Development of a cyclometalated iridium complex with specific intramolecular hydrogen-bonding that acts as a fluorescent marker for the endoplasmic reticulum and causes photoinduced cell death

Article abstract of DOI:10.1039/c4dt00845f

Cyclometalated iridium complexes have important applications as phosphorescent probes for cellular imaging due to their photophysical properties. Moreover, these properties also make them potential candidates as photosensitizers for photodynamic therapy (PDT) of tumors and skin diseases. Treatment of MCF7 breast carcinoma cells with a heteroleptic phosphorescent cyclometalated iridium(iii) complex C2 followed by confocal imaging indicates that the complex selectively localizes and exhibits high fluorescence in the endoplasmic reticulum. In an unprecedented approach, systematic alteration of functional groups or the metal core in C2 to synthesize a series of iridium(iii) complexes (C1-C10) and an organometallic rhenium complex C11 with an imidazolyl modified phenanthroline ligand has indicated the functional groups and their interactions that are responsible for this selective localization. Remarkably, the exposure of the cells treated with C2 to irradiation at 405 nm for one hour led to membrane blebbing and cell death, demonstrating a photosensitizing property of the compound. This journal is

Full text of DOI:10.1039/c4dt00845f

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Development of a cyclometalated iridium complex
with specific intramolecular hydrogen-bonding
that acts as a fluorescent marker for the
endoplasmic reticulum and causes photoinduced
cell death†
Cite this: Dalton Trans., 2014, 43,
17463
a
b
b
b
Soumik Mandal,‡ Dipak K. Poria,‡ Ritabrata Ghosh, Partho Sarothi Ray* and
Parna Gupta*
a
Cyclometalated iridium complexes have important applications as phosphorescent probes for cellular
imaging due to their photophysical properties. Moreover, these properties also make them potential can-
didates as photosensitizers for photodynamic therapy (PDT) of tumors and skin diseases. Treatment of
MCF7 breast carcinoma cells with a heteroleptic phosphorescent cyclometalated iridium(III) complex C2
followed by confocal imaging indicates that the complex selectively localizes and exhibits high
fluorescence in the endoplasmic reticulum. In an unprecedented approach, systematic alteration of func-
tional groups or the metal core in C2 to synthesize a series of iridium(III) complexes (C1–C10) and an
organometallic rhenium complex C11 with an imidazolyl modified phenanthroline ligand has indicated
the functional groups and their interactions that are responsible for this selective localization. Remarkably,
the exposure of the cells treated with C2 to irradiation at 405 nm for one hour led to membrane blebbing
and cell death, demonstrating a photosensitizing property of the compound.
Received 20th March 2014,
Accepted 19th September 2014
DOI: 10.1039/c4dt00845f
www.rsc.org/dalton
1
,2
of substituted phenyl pyridine and polypyridyl units. Pre-
sently, small-molecular dyes, fluorophore-conjugated anti-
bodies or fluorescent proteins are used commercially as
imaging agents for cellular organelles such as nuclei, mito-
chondria, endoplasmic reticulum (ER), Golgi complex and
Introduction
The cyclometalated heteroleptic iridium(III) complexes of poly-
pyridyl ligands are highly efficient in imaging of cells and
cellular organelles, and are important tools for studying
cellular function. The efficacy of the cyclometalated iridium(III)
complexes in this field has been demonstrated with a range
3
lysosomes. The cyclometalated iridium(III) complexes provide
a number of important advantages over these currently avail-
able imaging agents. These include enhanced chemical and
photochemical stability within the cellular environment, per-
meability, minimization of self-quenching due to the signifi-
cant Stokes’ shift, long-lived triplet excited state (τ ∼ μs)
compared to organic dyes (τ ∼ ns) that helps to eliminate the
short-lived autofluorescence of biological samples, and micro-
environment-sensitive emission characteristics which are less
a
Department of Chemical Sciences, Indian Institute of Science Education and
Research-Kolkata, Mohanpur Campus, Mohanpur, Nadia, West Bengal 741252,
India. E-mail: parna@iiserkol.ac.in; Fax: +91 3473279131; Tel: +91 3473279130
b
Department of Biological Sciences, Indian Institute of Science Education and
Research-Kolkata, Mohanpur Campus, Mohanpur, Nadia, 741252, India.
E-mail: psray@iiserkol.ac.in
†
Electronic supplementary information (ESI) available: Detailed synthesis;
4
,5
photophysical data (Table S1); pH dependent phosphorescence lifetime of damaging to cellular components. Kinetic inertness of the
complex C2 (Table S2); crystallographic parameters of C2; selected bond dis- low-spin octahedral d complexes contributes to both reducing
6
tances and angles of C2 (Table S3); cyclic voltammetric data of complexes C1–
the cytotoxicity of the heavy metal ions and enhancing the
1
C11 (Table S4); H NMR spectra of ligands and complexes (Fig. S1 and S3);
stability of the complexes. The high photostability of these
complexes allows the continuous monitoring of biological
ESI-MS spectra of ligands and complexes (Fig. S2 and S4); fluorescence spectra
of the complexes in acetonitrile and at pH 4, 7 and 9, exponential decay curve of
C2 (Fig. S5); pH dependent fluorescence spectrum of complexes C1–C11 events by fluorescence spectroscopy and microscopy. High
(
Fig. S6); DIC and confocal fluorescence images of live MCF7 cells not treated fluorescence lifetimes and tunable emission properties have
with C2 but exposed to photoirradiation at 405 nm for 30 min; the cells were
treated with DCFDA and fluorescence images were obtained at 529 nm after exci-
tation at 495 nm (Fig. S7). ESI videos 1 and 2. CCDC 967841. For ESI and crystal-
lographic data in CIF or other electronic format see DOI: 10.1039/c4dt00845f
6
7
8
resulted in their exploitation in sensors, OLEDs, OLECs, cat-
9
alysts for water splitting and dye-sensitized solar cells
1
0
(
DSSCs), together with successful usage as cellular imaging
1
1
‡
Authors contributed equally.
agents.
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A major challenge in the field of cellular imaging is the
selective staining of specific cell organelles or sub-cellular
compartments. The factors that influence the cellular uptake
and distribution within the microenvironment include per-
meability through the cell membrane, diffusion in the cellular
cytoplasm, the charge-interaction associated with the organel-
lar membranes during the uptake and interaction with orga-
nellar components through non-covalent forces, e.g. hydrogen
bonds, ionic bonds, van der Waals forces and hydrophobic
interactions, which results in specific localization. Within the
micro- or nano-environment of the cell, specific localization is
1
2
mostly regulated by pH, dielectric constant, hydrophobic and
1
3–16
hydrophilic interactions and redox potential.
Cyclometa-
lated iridium complexes have been reported as cellular
imaging probes for visualization of different organelles like
1
7,18
19
1,2,20
21
nucleoli,
Golgi complex, cytoplasm,
mitochondria,
2
2
23
lysosomes and endosomes and endoplasmic reticulum as
well as intracellular metal-containing molecules
2
1,24
and they
have also been reported to act as efficient photosensitizing
2
5
agents in cells. Therefore they have the potential of being
used for photodynamic therapy (PDT) which involves the
2
5,26
destruction of tumor cells by photoirradiation.
PDT uses
photosensitizing agents in a less invasive and more targeted
Scheme 1 Details of the synthesis of ligands 1–9 and complexes C1–
form of cancer treatment. In many cases, the photosensitizing C11, with procedures starting from 1,10-phenanthroline as the ligand
agents such as porphyrins, phthalocyanines or methylene and IrCl ·3H O as the metal precursor.
3
2
2
7
blue have been shown to generate reactive singlet oxygen
2
5,28
which causes cell death.
The iridium(III) complexes
also have great potential in the field due to their high phos- the sub-cellular microenvironment and the potential imaging
2
9
20
phorescence lifetime and cell permeability.
agent, resulting in specific localization and efficient fluore-
In order to rationally design cyclometalated iridium(III) scence emission characteristics. Taking these criteria into con-
complexes with specific subcellular localization properties, we sideration we selected the 2-phenyl-(1H-imidazo[4,5-f][1,10]-
have initially synthesized three simple imidazolyl-phenanthro- phenanthroline backbone as the polypyridyl ligand with sub-
line ligands and treated them with a cyclometalated iridium(III) stitutions at different positions of the appended phenyl ring
precursor to form phosphorescent iridium(III) complexes. (Scheme 1). We initially synthesized three complexes (C1–C3)
The complexes exhibit significant fluorescence intensity followed by eight other complexes (C4–C11). Complexes C1–
2 2
between pH 3 and 8 in a pH-dependent manner in vitro. Cellu- C10 have been synthesized by refluxing [Ir(C^N) Cl] in
lar imaging with these complexes showed differential fluore- dichloromethane–acetonitrile solution at 60 °C with ligand
scence intensity in cells, and remarkable selectivity for the ER 1–9, where systematic substitution of various functional
by one of the complexes. Alteration of the ancillary ligand in a groups have been done. C11 was obtained by the reaction of
5
systematic manner and synthesis of eight more complexes [Re(CO) Cl] with ligand 2 in toluene at 110 °C (Chart 1). The
1
enabled us to locate the precise functional moieties and synthesized complexes were purified and characterized by H
their interactions responsible for the ER localization and NMR (Fig. S1 and S2†), high-resolution mass spectrometry
fluorescence emission. A detailed analysis of the complexes (Fig. S3 and S4†), IR and elemental analysis. The photo-
revealed that presence of strong intramolecular hydrogen physical studies showed that the complexes have absorption
bonding interactions and cationic nature of the metal complex maxima at around 466–469 nm, 390–410 nm, 344–348 nm,
will contribute significantly to the specific localization and 321–334 nm and 272–276 nm in organic solvents. The absorp-
fluorescence intensity. Finally, the ER localized complex was tion bands with very high extinction coefficient at ca.
also found to have significant photosensitizing property, 270–350 nm have been assigned to spin-allowed ligand-
1
1
efficiently causing cell death induced by photoirradiation at centered IL transitions ( π → π*), from both the ppy and
05 nm. phen ligands. The low-energy absorption shoulders at ca.
80–410 nm have been assigned to an admixture of spin-
allowed metal-to-ligand charge-transfer (metal-to-ligand
4
3
Results
1
1
(
MLCT)(dπ–π* phen) and ligand-to-ligand ( LLCT)(π(ppy)–π*-
Synthesis and characterization of complexes C1–C11
phen)) processes. The very low intensity band around
3
3
The primary criteria for designing a cell-organelle specific 466–469 nm is assigned to spin-forbidden MLCT and LLCT
imaging agent involve the provision of interactions between transitions. C1–C11, upon excitation at 390 nm in aceto-
3
0
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Chart 1 Complexes at
a glance. Molecular structures of C1–C11
showing the cyclometalated core and the ancillary ligands. C1–C9 have
+
[
2 3
Ir(ppy) ] as the cyclometalated core whereas C11 contains [Re(CO) Cl]
as the metal core. C1–C9 contain various substitutions in the phenolic
ring of the ancillary ligand. C10 has benzo(h)quinoline instead of 2-phe-
nylpyridine attached to Ir(III).
Fig. 1 Confocal fluorescence images of MCF7 cells treated with C1–
C4. The same cells were transduced with CellLight ER-GFP. The cells
were imaged at λex = 405 nm and λem = 550–610 nm for the complexes
and at λex = 488 nm and λem = 490–540 nm for CellLight ER-GFP. Green
nitrile at 298 K, phosphoresces with λmax ∼ 570–600 nm with
phosphorescence quantum yield (ΦPL) and emission lifetime
(
e
τ ) in the range of 5.40–41.21% and 8.60–17.72 μs respectively
(Table S1 and Fig. S5†). These systems possess a large ligand-
(left panels) represents CellLight ER-GFP, red (central panels) represents
field stabilization of iridium owing to its high oxidation state, the complexes and the right panels represent merged images of both.
the presence of 5d orbitals and, most importantly, a high
ligand-field splitting energy due to the presence of two C^N
ligands. These enable the iridium to allow intersystem cross-
ing from the first singlet excited state to the triplet state
efficiently. Thus a long-lived triplet excited state (τ ∼ μs) and
sequence (CellLight ER-GFP from Life Technologies)
which specifically caused the localization of the GFP in the ER.
The fluorescence from C2 was found to completely and
specifically colocalize with the GFP fluorescence in the ER
whereas C1 and C3 did not show any colocalization with the
GFP fluorescence. This demonstrated that C2 acted as a cell
permeable, fluorescent imaging agent specifically marking the
ER. The CellLight ER-GFP exhibited fluorescence nonhomo-
geneously, and in only a subset of cells, whereas C2 showed
−
1
strong spin–orbit coupling (ζIr = 3909 cm ) are characteristics
3
1–33
of these complexes.
The photophysical parameters of the
complexes were quite satisfactory to explore their application
as cellular imaging agents.
C2 specifically localizes in the endoplasmic reticulum of cells
Initially, MCF7 breast carcinoma cells were treated with C1–C3 fluorescence in a majority of cells, suggesting the higher stain-
for 30 minutes following which fixed cells were imaged under ing efficiency of C2 compared to the expression vector-based
a confocal laser scanning microscope at 405 nm excitation system.
wavelength and 550–610 nm emission wavelength. Cells
treated with C1 and C3, which contained –OH groups in posi-
The 2′-OH group in the phenyl ring of C2 and its possible
tion 4′ and at positions 2′ and 3′ of the appended phenolic
interaction with the imidazolyl N plays a crucial role in ER
localization and fluorescence
ring respectively, showed diffused fluorescence throughout the
cytoplasm of the cells and lower fluorescence in the nuclei
(Fig. 1). In contrast, C2, with a –OH group at position 2′ of the Cyclometalated iridium(III) complexes with different ligand
phenolic ring (Scheme 1), showed specific and intense fluo- systems have shown localization in specific cellular organelles
rescence only in the perinuclear region of the cells, suggesting but the involvement of the functional groups of the ligands,
specific localization in the ER. The cells were also transduced and their chemical interactions, in this process has not been
1
,2,12,18–24
with a baculovirus-mammalian expression vector encoding elucidated.
We therefore attempted to systematically
Green Fluorescent Protein (GFP) fused with the calreticulin determine the precise functional group(s) and their inter-
ER insertion sequence and the KDEL tetrapeptide retention actions responsible for the specific localization by following a
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“mutational approach” of systematically altering the various
functional groups on the phenyl ring.
Firstly, in order to investigate the role of the 2′-OH group,
cells were treated with cyclometalated iridium(III) complex C4
containing the 2-phenyl-(1H-imidazo[4,5-f][1,10]phenanthro-
line) as an ancillary ligand, i.e. without any –OH groups. This
complex (C4) showed high intensity fluorescence (quantum
yield = 41.21%, τ = 14.57 μs) and considerable ER localization,
but the localization was less specific compared to C2, with
fluorescence exhibited in the nucleus and to some extent in
the cytoplasm (Fig. 1). This suggests that the specific localiz-
ation in the ER requires the presence of the –OH group at 2′
position of the appended phenyl ring. However, the other
groups in the complex also play a role in conferring the fluo-
rescence property. High fluorescence of C4 might be due to
the absence of fluorescence quenching by the –OH moiety.
We then further investigated the role of the 2′-OH group,
and its possible interaction with other groups in the complex.
In order to investigate whether the charged nature of the –OH
group and a possible intramolecular hydrogen bonding inter-
action (O–H⋯N) with the cyclic N of the imidazolyl group is
responsible for the ER localization and fluorescence, we syn-
thesized two more complexes (C5 and C6) by replacing –OH at
3
positions 2′ and 4′ by –OCH . C5 and C6 were found to have
fluorescence lifetimes of 10.34 and 10.52 μs respectively. They
also showed a very intense fluorescence with a quantum yield
of 23.73% (C5) and 16.31% (C6). The only difference between
Fig. 2 Confocal fluorescence images of MCF7 cells treated with C5–
C8. The same cells were transduced with CellLight ER-GFP. The cells
C6 and C2 is the absence of O–H⋯N interaction. On treating were imaged at λex = 405 nm and λem = 550–610 nm for the complexes
and at λex = 488 nm and λem = 490–540 nm for CellLight ER-GFP. Green
the cells with these complexes and imaging, C5 diffused nearly
(left panels) represents CellLight ER-GFP, red (central panels) represents
uniformly over the entire cell, including the cytoplasm (Fig. 2).
In the case of C6 although the complex did not localize in the
nucleus, it diffused more in the cytoplasm than C2. This
suggested that the O–H⋯N interaction is important for the
very specific localization observed in the case of C2.
the complexes and the right panels represent merged images of both.
Alteration of the cationic metal core, but not of the ligand
systems, abrogates the fluorescence of C2
To further study how substitutions at other positions in the
phenolic ring might influence the ER localization and fluo- Finally, we altered the crucial functional groups in the imid-
rescence emission of C2, a bulky tertiary butyl group was azolyl ring of C2 to estimate their contribution to the locali-
incorporated at positions 3′ and 5′ of the phenolic ring (C7) zation specificity and fluorescence properties of the compound.
and an –OCH
3
group was incorporated at position 3′ (C8). Therefore we have replaced the hydrogen atom of the –NH
These complexes were also found to have phosphorescence group in the imidazole ring with 4-methoxyphenyl group (C9).
lifetimes in the microsecond range (C7 = 11.66 μs, C8 = We also replaced the 2-phenylpyridine ligand with benzo(h)-
+
8
.60 μs). The quantum yield of the C7 (18.99%) was quite com- quinoline (C10) and the [Ir(ppy)
2
] core with a [Re(CO)
3
Cl] core
parable to complexes C1–C6 whereas C8 had a reduced (C11) to investigate the role of the ligand and the metal ion in
quantum yield of 6.74%. On treating the cells with these com- the properties of the molecule. Interestingly, the replacement
plexes, and subsequent imaging, it was found that the fluo- of the hydrogen in the –NH group did not abrogate the fluo-
rescence emission was strongly reduced in both the cases, rescence and localization of C9, but resulted in a decreased
being nearly absent in the case of C8 (Fig. 3). Also, there was fluorescence intensity (Fig. 3). This suggested that the O–H⋯N
no specific localization in the case of C7. This suggested that interaction in C9 is weaker in comparison with C2, due to the
substitution of other groups in the phenolic ring possibly loss in planarity of the imidazolyl and the phenyl rings. This is
caused structural distortions in the molecule which prevented due to the steric hindrance between the 4-methoxyphenyl
the O–H⋯N interaction between the 2′-OH and the cyclic N of group attached to the imidazolyl nitrogen and the ortho-hydro-
the imidazolyl group in the case of C8. On the other hand, gen of the appended phenyl group. Replacement of the
in C7, the introduction of a new O–H⋯O(–CH ) type intramole- 2-phenylpyridine ligand with benzo(h)quinoline did not
3
cular hydrogen bonding interaction might disturb the existing diminish the fluorescence emission or change the localization
O–H⋯N interaction and thus result in loss of fluorescence of C10 suggesting that the nature of the spectator ligand is not
intensity and specific localization.
significant for ER localization, provided it does not add any
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Fig. 3 Confocal fluorescence images of MCF7 cells treated with C9–
C11. The same cells were transduced with CellLight ER-GFP. The cells
were imaged at λex = 405 nm and λem = 550–610 nm for the complexes
and at λex = 488 nm and λem = 490–540 nm for CellLight ER-GFP. Green
(
left panels) represents CellLight ER-GFP, red (central panels) represents
the complexes and the right panels represent merged images of both.
specific functional moieties outside the C–H framework
+
(
Fig. 3). On the other hand, replacement of the [Ir(ppy)
2
] core
with a [Re(CO) Cl] core completely abrogated all fluorescence, Fig. 4 (a) Confocal fluorescence images of MCF7 cells treated with C2
3
indicating the importance of iridium(III) in determining the and Bafilomycin A. The same cells were transduced with CellLight
ER-GFP. The cells were imaged at λex = 405 nm and λem = 550–610 nm
properties of these cyclometalated complexes and also the
+
for the complexes and at λex = 488 nm and λem = 490–540 nm for Cell-
Light ER-GFP. (b) Confocal fluorescence images of MCF7 cells trans-
duced with CellLight Golgi-GFP or treated with MitoTracker,
LysoTracker or C2.
possible involvement of cationic [Ir(ppy) ] in the cellular
2
3
4–37
uptake process (Fig. 3).
Fluorescence and localization of C2 in cells are not
pH-dependent but are dependent on intermediate lipophilicity
As the fluorescence, uptake and specific localization of a com- in cells was dependent on pH by treating the cells with Bafilo-
pound would be influenced by the pH of the medium and the mycin A (BA), a macrolide antibiotic that specifically inhibits
lipophilicity of the compound, the pH dependence of the the vacuolar type ATPase (V-ATPase) in cells and thereby
2
9
fluorescence emission (Fig. S5 and S6†) as well as the lipo- increases the pH of subcellular organelles. It was found that
philicity of the complexes (Table S1†) were estimated. The pH BA did not affect the fluorescence intensity or ER localization
dependent fluorescence spectra (pH = 1–9) showed that all the of C2, thereby suggesting that the fluorescence of C2 in cells is
complexes exhibited pH-dependent fluorescence in vitro, with not pH-dependent (Fig. 4a). We also tested whether C2
highest fluorescence at acidic pH (Fig. S5 and S6†). C4 showed marked other organelles with acidic pH (Golgi complex and
the maximum fluorescence intensity at pH 4, as well as in the lysosomes, pH ∼ 6.5 and 5.5 respectively) or alkaline pH (mito-
organic solvent. Only C9 showed maximum fluorescence inten- chondria pH ∼ 8) by treating the cells with a Golgi marker
sity at basic pH (pH = 9). C2 also fluoresced with the highest (CellLight Golgi-GFP), a mitochondrial marker (MitoTracker)
intensity at acidic pH, and the fluorescence intensity decreased and a lysosome marker (LysoTracker) (Fig. 4b). The localization
with an increase in the pH, although there was considerable of C2 was distinct from any of these organelles, suggesting
fluorescence in the range of pH 4–8. (Table S2†). The that it specifically localizes to the ER rather than entering mul-
maximum fluorescence of C2 at acidic pH was surprising, con- tiple cellular organelles and exhibiting pH-dependent fluo-
sidering that the pH of the ER lumen is thought to be nearly rescence. This provides a better handle to design organelle-
3
8
neutral. Therefore we tested whether the fluorescence of C2 specific fluorescent markers than the previously reported
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Table 1 Lipophilicity of complexes C1–C11
a
Complex
Log P
C1
C2
C3
C4
C5
C6
C7
C8
C9
C10
C11
1.21
1.12
1.08
1.08
0.94
1.56
1.78
1.86
1.55
1.41
1.83
a
Partition coefficient
o w o w
P = C /C (C and C are the complex
concentrations in n-octanol and water, respectively); absorbance at
71 nm.
3
iridium(III) complexes which stain all acidic organelles in an
2
5
indiscriminate manner.
We also tested the lipophilicity of the compounds to esti-
mate their cellular uptake and distribution (Table 1). Highly
lipophilic compounds may be taken up easily through the cell
membrane, but will have poor diffusion in the aqueous cell
Fig. 5 (a) ORTEP diagram of C2 with 50% thermal ellipsoids and all
atoms numbered with hydrogen bonding interaction. Counter anions
and hydrogen atoms are omitted for clarity except for O–H and N–H
cytoplasm, and also low solubility in the cell culture medium, hydrogens. (b) Functionalized phenanthroline ligands showing bond
lengths and bond angles around the hydrogen bond.
making it less available for cellular uptake. Measurements of
partition coefficients showed that the increase in hydrophobic
interaction increased the log P value, and interestingly, C8
and C11, which were found to have the highest log P values,
Table 2 Crystallographic parameters of C2
showed the least cellular fluorescence. This might suggest Empirical formula
C41H28ClIrN O
848.36
Orthorhombic
1 1 1
P2 2 2
9.650(4)
15.353(6)
25.967(11)
3847(3)
4
0.71073
0.22 × 0.10 × 0.05
100
1.465
3.579
1.54–25.00
1672
−7 ≤ h ≤ 11
6
F.w.
a low cellular uptake as a possible reason for the low fluore-
Cryst syst
scence exhibited by C8 and C11, although many other factors,
Space group
such as interaction with the cellular microenvironment, might a (Å)
b (Å)
contribute to the loss of fluorescence. Conversely, C5, with the
c (Å)
lowest log P values, show the maximum fluorescence within
the cellular environment, but poor specific localization.
C2, with an intermediate lipophilicity, showed good uptake,
together with specific sub-cellular localization.
3
V (Å )
Z
λ (Å)
3
Crystal size (mm )
T (K)
−
3
ρcalcd (g cm
)
−
1
μ (mm
θ (°)
F(000)
)
Molecular structure of C2 shows the presence of hydrogen
bonding between 2′-OH of the phenyl ring and cyclic N of
imidazole
Index ranges
−
16 ≤ k ≤ 18
27 ≤ l ≤ 30
−
To understand the specific intramolecular hydrogen bonding
interactions in C2, the molecular structure of C2 was eluci-
dated by single crystal X-ray crystallography (Fig. 5a, Tables 2 R , wR (I > 2σ(I))
Data/restraints/params
GOF (F )
6787/1/152
0.964
2
1
2
0.0658, 0.1354
0.0986, 0.1489
R
W
1
, wR
2
(all data)
and S3†). The iridium(III) center is coordinated in a slightly
distorted octahedral fashion with the two N-donor groups of
ligand 2 and the metalated C atoms of the 2-phenylpyridine
ligands in a mutually cis arrangement. All bond lengths and
bond angles are within normal ranges. The average values
2
2
2
1/[σ (F
o
) + (0.0566P) + 0.0000P],
2 2
where P = (F
o
c
+ 2F )/3
of the Ir–C(ppy), Ir–N(ppy) and Ir–N(ligand 2) distances are formation correlates with the acceptor strength, and hetero-
3
9
2
.068 (3) Å, 2.059 (3) Å and 2.129 (10) Å respectively. The cyclic N is an efficient acceptor for this type of hydrogen
4
0
structure consists of an intramolecular hydrogen-bonding bonding interaction. Standard geometric parameters for
interaction between imidazolyl nitrogen N5 (as a heterocyclic ‘hydrogen bond’ formation (distance between H and the accep-
N-donor) and O1–H1A (as an acceptor). It has already tor atom = 2.30 Å) are well satisfied with 1.740 Å for O–H⋯N
4
1
been established that the probability of intramolecular ring distance, with the formation of a 6-membered conjugated
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ring (Fig. 5b). The structure suggests that C2 is unique among not exposed to photoirradiation, did not show cell death
these complexes as it contains strong intramolecular hydrogen (Fig. 6a). These observations suggested that C2 is an efficient
bonding interaction.
photosensitizing agent which can cause cell death with a
limited exposure to photoirradiation. We also tested whether
C2 could cause cellular toxicity by treating cells for prolonged
times (10 h, 22 h and 34 h) with 1 µM, 10 µM and 50 µM of
C2, and measuring cell viability by performing an MTT assay.
C2 acts as a photosensitizing agent to cause cell death by
limited photoirradiation
Cyclometalated iridium complexes have also been reported to
10 µM is the concentration that has been used for all the
act as efficient photosensitizing agents and induce cell death
2
3
imaging studies. We found that 10 µM of C2 does not cause
any reduction of cell viability till 22 h of treatment, and shows
a 50% decrease in cell viability only at 34 h of treatment
by photoirradiation. Therefore, we investigated whether C2
can also act as a photosensitizing agent and cause photo-
induced cell death. Cells treated with C2 were exposed to laser
irradiation at 405 nm for 1 h (2% AOTF laser power, 0.6 mW
intensity), and live cells were imaged during the time of
exposure. Cells treated with C2 and exposed to light at 405 nm
showed progressive membrane blebbing, contraction of cells
and generation of cellular processes, leading to nearly com-
plete cell death after 1 h of exposure (Fig. 6a and b and ESI
videos 1 and 2†). On the other hand, cells treated with C2, but
(
Fig. 6c). On the other hand, treatment with C2, followed by
photoirradiation for only 1 h, was sufficient to cause cell
death. This further reinforced the role of C2 as an efficient
imaging and photosensitizing agent with minimal auto-
nomous cytotoxicity.
C2 and photoirradiation cause apoptotic cell death by
generation of reactive oxygen species
To understand the mechanism of photoinduced cell death
caused by C2 we subjected the cells to four conditions: (1) C2
in the presence of photoirradiation, (2) C2 in the absence of
photoirradiation, (3) photoirradiation in the absence of C2
and (4) the absence of both C2 and photoirradiation (Fig. 7).
After the experimental treatment, the cells were stained with
Annexin V-FITC, which binds to externalized phosphatidyl
serine of cell membranes which is a marker of the early stage
of apoptosis, and propidium iodide (PI) which stains the DNA
in membrane permeabilized cells which is a marker of the late
stage of apoptosis. Only cells that were treated with both C2
and photoirradiation at 405 nm were found to be positive for
both Annexin V and PI staining, which suggests apoptotic cell
death. Also, the cells showed membrane blebbing and charac-
teristic changes in morphology, as observed in the DIC image.
Cells treated with C2 alone in the absence of photoirradiation
showed faint Annexin V staining whereas cells which were
either only photoirradiated for 1 hour or not treated at all did
not show any cell death. These observations demonstrated that
only photoirradiation of cells after uptake of C2 can cause
photoinduced cell death by apoptosis.
We then investigated whether the photosensitizing property
of C2 was mediated by the generation of reactive oxygen
species (ROS). Singlet oxygen has been reported to be gene-
rated on photoirradiation of Ir(III) complexes and suggested to
cause cell death although in vivo singlet oxygen generation has
2
5
not been demonstrated. To understand the photosensitizing
property of C2, we initially checked the generation of singlet
Fig. 6 (a) DIC and confocal fluorescence images of live MCF7 cells
treated with C2 and exposed to photoirradiation at 405 nm for 1 hour.
The upper panel shows the cells prior to photoirradiation whereas the
lower panel shows cells at the end of the photoirradiation period. The
1
oxygen ( O
2
) by the complex (5 μM) in organic solvents upon
photoirradiation at 366 nm measured by the change in UV/vis
spectra of 1,3-diphenylisobenzofuran (DPBF, 25 μM). DPBF is
1
central tile represents the field which was exposed to the laser whereas possibly the most well known
the surrounding tiles represent the fields not exposed. (b) Frames cap-
2
O acceptor, and the absor-
bance maximum of DPBF at 415 nm decreases by the reaction
with O . We observed a decline in DPBF absorbance on
tured from the video of the live imaging of the cells exposed to photoir-
radiation. Frames for both photoirradiated and non-photoirradiated cells
are at 30 minutes of exposure to photoirradiation. (c) MTT assay of
MCF7 cells exposed to no or three increasing concentrations of C2 and
incubated for 10, 22 and 34 hours.
1
42
2
photoirradiation in the presence of C2 in an organic solvent
1
(
methanol) (Fig. 8a). The O
2
quantum yield of the C2 (Φ =
0.21; Φmethylene blue = 0.5) was determined by comparing with a
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Fig. 7 DIC and fluorescence images of MCF7 cells treated with (1) C2
and exposed to photoirradiation at 405 nm for 1 hour, (2) treated with
C2 but not exposed to photoirradiation, (3) not treated with C2 but
exposed to photoirradiation and (4) not treated with C2 or photo-
irradiated. Following treatment the cells were stained with Annexin
V-FITC (right panels) and propidium iodide (central panel).
Fig. 8 (a) Comparative photooxidation of only 1,3-diphenylisobenzo-
furan (DPBF) (25 μM) in methanol (red dot), C2 (5 μM) + DPBF (black
square) and methylene blue (5 μM) + DPBF (blue triangle) in methanol
degassed by O
2
. Excitation at 366 nm for C2 and DPBF. (b) DIC and con-
methylene blue standard. However, the generation of singlet
focal fluorescence images of live MCF7 cells treated with C2 and
2
oxygen was not detected in DMSO–H O, as DPBF dimerizes in exposed to photoirradiation at 405 nm for 30 min. The cells were
aqueous media and thus becomes unreactive toward singlet treated with DCFDA and fluorescence images were obtained at 529 nm
4
3
after excitation at 495 nm. The central tile represents the field which
was exposed to the laser whereas the surrounding tiles represent the
fields not exposed.
2
oxygen in the H O-rich mixture. We then investigated the
generation of ROS in photoirradiated cells treated with C2.
Cells were treated with C2 and photoirradiated at 405 nm for
3
0 min, following which they were treated with 2′,7′-dichloro-
fluorescein diacetate (DCFDA) which fluoresces at 529 nm on
generation of ROS. It was observed that among the cells
treated with C2, only those cells that were photoirradiated and
showed the characteristic morphology of photoinduced cell
death demonstrated the DCFDA fluorescence, suggesting that
the cell death was induced by the generation of ROS (Fig. 8b).
Cells that were irradiated in the absence of C2 treatment did
not show any fluorescence with DCFDA (Fig. S7†).
18
agent, which would result in specific localization
and
efficient fluorescence emission properties. These interactions
may be influenced both by the properties of the imaging agent
and the cellular microenvironment such as pH of the cellular
compartment, intramolecular and intermolecular hydrogen
bonding interactions, sensitivity towards redox potential and
charge interactions during cellular uptake, and diffusion and
localization within the cell. In our attempt to design cyclome-
talated iridium complexes with imidazolyl modified phenan-
throline ligands based on these criteria, one particular
complex, C2, showed specific localization and high fluo-
rescence emission in the endoplasmic reticulum of cells.
Discussion and conclusion
Rational designing of a cell organelle-specific fluorescent Using an unprecedented “mutational” approach, systematic
marker would involve the provision of interactions between substitution of functional groups and elucidation of the crystal
the sub-cellular microenvironment and the potential imaging structure of C2 have provided insights into the intramolecular
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chemical interactions responsible for its photophysical pro- photoirradiation. These results suggest that the complex has
perties and specific subcellular localization. A specific hydro- enormous potential for application as a cellular imaging agent
gen bonding interaction between the 2′-OH group in the in cell biology as well as a photosensitizer for PDT in medi-
appended phenyl ring and the –NH group attached to the cine. Further studies need to be directed at elucidating the
imidazole ring was found to confer localization specificity to the mechanism by which C2 causes apoptotic cell death as a result
complex. Also, the highly stable resonance-assisted hydrogen of photoirradiation.
bonding resulted in enhanced π-delocalization within the
complex C2, and resulted in improved fluorescence due to
4
4
excited state intramolecular proton transfer (ESIPT). On the Experimental section
other hand, replacement of the –NH group with N-anisidine in
The starting materials IrCl ·3H O, 2-phenylpyridine, 1,10-
C9, which is expected to maintain the hydrogen bonding inter-
action, did not alter the ER specific localization, but reduced
the fluorescence intensity substantially. This is possibly due to
the distortion of planarity between the imidazolyl group and
the appended phenyl group, increasing the proportion of the
open form of the molecule relative to the closed form contribu-
ted by the intramolecular hydrogen bonding.
3
2
phenanthroline, aldehydes, and n-octanol were purchased
from Sigma-Aldrich and used without further purification. All
the solvents were dried by usual methods prior to use. The
cyclometalated iridium(III) chloro bridged dimer [Ir(ppy)
2 2
Cl]
was prepared according to the methods described in the
4
6
literature.
While changes in the functional groups in the phenyl
and imidazolyl rings only altered subcellular localization
Synthesis of ligands
4
7
The ligands 1–8 and 9 (–N-Ph has been replaced by N-p-
or reduced fluorescence emission, the replacement of the
4
8
+
anisidine) were synthesized with a similar procedure to that
reported earlier. Ligands 3, 5, 6, and 8 were not reported
earlier. See the ESI† for details of the synthesis and Fig. S1
and S3† for NMR and ESI-MS data.
[Ir(ppy)
2
]
with [Re(CO)
3
Cl] in C11 completely abrogated all
fluorescence, suggesting that the cationic nature of Ir(III) is
playing a crucial role in cellular uptake. On the other hand,
replacement of 2-phenylpyridine ligand with benzo(h)quino-
line did not substantially alter ER localization or fluorescence
emission suggesting that a change in the C–H framework
Synthesis of complexes
of the spectator ligand does not have a profound effect on sub- Complexes C2 and C4 were synthesized with a similar pro-
4
0
cellular localization.
cedure to that reported earlier. Complexes C1, C3, C5, C6,
The fluorescence emission of cyclometalated iridium(III) C7, C8, C9, C10 and C11 were not reported earlier. See the ESI
complexes is known to be pH dependent based on the ancil- for details of the synthesis and Fig. S2 and S4† for NMR and
4
5
lary ligand system. In our study, in vitro fluorescence of the ESI-MS data.
complexes was found to be pH-dependent, with maximum
fluorescence of most of the complexes being in the acidic pH
Physical measurements
range. However, the high fluorescence of C2 in the ER, which IR spectra were obtained on a Perkin-Elmer Spectrum RXI
is reported to have neutral pH, and the absence of a decrease spectrophotometer with samples prepared as KBr pellets.
in fluorescence intensity under Bafilomycin A treatment Elemental analyses were performed on a Perkin-Elmer 2400
showed that the fluorescence of C2 in cells was not pH depen- series II CHN series. Electronic spectra were recorded on a
1
dent. This is unlike the properties of cyclometalated Ir(III) com- U-4100, HITACHI spectrometer. H NMR spectra were obtained
plexes reported previously, which specifically stained acidic on a Jeol 400 MHz NMR spectrometer using TMS as the
2
5
organelles. This provides a better tool to design organelle- internal standard. Electrochemical measurements were made
specific fluorescent markers which might be targeted to using a PAR model 273 potentiostat. A platinum disk working
specific sub-cellular compartments or organelles by substitut- electrode, a platinum wire auxiliary electrode and an aqueous
+
ing different functional groups. Also, the intermediate lipo- Ag/Ag reference electrode were used in a three electrode con-
philicity of the complex appears to have contributed to the figuration. Electrochemical measurements were made under a
efficient uptake and subcellular localization, suggesting that dinitrogen atmosphere. All electrochemical data were collected
efficient diffusion through the cytoplasm might be beneficial at 298 K and are uncorrected for junction potentials. Mass
for targeting to specific organelles.
spectra were recorded on a Q-Tof Micromass spectrometer by
Furthermore, the complex has been found to be an efficient positive-ion mode electrospray ionization. Fluorescence
photosensitizing agent, causing extensive photoinduced cell spectra were taken on a HORIBA JOBINYVON spectrofluorom-
death upon exposure to light at 405 nm for one hour. Ir(III) eter Fluoromax 4. Quantum yield data reported here were
2 4
complex functionalized nanoparticles have been reported to measured relative to quinine sulfate in 0.1 M H SO (λex =
have applications in PDT and a recent study has shown an 350 nm, Φ = 0.577). The integration of the emission spectra
iridium(III) complex with the N,N-diethylamino group in the was obtained from the Fluoromax-4 instrument directly. The
2
1,23–25
ancillary ligand
to cause photoinduced necrosis-like cell photoluminescence spectra were measured on a Horiba Jobin
death. However, C2 was found to cause photoinduced apopto- Yvon spectrometer equipped with a 150 W Xe lamp. The life-
tic cell death and very little cytotoxicity in the absence of time measurements were done by exciting the sample using a
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pulsed Xe lamp. Lifetime measurements were performed with oxidation of DPBF was monitored at intervals of 5 s. The
−
5
dry deaerated dichloromethane solution (conc. 10 M). The quantum yields of singlet oxygen generation (Φ ) were calcu-
Δ
first order exponential decay curve was fitted to obtain the lated by a relative method using methylene blue (in methanol)
5
2
phosphorescence lifetime.
(Φ
Δ
= 0.50) as the reference. The following equation was
used:
X-ray crystallography
Φ
ΔðC2Þ
¼ ΦΔðstdÞðmC2 ꢀFstdÞ=ðmstd ꢀFC2Þ
Crystal data were collected on a Bruker SMART APEXII CCD
area-detector diffractometer using graphite monochromated where subscripts ‘C2’ and ‘std’ designate the iridium complex
Mo Kα radiation (λ = 0.71073 Å). For the crystal, X-ray data and methylene blue, respectively, m is the slope of a plot of
reduction was carried out using the Bruker SAINT program. differences in changes in absorbance of DPBF (at 415 nm)
The structures were solved by direct methods using the with the irradiation time and F is the absorption correction
SHELXS-97 program and refinement was done using the factor, which is given by F = 1–10− (absorbance at the
SHELXL-97 program. Selected crystal data and data collection irradiation wavelength).
A
parameters for all the complexes are given in Table 1. X-ray
data reduction, structure solution and refinement were done
using the SHELXL-97 program package.
4
9
Cellular imaging
MCF7 human breast carcinoma cells were maintained in
DMEM with 10% FBS. Cells were treated with 10 µM of the
5
0
Partition coefficient determination
The lipophilicity of complexes C1–C11 was determined by complexes in 0.1% DMSO in DMEM medium. Cells were
the “shake flask” method using a pH 7.4 phosphate buffer treated for 30 minutes, following which they were washed with
(0.129 M NaCl) and n-octanol as solvents. Each compound was PBS (pH 7.4) and fixed with 4% paraformaldehyde. Fixed cells
dissolved in the phase in which it is most soluble, resulting in were imaged in a Zeiss LSM710 confocal laser scanning micro-
typical concentrations of 50–350 μM. Duplicate determinations scope at 405 nm excitation wavelength and 550 nm–610 nm
using three different solvent ratios were performed for each emission wavelengths. For identifying the ER, cells were also
complex. Following the mixing and phase separation, each transduced with a baculovirus-mammalian expression vector
phase was analyzed for solute content, and the concentration encoding Green Fluorescent Protein (GFP) fused with the calre-
was determined using spectrophotometric methods. All the ticulin ER insertion sequence and the KDEL tetrapeptide
n-octanol–water partition coefficients were determined by retention sequence (CellLight ER-GFP from Life Technologies)
UV/vis spectroscopy. Octanol and buffer solutions were presa- according to the manufacturer’s instructions. A similar treat-
turated. Fifty rotations were performed by hand, followed ment was also performed for identifying the Golgi complex
by 1 h of settling time. Equilibration and absorption measure- (CellLight ER-GFP from Life Technologies). Mitochondria and
ments were made at 20 °C.
lysosomes were stained with MitoTracker and LysoTracker dyes
Life Technologies) respectively according to the manufac-
turer’s instructions. For the inhibition of V-type ATPases, cells
(
Study of pH-dependent fluorescence
Tris buffer solution (500 ml, 0.1 M) was prepared and a series were treated with 0.5 µM Bafilomycin A1 (Sigma Life Sciences)
of buffer solutions having pH in the range 2–9 was prepared by for 1 hour prior to treatment with the complex. For live cell
adding 1 M HCl/NaOH aqueous solution to the Tris solution. imaging, cells were treated with the complex 10 minutes prior
The pH of the resulting solutions was recorded using a pH to the initiation of live cell imaging. Live imaging at 405 nm
meter. 31 × 10− M stock solutions of all the complexes were was done for 1 hour. For checking apoptotic cell death, cells
prepared in dry DMSO, and in the fluorescence cuvette, 3.0 ml were stained with Annexin-V FITC and propidium iodide (PI)
buffer solution and 0.1 ml complex solution in DMSO were after treatment with C2 and photoirradiation. For checking
5
mixed and fluorescence emission was recorded at 25 °C.
ROS generation, cells were treated with 2′,7′-dichlorofluore-
scein diacetate (DCFDA), a fluorogenic dye that fluoresces in
the presence of ROS with excitation and emission maxima at
1
Singlet oxygen ( O
2
) quantum yield
3
Quantum yields for singlet oxygen generation in CH OH were 495 nm and 529 nm respectively.
determined by monitoring the photooxidation of 1,3-dipheny-
lisobenzofuran (DPBF) sensitized by the Ir complexes. Values
of the triplet photosensitizers were calculated according to a
Cell viability assay
5
1
modified literature method. The DPBF and samples with MCF7 cells were treated with 1 µM, 10 µM and 50 µM of the
DPBF were irradiated at 405 nm and methylene blue was irra- complex and kept for 10 hours, 22 hours and 34 hours. At the
2
5
diated at 575 nm. The light source of a spectrofluorometer end of these incubation times, cells were washed with PBS and
was used as an irradiation light source (150 W Xe source) to 0.5 mg ml− of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetra-
obtain constant monochromatic light. The absorbance of zolium bromide (MTT from Sigma Life Sciences) was added to
DPBF was adjusted to around 1.0 at 415 nm in oxygen satu- the medium. Cells were incubated for 3 hours following which
1
3
rated CH OH, and the absorbance of the sensitizers was cells were lysed with 0.1 N HCl in isopropanol and absorption
adjusted to 0.2–0.3 at the irradiation wavelength. The photo- readings were taken at 595 nm.
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2
1 S. P.-Y. Li, C. T.-S. Lau, M.-W. Louie, Y.-W. Lam,
S. H. Cheng and K. K.-W. Lo, Biomaterials, 2013, 34,
7519.
Acknowledgements
Soumik Mandal and Dipak K. Poria are thankful to CSIR, New
Delhi for Senior Research Fellowship. This work is supported 22 P. Steunenberg, A. Ruggi, N. S. van den Berg, T. Buckle,
by DST, New Delhi through research grants ST/FT/CS-057/2009
awarded to Parna Gupta and Wellcome Trust-DBT India
J. Kuil, F. W. B. van Leeuwen and A. H. Velders, Inorg.
Chem., 2012, 51, 2105.
Alliance Intermediate Fellowship WT500139/Z/09/Z awarded to 23 R. Cao, J. Jia, X. Ma, M. Zhou and H. Fei, J. Med. Chem.,
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Bangalore for helpful discussions.
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