-1 → LUMO (f = 1.0447) for Ir2, and H-3 → LUMO (f
= 0.1782) for Ir3 are considered as major contributions to the
MLCT absorptions (the weaker absorption bands above 400
nm) of their complexes. The stronger absorption bands
between 400 and 360 nm are attributed to the intraligand (IL)
transitions of sip-type ligands (πsip → π*sip). Due to the
different MO population of Ir4, some strong IL transitions
appear at longer wavelength, giving its MLCT band partial
intraligand character. As a result, Ir4 has a longer absorption
wavelength in comparison to the other three complexes.
Natural transition orbital (NTO) calculations were also
performed to explore the characters of the above optical
transitions. Cartesian coordinates of the optimized structures
of Ir1−Ir4 for the ground state (S0, DFT), first excited singlet
state (S1, TDDFT), and first excited triplet state (T1,
TDDFT) are given in Tables S2−S5. As indicated by the
NTOs (Table 2), the holes in all complexes were primarily
localized on the N^N ligands and the d orbital of the Ir(III)
ion. The electron-density distribution calculations show that
the HOMO → LUMO transition played the major role (99.4−
100%) in generating the S1 excited state and the substituent
groups had an apparent effect on the hole energy. Therefore, it
is important to analyze how structural modifications influenced
the HOMO and LUMO energy levels. As displayed in the
ground-state energy diagram (Figure 3), an electron-withdrawing group (−NO2 in Ir2) lowered the energy level of both
the HOMO and LUMO, while an electron-donating group
(−OMe in Ir3 and −NMe2 group in Ir4) raised the energy
level of the HOMO and LUMO. The HOMO−LUMO energy
gaps in Ir2 and Ir4 are apparently smaller than those in Ir1 and
Ir3. In view of the direct relationship between the frontier
molecular orbital energy level and the redox potential, the
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electrochemical properties of complexes Ir1−Ir4 were then
investigated.
Electrochemistry. To confirm the effect of substituent
groups on the HOMO−LUMO gaps in Ir1−Ir4, ground state
redox potentials of Ir1−Ir4 were investigated in degassed
anhydrous CH3CN solutions by CV using the Fc+/Fc couple
as the internal standard. Ir1−Ir3 showed an irreversible couple
from 0.78 to 0.84 V (Table 1 and Figure S3), which was
assigned to Ir(IV)/Ir(III) oxidation.40,51,52 For Ir4, however,
two oxidation couples were observed. This experimental result
could be explained by the DFT calculations. As illustrated in
Figure 3, the HOMO of Ir4 is composed of the π orbital of
N^N ligand and its energy level is obviously higher than those
of Ir1−Ir3. The occupied d orbital of Ir(III) in Ir4 mainly
populates on HOMO-1, with an energy level close to that of
the HOMO of Ir1−Ir3 and a similar orbital population.
Therefore, the two oxidation couples at 0.50 and 0.76 V were
attributed to be the oxidation of the N^N ligand and Ir(III)
ion, respectively.
Three reduction couples (from −1.12 to −1.42 V, from
−1.52 to −1.62 V, and from −2.04 to −2.14 V) were observed
for Ir1−Ir4, representing the reduction of sip and two ppy
ligands, comparable to the case for similar Ir(III) complexes.51−53 The trend of the electrochemical energy gaps
(Egap) of these complexes matches well with the trend of the
calculated HOMO−LUMO gaps (ΔE), although there appears
to be some discrepancy in the absolute values of ΔE with Egap.
Therefore, the DFT calculations clearly explained the trend in
energy level shifts upon modification by different substituent
groups. A similar discrepancy between the calculated HOMO−
LUMO gaps and the electrochemical energy gaps has been
reported in other Ir(III) complexes.54,55
Photoluminescence. Photoluminescent properties are of
great importance to Ir(III) complexes in many application
fields, such as organic light-emitting diodes (OLEDs), solar
energy conversion, luminescent biological labeling, etc.56
Because the emission of Ir(III) complexes typically originates
from the excited triplet state, the emission characteristics of
Ir1−Ir4 at room temperature were investigated to understand
the effects of substitution on the excited triplet states with the
assistance of DFT calculations.
The emission spectra of Ir1−Ir4 in water are displayed in
Figure S4, and the emission band maxima are summarized in
Table 2, together with theoretical data obtained by excited
triplet state DFT calculations and NTO analysis. In order to
intuitively reflect the photoluminescent properties and relative
intensity of each compound, all compounds used the exact
same measurement conditions, such as compound concentration, excitation wavelength, slit width, detector voltage, etc.,
and the resulting emission spectra were not normalized. The
emission spectrum of Ir1 (single peak at 600 nm) was
structureless, which is a characteristic of 3 CT state
(3MLCT/3LLCT) emission.57−59 The theoretical emission
wavelength obtained by TDDFT calculations (611 nm) was in
good accordance with the experimental data. The spin-density
distributions shown in Table 2 for Ir1 agree well with the
NTOs representing the excitation from S0 to T1 (99.2% for
3
MLCT/3LLCT states). In contrast, the emission spectra of
Ir2−Ir4 were apparently red shifted in comparison to Ir1 and
exhibited clear vibronic structures (shoulder peaks at 685, 718,
and 697 nm for Ir2−Ir4, respectively), suggesting that the
emission of these three complexes emanated from the
intraligand (IL, ligand-centered 3π,π* state) transition.53
Article
NTOs obtained by the TDDFT calculations also supported
this attribution (Table 2). The spin density of S0 to T1
excitation for Ir2−Ir4 was mainly distributed on the sip-type
ligands for both holes and electrons, and the theoretical
contributions for these 3IL transitions were 97.9−100%. These
3
IL transitions have been proven to be very important for a
metal-based PDT agent to sensitize 1O2, such as TLD1433.32
The advantage of TDDFT calculations is that they can
provide specific information about the excited-state transitions
shown above. However, the calculation results of the excitation
energy deviated greatly from the experimental data. Therefore,
an additional calculation based on the ΔSCF approach45 was
performed, which provides more accurate results because of
the inclusion of orbital relaxations. As shown in Table 2, the S0
to T1 excitation energies obtained by the ΔSCF approach were
much closer to the experimental data, providing support for
the rationality of theoretical calculation results. The above
studies of fluorescence properties and calculations of excited
states have revealed the luminescent nature of this class of
compounds and how the substituents affected their luminescent properties, which could have important implications for
the design of Ir(III)-based cellular photoluminescent dyes and
photosensitizers for PDT.
Singlet Oxygen Quantum Yield. To estimate the
efficiency of 1O2 generation, which is essential for PDT, the
1
O2 generation quantum yields (ΦΔ) for all Ir(III) complexes
were measured by monitoring the time-dependent absorbance
of DPBF at 409 nm (Table 1 and Figure S5). The ΦΔ values
for Ir1 and Ir3 were found to be 0.85 and 0.95, indicating a
high 1O2 generation efficiency. Ir3 has a higher ΦΔ value than
Ir1. On one hand, this may be due to the electron-withdrawing
effect of the methoxy group. It have also been observed
recently that ΦΔ values of similar Ir(III) complexes increased
greatly by the introduction of electron-donating substituents.40
On the other hand, the higher triplet quantum yield (stronger
emission intensity) of Ir3, as discussed above in the
photoluminescence section (Figure S4), might also contribute
to its higher ΦΔ value, because the efficiency of 1O2 generation
was considered to be relative to the triplet excited state energy
and quantum yield.40 Therefore, it would not be surprising that
Ir2 has a very low 1O2 generation efficiency (ΦΔ = 0.37),
because its low emission intensity (Figure S4) and strongly
electron withdrawing substituent (−NO2). Although Ir4 has
an electron-donating substituent (−NMe2), its triplet excited
state energy level and quantum yield are much lower than
those of Ir1 and Ir3. The 1O2 generation efficiency of Ir4 (ΦΔ
= 0.57) suggested that the triplet quantum yield played a more
important role than the electronic effect of the substituent
group. These studies suggested that the 1O2 generation
efficiency is tunable via simple substitution on the sip ligand,
and the higher ΦΔ values might result in a better performance
of Ir1 and Ir3 in cell imaging and PDT. Similarly, the 1O2
generation quantum yields in aqueous solution ΦΔ(aq) were
also determined (Table 1), using DHPA instead of DPBF. All
the tested ΦΔ(aq) values are higher than those of the reported
cyclometalated Ir(III) complexes.60
Two-Photon-Absorption (TPA) Cross Sections. To
examine the TPA properties of Ir1−Ir4, the TPA cross
sections (δ) of these compounds were also investigated, using
RB as the standard. The TPA cross sections of the complexes
were 113−168 GM (1 GM = 10−50 cm4 s photon−1), as shown
in Table 1. As suggested by Furuta, δ values should be higher
than 0.1 GM for optical imaging applications in live
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specimens.61 The δ values of Ir1−Ir4 not only meet the
requirements mentioned above but are also 2-fold higher than
those of similar mononuclear Ir(III) complexes.60 However,
the δ values of Ir1−Ir4 are lower than those of some dinuclear
Ir(III) complexes.37
Cellular Localization by Two-Photon Confocal Laser
Scanning Microscopy. After confirming that Ir1−Ir4 can
efficiently produce 1O2 upon light irradiation, we investigated
their cellular uptake and cellular localization. The cellular
localization of Ir1−Ir4 was investigated by confocal laser
scanning microscopy (CLSM) on human lung cancer cells
A549 in PBS containing 0.1% (v/v) DMSO. As shown in
Figure 4, all Ir(III) complexes showed strong red luminescence
Article
In Vitro Photodynamic Therapy Studies by One- and
Two-Photon Technology. The toxicities of Ir1−Ir4 on
three human tumor cell lines (A549, MCF7 and 22Rv1) were
first investigated in the dark to assess whether the compounds
were nontoxic, which is an important property for a PDT
agent. It is encouraging that all compounds were found to be
nontoxic toward these cells in the absence of light (IC50 > 160
μM, Table 3). The phototoxicities of the Ir(III) complexes on
the same tumor cell lines were then investigated. For OP-PDT
(450 nm, irradiated for 10 min and incubated for 48 h), all
Ir(III) compounds became highly toxic. Cells untreated by
Ir(III) complexes were also exposed to the same irradiation
procedure and found not to be affected. Among the three
different tumor cell lines studied, all Ir(III) complexes showed
a better inhibitory effect on A549 cells (Table 3), in
comparison to MCF7 and 22Rv1 cells. Ir3 has the highest
inhibitory activity for the three tested cell lines among the four
Ir(III) complexes. The photocytotoxicity of Ir3 to A549 cells
tested by the one-photon excitation (OPE) technology
reached an IC50 value as low as 1.35 μM (PI-1 = 179).
Under similar experimental conditions, the clinically approved
PDT agent 5-aminolevulinic acid (5-ALA) displayed a lower
phototoxicity and a lower PI in comparison to the Ir(III)
complexes studied in this work. [Ir(tpy)(pbpz)Cl]+ has the
highest activity and PI among the four Ir(III) complexes.64
Although the OPE IC50 value of [Ir(tpy)(pbpz)Cl]+ is lower
than that of Ir3 in this study, its dark cytotoxicity is much
higher. Therefore, PI-1 for Ir3 is over 2-fold that of
[Ir(tpy)(pbpz)Cl]+.
The TP-PDT experiments were then performed under the
same cell conditions, except for the 800 nm laser light source.
All Ir(III) complexes showed higher phototoxicity toward the
three tested tumor cell lines (Table 3). The two-photon
excitation (TPE) phototoxicity index values (PI-2) for all
Ir(III) complexes were also apparently larger in comparison to
those irradiated by 450 nm light (Table 3). Cisplatin, as a
traditional chemotherapeutic drug without PDT activity,
showed no significant difference in toxicity (PI = 1.00−
1.15), under dark, OPE, or TPE conditions. As the most
promising compound of the series, Ir3 showed a very high
photocytotoxicity to A549 cells (IC50 value as low as 0.96 μM,
PI-2 = 253), as tested by TPE technology. This exciting
experimental result was obtained by simply improving an
experimental operationthe use of 384-well plates. Usually,
96-well plates are used for in vitro PDT experiments. However,
the TP laser is concentrated, and it is difficult to cover the
entire well area. Therefore, the cell viability estimation is more
difficult, and a precise cell viability test has to be performed
only in the irradiated area.22 This limitation can nevertheless
be overcome by using 384-well plates and a low-magnification
objective, which allow scanning the whole well with the laser.65
By plating the same number of cells in the 96-well plate into
the 384-well plate, the number of cells per volume increased
significantly, which allowed the experimental results to show
the effect of light penetration on cell viability. In this study, the
superiority in phototoxic properties of Ir1−Ir4 irradiated by
800 nm light to those irradiated by 450 nm light should be
attributed to the excellent penetration of infrared light. The
IC50 values toward three different tumor cell lines correlate
well with their efficiency in 1O2 generation (Ir3 > Ir1 > Ir4 >
Ir2). Surprisingly, the dark toxicity of Ir2 is very low (418−
704 μM), giving it a higher than expected PI value. This could
be considered as an advantage for the PDT properties of Ir2.
Figure 4. Cellular localization of Ir1−Ir4 by two-photon confocal
laser scanning microscopy on human cancer cells A549 in PBS
containing 0.1% (v/v) DMSO: MTG, mitochondrion dye, green
channel; Ir(III), Ir1−Ir4, red channel; DAPI, nucleus (DNA) dye,
blue channel; BF, bright field; Merged, overlay of the four channels on
the left.
within the cells under TP excitation (800 nm). The specific
cellular target of Ir1−Ir4 was further confirmed by
colocalization assays. The signals of Ir1−Ir4 overlapped well
with the that of commercial mitochondrion-specific dye MTG.
In contrast, a poor correlation coefficient was found when the
nucleus dye DAPI was used. Therefore, mitochondria are the
main target of Ir1−Ir4 in A549 cells. In order to confirm this
conclusion, Pearson correlation coefficients (R) have been
calculated for the colocalization between MTG and Ir(III)
complexes (Figure S6). The values of R reached 0.91−0.94 for
Ir1−Ir4, suggesting a very high colocalization. The fact that
Ir1−Ir4 target on mitochondria may be beneficial for their
PDT activity, because a mitochondria-targeted Ir(III) complex
has been found to exhibit an improved PDT effect in
comparison to a lysosome-targeted Ir(III) complex with
similar photophysical properties and 1O2 quantum yields,
especially under hypoxia conditions.62
Moreover, as displayed in the Z-stack images (Figure S7), all
Ir(III) complexes gave clear fluorescence photos of the entire
cells at 12 increasing depths. It can be assumed, not arbitrarily,
that a 3D image of the mitochondria in the whole cell can be
obtained by setting a small enough scanning interval.63
Therefore, these Ir(III) complexes have good potential as
highly sensitive cellular mitochondrial dyes and PDT agents.
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The IC50 values in the dark (incubated for 24 h). bThe IC50 values under one-photon excitation (450 nm, irradiated for 10 min and incubated for 48 h). cThe IC50 values under two-photon excitation
(800 nm, irradiated for 10 min and incubated for 48 h). dPI-1 is the phototoxicity index for one-photon excitation, which is the ratio between the IC50 values in the dark and those under one-photon
excitation. ePI-2 is the phototoxicity index for two-photon excitation, which is the ratio between the IC50 values in the dark and those under two-photon excitation. fMeasured IC50 values were adjusted
to account for the fact that four molecules of ALA are required for the formation of the active species protoporphyrin IX. gFrom ref 64.
Although the excellent phototoxicity of these Ir(III)
complexes, under both OPE and TPE conditions, could be
largely attributed to their high 1O2 yield, we have also tested
other possible antitumor mechanisms related to DNA binding.
The DNA-binding properties of Ir1−Ir4 have been studied by
absorption spectral titration, DNA thermal denaturation, and
DNA viscosity measurements. With increasing amounts of CTDNA, the absorption spectra of Ir1−Ir4 showed slight changes
in both absorbance (hypochromic effect less than 5%) and
wavelength of absorption peaks (red shift less than 3 nm), as
displayed in Figure S8. Negligible differences have been found
in the melting point of DNA (Tm) in the absence and presence
of Ir(III) complexes ([DNA]/[Ir] = 10/1), as illustrated in
Figure S9. The viscosity of DNA also showed slight changes in
the presence of Ir(III) complexes in the tested concentration
range (Figure S10). All of these results suggested that Ir1−Ir4
have very low DNA affinity. Therefore, they are unlikely to
have a significant inhibitory effect on DNA-related enzymes.
This may explain why these compounds showed very low dark
toxicity and their selectivity for mitochondria instead of the
nucleus, where the DNA content is obviously high.
■
CONCLUSIONS
Since TLD1433 has entered a human clinical trial, more
attention should be paid to the pharmaceutical issue in the
research of PDT agents, in addition to the pursuit of high
activity. In this study, four cyclometalated Ir(III) complexes
that can specifically accumulate in mitochondria have been
designed and prepared by an optimized synthetic route with
apparently higher yield, productivity, and efficiency in
comparison to the traditional methods. With the help of a
spectroscopic study and DFT/TDDFT quantum chemistry
theoretical calculations, their ground state and excited singlet
and triplet state properties have been understood in detail. Ir1
and Ir3 have high 1O2 production quantum yields, impressive
TP absorption properties, and a low dark cytotoxicity.
Importantly, they could induce cell apoptosis upon TP
irradiation with very high PIs in comparison to similar
complexes. These Ir(III) complexes, especially Ir3, hold
great promise as TP mitochondrial dye and PDT agents.
Overall, in this study, TP-PDT agents with high antitumor
activity and PI were successfully designed by the combination
of a substituent group which can amplify ROS and an Ir(III)
complex with good TP-PDT properties. The results suggest
that the complexes have enormous potential for application as
a mitochondrion-specific dye in cell biology as well as a
photosensitizer for TP-PDT in medicine. Studies using Ir3 as
in vivo TP-PDT agents and further modification of the ppy
ligands to improve the PDT activity and PI are currently
underway.
■
ASSOCIATED CONTENT
sı Supporting Information
*
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.inorgchem.0c01509.
Optimized structures and frontier molecular orbitals,
simulated and experimental electronic absorption
spectra, cyclic voltammetry, emission spectra in aqueous
solution, time-dependent changes in absorption spectra
of DPBF in the presence of Ir(III) complexes, Pearson’s
correlation coefficient (R) analysis (PCCA) between
Ir(III) complexes and MTG by CLSM on human cancer
a
6.49 ± 0.43 (31.6)
11.7 ± 0.96 (35.7)
4.51 ± 0.23 (41.5)
7.13 ± 0.52 (36.9)
1.09 ± 0.06 (1.04)
97.5 ± 7.48 (1.66)
8.52 ± 0.68 (24.1)
13.6 ± 0.83 (30.7)
6.36 ± 0.52 (29.4)
9.72 ± 0.76 (27.1)
1.04 ± 0.06 (1.09)
32.7 ± 2.94 (4.95)
205 ± 17.8
418 ± 34.6
187 ± 13.6
263 ± 19.0
1.13 ± 0.07
162 ± 7.28
5.53 ± 0.42 (40.5)
8.43 ± 0.85 (49.1)
2.84 ± 0.14 (57.7)
7.61 ± 0.81 (28.4)
1.04 ± 0.05 (1.00)
124 ± 6.81 (1.47)
7.86 ± 0.56 (28.5)
9.72 ± 0.72 (47.6)
4.31 ± 0.27 (38.1)
9.24 ± 0.37 (23.4)
1.01 ± 0.06 (1.04)
38.7 ± 3.13 (4.70)
224 ± 20.6
463 ± 51.7
164 ± 10.3
216 ± 18.5
1.05 ± 0.06
182 ± 9.43
3.73 ± 0.26 (97.0)
6.38 ± 0.72 (110)
0.96 ± 0.08 (253)
3.86 ± 0.42 (81.3)
1.13 ± 0.09 (1.10)
116 ± 5.42 (1.51)
5.37 ± 0.36 (67.4)
8.76 ± 0.69 (80.4)
1.35 ± 0.14 (179)
5.91 ± 0.06 (53.1)
1.08 ± 0.08 (1.15)
34.1 ± 2.30 (5.13)
0.6 ± 0.1 (94.3)
362 ± 28.2
704 ± 51.6
242 ± 18.2
314 ± 26.4
1.24 ± 0.10
175 ± 8.40
56.6 ± 0.2
dark
TPE (PI-2)
OPE (PI-1)
dark
compound
Ir1
Ir2
Ir3
Ir4
cisplatin
5-ALAf
[Ir(tpy)(pbpz)Cl]+ g
OPE (PI-1)d
OPE (PI-1)
d
b
a
e
c
A549
d
b
a
Table 3. (Photo)cytotoxicity (IC50, μM) of Ir1−Ir4 toward Three Human Tumor Cell Lines
MCF7
c
TPE (PI-2)
e
dark
a
b
22Rv1
TPEc (PI-2)e
Inorganic Chemistry
I
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ACKNOWLEDGMENTS
This work was supported by National Natural Science
Foundation of China (21662039, 21907044), Yunnan
Provincial Science and Technology Department (2018FB022,
2019FB124), Ten Thousand Talents Project of Yunnan
Province (YNWR-QNBJ-2018-057), and the Program for
Changjiang Scholars and Innovative Research Team in
University (IRT17R94). F.G. thanks the High Performance
Computing Center of Yunnan University for computational
support. D.L. acknowledges the support of grants from the
Hundred Talent Program of Kunming Medical University.
cells A549, Z-stack images of A549 stained by Ir(III)
complexes, DNA-binding experiments, and experimental
and computational absorption spectral data and
Cartesian coordinates of the optimized structures of
Ir(III) complexes for S0, S1, and T1 states (PDF)
■
Article
AUTHOR INFORMATION
Corresponding Authors
Dandan Liu − School of Pharmaceutical Sciences and Yunnan
Key Laboratory of Pharmacology for Natural Products,
Kunming Medical University, Kunming 650500, Yunnan,
People’s Republic of China; Email: 709249906@qq.com
Feng Gao − Key Laboratory of Medicinal Chemistry for Natural
Resource, Ministry of Education, Yunnan Research &
Development Center for Natural Products, School of Chemical
Science and Technology, Yunnan University, Kunming 650091,
Yunnan, People’s Republic of China; orcid.org/0000-00017490-4887; Email: gaofeng@ynu.edu.cn
■
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Authors
Xu-Dan Bi − Key Laboratory of Medicinal Chemistry for
Natural Resource, Ministry of Education, Yunnan Research &
Development Center for Natural Products, School of Chemical
Science and Technology, Yunnan University, Kunming 650091,
Yunnan, People’s Republic of China
Rong Yang − Key Laboratory of Medicinal Chemistry for
Natural Resource, Ministry of Education, Yunnan Research &
Development Center for Natural Products, School of Chemical
Science and Technology, Yunnan University, Kunming 650091,
Yunnan, People’s Republic of China
Yue-Chen Zhou − Key Laboratory of Medicinal Chemistry for
Natural Resource, Ministry of Education, Yunnan Research &
Development Center for Natural Products, School of Chemical
Science and Technology, Yunnan University, Kunming 650091,
Yunnan, People’s Republic of China
Daomei Chen − National Center for International Research on
Photoelectric and Energy Materials, School of Materials and
Energy, Yunnan University, Kunming 650091, People’s Republic
of China
Guo-Kui Li − Key Laboratory of Medicinal Chemistry for
Natural Resource, Ministry of Education, Yunnan Research &
Development Center for Natural Products, School of Chemical
Science and Technology, Yunnan University, Kunming 650091,
Yunnan, People’s Republic of China
Yuan-Xiao Guo − Key Laboratory of Medicinal Chemistry for
Natural Resource, Ministry of Education, Yunnan Research &
Development Center for Natural Products, School of Chemical
Science and Technology, Yunnan University, Kunming 650091,
Yunnan, People’s Republic of China
Meng-Fan Wang − Key Laboratory of Medicinal Chemistry for
Natural Resource, Ministry of Education, Yunnan Research &
Development Center for Natural Products, School of Chemical
Science and Technology, Yunnan University, Kunming 650091,
Yunnan, People’s Republic of China
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.inorgchem.0c01509
Notes
The authors declare no competing financial interest.
J
https://dx.doi.org/10.1021/acs.inorgchem.0c01509
Inorg. Chem. XXXX, XXX, XXX−XXX
Inorganic Chemistry
pubs.acs.org/IC
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