Phosphorescent biscyclometallated iridium(iii) ethylenediamine complexes functionalised with polar ester or carboxylate groups as bioimaging and visualisation reagents

Article abstract of DOI:10.1039/c4dt02890b

We report the synthesis, characterisation and photophysical properties of new phosphorescent biscyclometallated iridium(iii) ethylenediamine (en) complexes functionalised with polar ester or carboxylate groups [Ir(N^C)₂(en)]ⁿ(X) (n = +1, X = Cl^-, HN^C = methyl 4-(2-pyridyl)benzoate Hppy-COOMe (1a), methyl 2-phenyl-4-quinolinecarboxylate Hpq-COOMe (2a); n = -1, X = Li⁺, HN^C = 4-(2-pyridyl)benzoate Hppy-COO^- (1b), 2-phenyl-4-quinolinecarboxylate Hpq-COO^- (2b)). In aqueous solutions, the carboxylate complexes 1b and 2b displayed emission quenching (ca. 7 and 74 fold, respectively) and lifetime shortening upon protonation, and their pK_a values were determined to be 5.13 and 3.46, respectively. The pq complexes 2a and 2b exhibited hypsochromic shifts in their emission maxima and a significant increase in emission intensity (ca. 84 and 15 fold, respectively) upon nonspecific binding to the protein bovine serum albumin (BSA). Inductively coupled plasma-mass spectroscopy (ICP-MS) and laser-scanning confocal microscopy (LSCM) results revealed that the ester complexes 1a and 2a were efficiently internalised by the human cervix epithelioid carcinoma (HeLa) cells through energy-requiring pathways and subsequently localised in endosomes and mitochondria, respectively. They showed good biocompatibility in the dark, but became significantly cytotoxic upon photoirradiation due to the generation of singlet oxygen. In contrast, in aqueous solutions of physiological pH, the carboxylate complexes 1b and 2b existed as the anionic form and hardly entered cells due to limited membrane permeability, as evidenced by the intense emission surrounding the plasma membrane of the cells. They showed negligible cytotoxicity and the cell viability remained over 95% for an incubation period of 24 hours. In view of the low cytotoxicity and strongly emissive nature of the hydrophilic ppy-COO^- complex 1b in an aqueous medium, the potential application of the complex as a visualisation reagent has been demonstrated using zebrafish (Danio rerio) as an animal model. This journal is

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Phosphorescent biscyclometallated iridium(III)
ethylenediamine complexes functionalised with
polar ester or carboxylate groups as bioimaging
and visualisation reagents†
a
a
a
a
Tommy Siu-Ming Tang, Kam-Keung Leung, Man-Wai Louie, Hua-Wei Liu,
b
a
Shuk Han Cheng and Kenneth Kam-Wing Lo*
We report the synthesis, characterisation and photophysical properties of new
phosphorescent biscyclometallated iridium(III) ethylenediamine (en) complexes
n
−
functionalised with polar ester or carboxylate groups [Ir(N^C)
HN^C methyl 4-(2-pyridyl)benzoate Hppy-COOMe 1a), methyl 2-phenyl-4-
quinolinecarboxylate Hpq-COOMe (2a); n = 1, X = Li , HN^C = 4-(2-pyridyl)benzoate
2
(en)] (X) (n = +1, X = Cl ,
=
(
+
−
−
−
Hppy-COO
solutions, the carboxylate complexes 1b and 2b displayed emission quenching (ca. 7
and 74 fold, respectively) and lifetime shortening upon protonation, and their pK
values were determined to be 5.13 and 3.46, respectively. The pq complexes 2a and
exhibited hypsochromic shifts in their emission maxima and a significant increase
(
1b), 2-phenyl-4-quinolinecarboxylate Hpq-COO
(
2b)).
In aqueous
a
2
b
in emission intensity (ca. 84 and 15 fold, respectively) upon nonspecific binding to the
protein bovine serum albumin (BSA). Inductively coupled plasma-mass spectroscopy
(
ICP-MS) and laser-scanning confocal microscopy (LSCM) results revealed that the
ester complexes 1a and 2a were efficiently internalised by human cervix epithelioid
carcinoma (HeLa) cells through energy-requiring pathways and subsequently localised
in endosomes and mitochondria, respectively. They showed good biocompatibility in
the dark, but became significantly cytotoxic upon photoirradiation due to the
generation of singlet oxygen. In contrast, in aqueous solutions of physiological pH, the
carboxylate complexes 1b and 2b existed as an anionic form and hardly entered cells
due to limited membrane permeability, as evidenced by intense emission surrounding
the plasma membrane of the cells. They showed negligible cytotoxicity and the cell
viability remained over 95% for an incubation period of 24 hours. In view of the low
−
cytotoxicity and strongly emissive nature of the hydrophilic ppy-COO complex 1b in
aqueous medium, the potential application of the complex as a visualisation reagent
has been demonstrated using zebrafish (Danio rerio) as an animal model.
Introduction
Visualisation of cellular compartmentation has substantially facilitated the understanding of structures, functions and dynamics of
1
2
3
4
cells. The design of cellular imaging reagents has relied mainly on organic dyes, lanthanide chelates and quantum dots. In the
past decade, phosphorescent transition metal complexes have emerged as excellent luminophores for biological labelling and
imaging due to their interesting photophysical properties: intense, longꢀlived and tunable phosphorescence, good photostability
5
and high sensitivity toward environment variables such as pH, hydrophobicity and rigidity. Phosphorescent biscyclometallated
iridium(III) diimine complexes [Ir(N^C) (diimine)] (where N^C = cyclometallating ligands) are amongst the most widely studied
systems. They have been modified with various reactive functionalities and biologicallyꢀrelevant moieties to serve as organelleꢀ
specific imaging reagents and molecular probes for different intracellular species. Similar to diimine ligands, diamines can also
+
2
5,6
5,6
+
be coordinated to the iridium(III) centre to afford robust cyclometallates [Ir(N^C) (diamine)] that show strong phosphorescence
2
upon excitation; for example, we have recently reported a series of highly emissive cyclometallated iridium(III) ethylenediamine
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is
+
n+
(
en) complexes [Ir(N^C) (en)] and their polyamine counterparts [{Ir(N^C) } (bPEI)]
(where bPEI
= branched
2
2 n
7
poly(ethyleneimine)). We believe that the en complexes allow a higher control of emission energy tuning since the chromophore
3
is mainly localised in the [Ir(N^C) ] moiety. Their emissiveꢀstate nature can vary from triplet intraligand ( IL) (π
→
π*) (N^C) to
2
3
metalꢀtoꢀligand chargeꢀtransfer ( MLCT) (dπ(Ir)
→ π*(N^C)). More importantly, they maintain high stability under physiological
conditions and their cellular uptake and cytotoxic activity can be readily controlled through a judicious choice of N^C ligands. All
these features facilitate the design of biscyclometallated iridium(III) en complexes as effective cellular imaging reagents.
However, our experience is that many iridium(III) cyclometallates are highly lipophilic, resulting in too efficient cellular uptake
and extensive hydrophobic interactions with biomolecules in the cytosol, limiting their potential in staining specific cellular
8
compartments. Since the aromatic scaffold of N^C ligands substantially contributes to the hydrophobicity of the en complexes, it
is conceivable that an increase in the polarity of N^C ligands will reduce the cellular uptake rate and enable more selective
subcellular localisation of the complexes.
Herein, we describe the synthesis, characterisation and photophysical properties of new phosphorescent biscyclometallated
n
−
iridium(III) en complexes functionalised with polar ester or carboxylate groups [Ir(N^C) (en)] (X) (
n
= +1, X = Cl , HN^C =
2
+
methyl 4ꢀ(2ꢀpyridyl)benzoate HppyꢀCOOMe (1a), methyl 2ꢀphenylꢀ4ꢀquinolinecarboxylate HpqꢀCOOMe (2a);
n
=
−
1, X = Li ,
−
−
HN^C = 4ꢀ(2ꢀpyridyl)benzoate HppyꢀCOO (1b), 2ꢀphenylꢀ4ꢀquinolinecarboxylate HpqꢀCOO (2b)) (Scheme 1). The pHꢀinduced
changes in the absorption and emission properties of the carboxylate complexes 1b and 2b have been investigated and their pKa
values have been determined. Also, the emission changes of all the complexes upon binding to the protein bovine serum albumin
(
BSA) have been studied. The cellular uptake efficiency, dark and photoinduced cytotoxicity and intracellular localisation of the
complexes have also been investigated using human cervical epithelioid carcinoma (HeLa) cells. Additionally, the potential
−
application of the hydrophilic ppyꢀCOO complex 1b as a visualisation reagent has been demonstrated using zebrafish (Danio
rerio) as an animal model.
Results and discussion
Design and synthesis of the complexes
In this work, we have introduced an ester or carboxylate to the cyclometallating ligands ppy and pq and obtained the iridium(III)
ester complexes [Ir(N^C) (en)](Cl) (N^C = ppyꢀCOOMe (1a), pqꢀCOOMe (2a)) and their carboxylate counterparts
2
−
−
[
Ir(N^C) (en)](Li) (N^C = ppyꢀCOO (1b), pqꢀCOO (2b)) (Scheme 1). We anticipate that the incorporation of ester and
2
carboxylate functionalities into the cyclometallating ligands will allow direct perturbation of the photophysical properties, cellular
uptake, cytotoxicity and subcellular distribution of the complexes. The HN^CꢀCOOMe ligands were synthesised from the
esterification of HN^CꢀCOOH in a mixture of concentrated H SO and MeOH. The dinuclear precursor complexes [Ir (N^Cꢀ
2
4
2
9
COOMe) Cl ] were prepared from the reaction of IrCl ꢁnH O and the HN^CꢀCOOMe ligands. The cationic ester complexes 1a
and 2a were obtained from the reaction of the corresponding iridium(III) dimers and excess en.
4
2
3 2
7
,10
Hydrolysis of the ester
complexes with LiOH led to the formation of the anionic carboxylate complexes 1b and 2b. All the complexes were characterised
1
by ESIꢀMS, IR and H NMR spectroscopy and gave satisfactory elemental analyses.
n
N
H
2
C
C
N
I r
N
N
H
2
X
CHOO C
OO C
3
N
C
N
C
N
N
C
N
C
=
C
CO O CH3
CO O
1b
1
a
2
a
2b
-
-
n
=
=
+1
+1
1
1
+
-
-
+
X
Cl
Cl
L i
L i
Scheme 1: Structures of the iridium(III) complexes.
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Electronic absorption and emission properties
The electronic absorption data and spectra of all the complexes in MeOH at 298 K are shown in Table S1 and Figure 1a,
respectively. The ppy complexes 1a and 1b displayed intense absorption bands at ca. 270 – 370 nm, moderate bands at 370 nm
(Ir) → π*(N^C))
Due to the more conjugated pq ligands, the IL
>
1
1
and weak tailing beyond 470 nm, which have been attributed to spinꢀallowed IL (π → π*) (N^C), MLCT (d
π
3
7,8a,11,12
and spinꢀforbidden MLCT (d
π(Ir) → π*(N^C)) transitions, respectively.
1
1
and MLCT absorption features of complexes 2a and 2b occurred at longer wavelengths.
Upon irradiation, all the complexes exhibited intense and longꢀlived emission in fluid solutions at ambient temperature and in
lowꢀtemperature glass. The photophysical data of the complexes are presented in Table 1 and the emission spectra in degassed
MeOH at 298 K are shown in Figure 1b. As expected, the emission energies were highly dependent on the cyclometallating
ligands. The ppy complexes displayed greenishꢀyellow to yellow emission while the pq complexes showed orangeꢀred to red
emission. Also, the electronꢀwithdrawing ester and carboxylate substituents significantly redꢀshifted the emission maxima of the
7
ppy and pq complexes compared with their unsubstituted counterparts [Ir(N^C) (en)](PF ) (N^C = ppy and pq), respectively. In
2
6
fluid solutions at 298 K, the ppy complexes 1a and 1b displayed slightly structured emission bands with small solvatochromic
shifts. In lowꢀtemperature glass, they showed highly structured emission features with longer emission lifetimes (Figure 1c and
3
3
Table 1), suggestive of a IL (π → π*) (N^C) emissive state that is probably mixed with some MLCT (d
π
(Ir) → π*(N^C))
7,8a,11,12
character.
In contrast, the pq complexes 2a and 2b displayed broad and featureless emission bands. Upon increasing the
polarity of the solvents, the peak maxima underwent large bathochromic shifts with reduced emission quantum yields and lifetimes
3
7,8a,11,12
(
Table 1), indicative of substantial MLCT (d
π
(Ir) → π*(N^C)) character in their emissive states.
However, the mixing of
3
some IL (π → π*) (N^C) characters into the states cannot be ruled out.
Table 1: Photophysical data of the iridium(III) complexes.
a
Complex
Medium
CH CN
MeOH
T
/K
λem/nm
το
/ꢀ
s
Φem
1
2
a
a
3
298
298
298
298
77
298
298
298
298
298
77
298
298
298
77
298
298
298
77
530, 566 sh
540, 577 sh
550
3.56
2.28
0.50
0.49
6.32
0.30
0.09
0.02
0.019
0.023
5.13
2.37
1.95
1.06
6.20
0.26
0.023
0.021
7.95
0.60
0.30
0.06
0.056
H O
Buffer
2
b
552
c
Glass
530 (max), 570, 626 sh
636 sh, 660
679
680
681
CH
Cl
0.01
2
2
CH CN
0.003
0.0008
0.0004
0.0006
3
MeOH
d
H O/DMSO
2
e
Buffer/DMSO
678
c
Glass
635, 699 sh, 711 sh
527, 554 sh
528, 560 sh
529, 559 sh
518 (max), 556, 607 sh
620, 655 sh
629, 657 sh
629, 663 sh
1
b
b
MeOH
0.56
0.36
0.25
H O
2
b
Buffer
Glass
c
2
MeOH
0.055
0.003
0.003
H O
Buffer
2
b
c
Glass
592, 644 sh, 695 sh
b
a
c
The lifetimes were measured at the emission maxima. Potassium phosphate buffer (50 mM, pH 7.4). EtOH/MeOH (4:1,
v/v). H O/DMSO (99:1, v/v). Potassium phosphate buffer (50 mM, pH 7.4) containing 20% DMSO.
d
e
2
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(a)
(b)
(c)
5
4
3
2
1
0
ε
300
500
500
400
500
600
800
800
Wavelength / nm
600
700
Wavelength / nm
600
700
Wavelength / nm
Figure 1: (a) Electronic absorption and (b) emission spectra of complexes 1a (black), 2a (green), 1b (red) and 2b (blue) in MeOH at 298 K and (c) EtOH/MeOH (4:1, v/v)
at 77 K.
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pH-dependence studies
The effects of pH on the electronic absorption and photophysical properties of the carboxylate complexes 1b and 2b in aqueous
solutions have been investigated by absorption and emission titrations. The pH values of the solutions were tuned from high to
low so as to mimic the pH changes during the uptake of the complexes from slightly basic physiological environment into slightly
−
acidic intracellular environment. We found that acidification led to minor changes in the absorption features of the ppyꢀCOO
−
complex 1b, but resulted in a decrease of the absorbance at 271 and 345 nm of the pqꢀCOO complex 2b and the formation of a
new band at 354 nm (Figure S1). In contrast, the emission response of both complexes toward decreasing pH was similar; they
displayed reduced emission intensity, bathochromicallyꢀshifted emission bands and shortened lifetimes upon protonation (Figures
2
a, 3a and S2). A decrease of pH from 6.5 to 4.2 reduced the intense emission of complex 1b at 531 nm by ca. 7 fold (Figure 2b),
while acidification from pH 4.3 to 1.9 resulted in ca. 74ꢀfold decrease in the emission intensity of complex 2b at 646 nm (Figure
b). In view of the broad and featureless emission profile of the protonated complexes, we have tentatively ascribed the
3
3
−
acidificationꢀinduced emission changes to a lower degree of IL character in the emissive states because protonation of the COO
moiety favours chargeꢀtransfer from the iridium centre to the cyclometallating ligands. The pKa values of complexes 1b and 2b
were determined to be 5.13 and 3.46 by fitting the intensity changes of emission maxima at 531 and 646 nm, respectively, to the
−
HendersonꢀHasselbalch equation (Figures 2b and 3b, insets). The smaller pKa value of the pqꢀCOO complex 2b than that of the
−
−
ppyꢀCOO complex 1b is due to the higher degree of πꢀconjugation of the pq moiety, which stabilises the COO group better than
the ppy moiety does. The titration results indicated that both complexes 1b and 2b existed in the fully deprotonated form over the
physiological pH range and the more intense emission and longer lifetimes associated with the IL states are advantages for
3
bioimaging applications.
(a)
Decreasing pH
500
600
700
Wavelength / nm
(b)
1.0
0.8
0.6
0.4
0.2
0.0
5
5
4
4
.6
ο
.2
.8
.4
-
0.8 -0.4 0.0 0.4 0.8 1.2
log [(
I
max
−
I)
/
(I
−
I
min)]
12
10
8
6
4
2
pH
Figure 2: (a) Emission spectral traces of complex 1b (50
λ
ꢀ
emission titration curve monitored at em = 531 nm (inset: plot of pH versus log [(Imax
M) in aerated 10 mM KOH(aq) and 100 mM KCl(aq)/DMSO (9:1, v/v) at 298 K upon decreasing the pH and (b)
I)/(I
−
− Imin)]).
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Decreasing pH
500
600
700
800
Wavelength / nm
(b)
1.0
0.8
0.6
0.4
0.2
0.0
4
3
3
2
.0
.6
.2
.8
ο
-
0.8 -0.4 0.0 0.4 0.8
log [(
I
max
−
min
I)/(I − I )
1
2
10
8
6
4
2
pH
Figure 3: (a) Emission spectral traces of complex 2b (50
emission titration curve monitored at
ꢀ
em = 646 nm (inset: plot of pH versus log [(Imax I)/(I
M) in aerated 10 mM KOH(aq) and 100 mM KCl(aq)/DMSO (9:1, v/v) at 298 K upon decreasing the pH and (b)
−
λ
− Imin)]).
Protein-binding studies
The possible binding interactions of all the complexes with the protein BSA have been investigated by emission titrations.
Although the ppy complexes 1a and 1b showed no significant changes in the emission properties in the presence of BSA, the
emission maxima of the pq complexes 2a and 2b were shifted to shorter wavelengths with large emission enhancement of ca. 84
and 15 fold at 651 and 606 nm, respectively, at [BSA]:[Ir] = 2.5 (Figures 4 and S3). The emission lifetime of complex 2a was
elongated from 0.02 to an average of 0.68 ꢀs while that of complex 2b from 0.02 to an average of 1.11 ꢀs. These changes have
3
been attributed to the high environmentꢀsensitivity of MLCT emissive states of the pq complexes. When the complexes are in
close proximity to the protein surface or bind to the hydrophobic pockets, the increased hydrophobicity and rigidity of the local
environment enhance the emission intensity and extend the emission lifetimes. Another possible reason is that the complexes are
well protected in the interior of the protein and a lower exposure to the aqueous surrounding could account for the less efficient
1
3
quenching by the oxygen molecules. A similar observation has been noticed in other luminophoreꢀlabelled protein systems.
Nevertheless, this proteinꢀinduced emission enhancement is an advantage for live cell imaging because when the pq complexes
accumulate in specific cellular compartments, their hydrophobic interactions with the surrounding protein molecules would lead to
intense emission for visualisation.
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(
a)
increasing
[BSA]:[Ir]
600
700
800
Wavelength / nm
(b)
increasing
[BSA]:[Ir]
5
00
600
700
800
Wavelength / nm
Figure 4: Emission spectral traces of (a) complex 2a (10
ꢀ
M) and (b) complex 2b (10
ꢀ
M) at [BSA]:[Ir] ratios of 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.5, 2.0 and 2.5 after
incubation for 24 h at 298 K.
Lipophilicity
The lipophilicity of all the complexes, as a pivotal physiological parameter to determine their permeability across biological
membranes, has been measured using the shakeꢀflask method involving ꢀoctanol and phosphateꢀbuffered saline (PBS) and the
n
results are listed in Table 2. As expected, the lipophilicity of the complexes strongly depended on the nature of the
cyclometallating ligands and their substituents. The log Po/w values of the ppy complexes 1a and 1b (0.62 and –1.83, respectively)
were lower than those of their pq counterparts 2a and 2b (1.10 and –0.45, respectively) due to the higher πꢀconjugation of the pq
unit. Also, the ester complexes 1a and 2a exhibited substantially higher lipophilicity than the carboxylate complexes 1b and 2b
reflecting the extraordinarily high polarity of COO moiety, which remained as the deprotonated form in PBS at pH 7.4.
,
−
Cytotoxicity and cellular uptake studies
Live cell imaging requires a biological probe to exert minimum perturbation on the viability and morphology of the cell samples.
The cytotoxic activity of all the complexes toward HeLa cells has been investigated using the (3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀ
diphenyltetrazolium bromide (MTT) assay, in which the dose dependence of surviving cells after exposure to the complexes for 6
and 24 h was evaluated. Under our experimental conditions, only the pqꢀCOOMe complex 2a exhibited noticeable cytotoxicity
and its IC50 values (ca. 25 and 14
those of the other complexes (> 200
lipophilic amongst the four complexes, it is likely that the lipophilicity plays a key role on the cytotoxicity of the complexes (Table
). Based on the IC50 values, the concentrations of the complexes used in the imaging studies ([1a], [1b] and [2b 100 M; [2a
10 M, see below) were sufficiently low so that the complexes can be considered to be noncytotoxic to HeLa cells within an
incubation period of 6 h. The cellular uptake of all the complexes by HeLa cells has been determined by ICPꢀMS measurements.
ꢀ
M for 6 and 24 h incubation, respectively) were found to be at least 8ꢀ and 14ꢀfold lower than
ꢀ
M) after 6 and 24 h incubation, respectively (Table 2). Since complex 2a is the most
2
]
≤
ꢀ
]
≤
ꢀ
Upon incubation with the complexes (10
ꢀ
M) at 37
°
C for 6 h, an average HeLa cell (volume = 3.4 pL) contained 3.52 to 4.14
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fmol of iridium (Table 2). The cells loaded with the ester complexes 1a and 2a revealed higher iridium concentrations (ca. 1.2
fold) than those treated with the carboxylate complexes 1b and 2b, which is consistent with their lipophilicity. Also, the iridium
concentration of all the complexes associated with the cells (1.04 to 1.22 mM) was much higher than that in the medium (10
providing evidence on cellular accumulation of the complexes.
ꢀM),
Table 2: Lipophilicity (log Po/w), cytotoxicity and cellular uptake efficiency of the iridium(III) complexes.
a
b
c
Complex
Lipophilicity
IC /
ꢀ
M , 6 h (24 h)
> 200 (> 200)
25.3 5.02 (14.3
Amount of iridium
4.04 (1.19)
4.14 (1.22)
3.65 (1.08)
3.52 (1.04)
5
0
1
2
1
a
a
b
b
0.62
1.10
±
±
0.01
0.08
±
±
0.23)
−
−
1.83
0.45
±
±
0.05
0.01
> 200 (> 200)
> 200 (> 200)
2
a
log Po/w is defined as the logarithmic ratio of the concentration of the iridium(III) complexes in the
n
ꢀoctanol (saturated with
ꢀoctanol) layers. Cytotoxicity of the complexes toward HeLa cells upon
°C for 6 and 24 h. Number of mole [fmol] (data in parentheses: concentration [mM]) of iridium
b
PBS) to that in the PBS (pH 7.4 and saturated with
n
c
incubation in the dark at 37
associated with an average HeLa cell upon incubation with the complexes (10 ꢀM) at 37 °C for 6 h.
Live cell confocal imaging and inhibition studies
By virtue of their interesting phosphorescence properties, the potential applications of the complexes as live cell imaging reagents
have been studied. The subcellular localisation of the complexes internalised by HeLa cells has been studied by LSCM (Figure 5).
The ester complexes 1a and 2a gave very strong emission after cellular uptake. Treatment of HeLa cells with the ppyꢀCOOMe
complex 1a revealed sharp vesicles staining throughout the cytoplasm (Figure 5), which became more evident upon increasing the
incubation concentration and duration (Figure S4). Since
Zꢀscan images have excluded the possibility of precipitation or
adherence to the external cell surface (Figure S5), we believe that the granular staining resulted from the aggregation of the
complex within specific hydrophobic organelles, which appeared to be endosomes. To confirm the localisation of complex 1a
,
–
1
HeLa cells pretreated with Alexa Fluor 633ꢀconjugated transferrin (50
incubated with the complex (50 M, 6 h, λex = 405 nm, λem = 500
a showed substantial costaining (Pearson’s colocalisation coefficient: 0.88), indicating that the majority of the complex was
ꢀ
g mL , 1.5 h, λex = 633 nm, λem = 650
−
700 nm) were
ꢀ
− 600 nm). The overlaid confocal microscopy images in Figure
6
accumulated at endosomes after uptake. On the contrary, images of HeLa cells loaded with the pqꢀCOOMe complex 2a revealed
that the complex was diffusely distributed in the cytoplasm (Figure 5). The nucleus gave no emission, which indicates negligible
nuclear uptake and/or very effective nuclear export. The complex was concentrated in specific compartments of the cells, which
appeared to be mitochondria on morphological grounds. Thus, HeLa cells pretreated with complex 2a (10
ꢀM, 1.5 h, λex = 405
nm, λem = 650 750 nm) were incubated with MitoTracker Green FM (300 nM, 15 min, λex = 488 nm, λem = 500 – 550 nm). The
−
mitochondria of the cells were considerably costained with the complex and the fluorescent dye (Pearson’s colocalisation
coefficient: 0.86) as shown in Figure 6b. Related cationic transition metal complexes with lipophilic ligands have been observed
7
,12c,14
−
to exhibit similar mitochondriaꢀtargeting behaviour.
The emission intensity of HeLa cells loaded with the ppyꢀCOO
complex 1b was remarkably weaker than those incubated with the ester complexes (Figure 5), although this complex showed the
−
highest quantum yield in aqueous medium (Table 1). HeLa cells treated with the pqꢀCOO complex 2b gave no emission even at a
high concentration (100
ꢀM) of the complex was used (Figure 5). We have ascribed these observations to the limited cellular
−
uptake of the carboxylate complexes due to their lower lipophilicity. Interestingly, HeLa cells loaded with the ppyꢀCOO complex
1
1
b
b
displayed plasma membrane staining and weak cytoplasmic emission (Figure 5). Images of HeLa cells treated with complex
−
1
(50
ꢀ
M, 6 h, λex = 405 nm, λem = 500
− 600 nm) and CellMask Deep Red Plasma Membrane Stain (12.5 ꢀg mL , 10 min, λex
=
633 nm, λem = 650
−
700 nm) in Figure 6c have confirmed the localisation of the complex on cell surface (Pearson’s
colocalisation coefficient: 0.95). The slow cellular internalisation process is most likely a result of the charge repulsion between
the COO groups of the complex and the phospholipid bilayers of the cells.
−
In view of the efficient cellular uptake of the ester complexes 1a and 2a, the internalisation mechanism of these complexes has
been investigated by temperatureꢀdependence and inhibition studies. Confocal microscopy images showed that incubation of the
cells with the complexes at 4
the cells through energyꢀrequiring processes, such as endocytosis. Additionally, treatment of the cells with the metabolic inhibitor
carbonyl cyanide 3ꢀchlorophenylhydrazone (CCCP) (10 M, 30 min) in a glucoseꢀfree medium at 37 C before incubation with
the complexes also resulted in reduction in intracellular emission intensity (Figure 7).
Since CCCP induces mitochondrial membrane potential depolarization, inhibits oxidative phosphorylation and subsequently leads
°C resulted in a significant reduction in uptake (Figure 7), highlighting that the complexes entered
ꢀ
°
a
1
5
to decreased ATP production and a slower metabolic rate, the observed decrease in uptake efficiency is in accordance with an
energyꢀrequiring mechanism.
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1
a
2a
1b
2b
Figure 5: Fluorescence (top), overlaid (middle) and brightfield (bottom) LSCM images of HeLa cells incubated with complexes 1a (50
50 M, 6 h) and 2b (100 M, 6 h) at 37 C ( ex = 405 nm).
ꢀM, 6 h), 2a (10 ꢀM, 1.5 h), 1b
(
ꢀ
ꢀ
°
λ
Complex
Organelle Stains
Overlaid
(a)
(b)
(c)
Figure 6: LSCM images of HeLa cells treated with (a) complex 1a (50
ꢀ
M, 1.5 h,
M, 6 h,
λ
ex = 405 nm,
λ
λ
em = 500
em = 650
600 nm) and CellMask Deep Red Plasma Membrane Stain (12.5 ꢀ
−
−
600 nm) and Alexa Fluor 633-conjugated transferrin (50
750 nm) and MitoTracker Green FM (300 nM, 15 min, ex = 488
g mL , 10 min,
ꢀg
–1
mL , 1.5 h,
nm, em = 500
ex = 633 nm,
λ
ex = 633 nm,
550 nm) and (c) complex 1b (50
em = 650 700 nm) at 37 C, respectively.
λ
em = 650
−
700 nm), (b) complex 2a (10
ꢀ
λ
ex = 405 nm,
λ
–
1
λ
−
λ
ꢀ
M, 6 h, ex = 405 nm,
λ
λ
em = 500
−
λ
−
°
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3
7 °C
4 °C
CCCP
1
2
a
a
Figure 7: LSCM images of HeLa cells incubated with complex 1a (100
after pretreatment with CCCP (10 M, 30 min) (right) ( ex = 405 nm).
ꢀ
M) (top) and complex 2a (10
ꢀ
M) (bottom) for 1.5 h at 37 °C (left), 4 °C (middle) and 37 °C
ꢀ
λ
Singlet oxygen generation and photoinduced cytotoxicity
The triplet emissive states of many iridium(III) complexes are effectively quenched by molecular oxygen to generate cytotoxic
1
16
1
1
O2. The O generation efficiency of all the complexes in this work has been studied. The quantum yields for O production
2
2
(
Φ ) of the complexes have been determined in aerated DMSO using a steadyꢀstate method with diphenylisobenzofuran (DPBF)
△
1
17
1
as a O scavenger and the results are listed in Table 3. The conversion rates of DPBF to 1,2ꢀdibenzoylbenzene by O are
2
2
illustrated in Figure S6. The
Φ△
value of complex 2a (0.082) is noticeably lower than those of other complexes (0.21 – 0.29)
(
Table 3), which can be attributed to its shorter emission lifetime (Table 1). It is reasonable that a shortꢀlived excited state reduces
1
collisional tripletꢀtriplet energy transfer and/or electron transfer from the complexes to molecular oxygen. On the basis of the O
2
generation capability, the photocytotoxic properties of all the iridium(III) complexes toward HeLa cells have been investigated by
the MTT assay. Without irradiation, complexes 1a 1b and 2b were basically noncytotoxic while complex 2a exhibited an IC50
value of 8.6 0.01 M (Table 4). However, upon irradiation at 365 nm for 1 h, the IC50 values of the ester complexes 1a and 2a
dropped substantially to 17.3 0.02 and 0.5 0.01 M, respectively (Table 4 and Figure 8). The IC50 values of the carboxylate
complexes 1b and 2b were still > 200 M (Table 4). The low dark and photoinduced cytotoxicity of polar carboxylate complexes
should be a result of their poor membrane permeability and uptake efficiency. Although a proportion of the ppyꢀCOO complex
,
±
ꢀ
±
±
ꢀ
ꢀ
−
1
1b was found to reside on the cell surface, the O generated by the complex may not be capable of damaging plasma membrane
2
effectively. In contrast, the ester complexes 1a and 2a were efficiently taken up by cells and subsequently localised in endosomal
1
and mitochondrial regions, respectively. Thus, the accumulation of O is believed to impose damage on these vital intracellular
2
organelles and elicit cell death. Similar photocytotoxic effects have been observed for other related organometallic singlet oxygen
1
2c,18
sensitisers.
Table 3: Photophysical data and singlet oxygen generation quantum yields of the iridium(III) complexes in aerated DMSO at 298 K.
a
b
c
Complex
λem/nm
το
/ꢀ
s
Φꢁ
1
2
1
a
a
b
b
543, 578 sh
688
528, 550 sh
594
0.326
0.037
0.227
0.416
0.29
0.082
0.27
0.21
2
a
b
c
λex = 365 nm. The lifetimes were measured at the emission maxima. Methylene blue was used as the reference for the
determination of singlet oxygen generation quantum yields.
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Table 4: Photocytotoxicity of the iridium(III) complexes toward HeLa cells.
a
a
Complex
IC /
(
ꢀ
M
IC /ꢀM
50
5
0
in the dark)
> 200
8.6
(upon irradiation)
17.3 0.02
0.5 0.01
1
2
1
a
a
b
b
±
±
> 200
> 200
±
0.01
> 200
> 200
2
a
HeLa cells pretreated with various concentrations of the iridium(III) complexes for 6 h were incubated in the dark or irradiated
at 365 nm with a 6 W UVꢀA lamp (Spectroline, USA) for 1 h. MTT assays were conducted after further incubation for 24 h.
The IC50 values were determined from the dose dependence curves.
(a)
1
20
00
IC > 200 ꢀM
5
0
1
8
6
4
2
0
0
0
0
0
IC = 17.3
± 0.02 ꢀM
50
10
100
[
1a] / ꢀM
(b)
1
20
00
IC = 0.5
±
0.01 ꢀM
IC50 = 8.6
±
0.01 ꢀM
5
0
1
8
6
4
2
0
0
0
0
0
0
.1
1
10
M
100
[2a] / ꢀ
Figure 8: Dose-response curves of (a) complex 1a and (b) complex 2a toward HeLa cells for the determination of dark (triangles) and photoinduced (squares)
cytotoxicity.
Live zebrafish imaging
−
Given the high hydrophilicity and negligible in vitro cytotoxicity of the strongly emissive ppyꢀCOO complex 1b, it is interesting
to exploit the possibility of using the complex as a visualisation agent for live animals. Zebrafish (Danio rerio) in embryoꢀlarval
stages was chosen as a model due to its transparence for luminescence bioimaging. The hydrophilic complex 1b was dissolved in
sterilised PBS (pH 7.4) at 2 mM and introduced to the cardiovascular system of a 48 hourꢀpostfertilization (hpf) zebrafish larva via
intravascular loading. The distributions of the complex inside the larva at three postinjection time points (5 min, 24 h and 48 h)
were visualised using fluorescence confocal microscopy (Figure S7). The loaded complex was translocated to other parts of the
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larva body through blood circulation, as indicated by intense luminescence observed in the notochord, spinal cord and brain
ventricles. As shown in the magnified images (Figure 9), the segmented repetitive networks of vessels along the trunk and the
complicated vessel system around head space of the larva were visualised upon irradiation. Negligible luminescence was observed
after 24 h (Figure S7). Based on these observations, we believe that most of the loaded complex molecules remained inside the
blood vessel system (i.e., in the 24 h postꢀinjection) before their clearance from the body through renal excretion. Also, the
complex did not adhere to the inner surface of blood vessels and was not retained in other tissues and organs of the zebrafish
−
larvae, causing negligible toxic effects to the animal. Thus, these results suggest that the ppyꢀCOO complex 1b retained its
strongly emissive nature and membrane impermeability inside vascular tissues of transparent zebrafish larvae, resulting in the
exclusive staining of circulation system.
a
b
c
B
Figure 9: Fluorescence microscopy images of a selected 48 hpf zebrafish larva that was microinjected with complex 1b (2 mM, 40 nL) (5 min after microinjection): (a)
the intravital image, (b) segmented repetitive networks of vessels along the trunk of the larva and (c) blood vessels distributed on the head space of the larva.
Conclusions
In summary, we present four biscyclometallated iridium(III) en complexes functionalised with polar ester or carboxylate groups.
3
3
The phosphorescence of the complexes originated from emissive states with mixed IL and MLCT character. We found that the
carboxylate complexes 1b and 2b existed as the deprotonated form in physiological pH and displayed intense emission. The pq
complexes 2a and 2b exhibited significant emission enhancement upon binding to the protein BSA and it is likely that the
interaction is hydrophobic in nature. Additionally, the nature and polarity of cyclometallating ligands play a pivotal role in
lipophilicity which subsequently controls the cellular uptake efficiency, cytotoxicity and subcellular distribution of the complexes.
The cytotoxicity of the lipophilic ester complexes 1a and 2a toward HeLa cells was largely increased upon irradiation due to the
1
generation and accumulation of the iridium(III)ꢀsensitised O in the cells. In contrast, the carboxylate complexes 1b and 2b
2
showed negligible cytotoxicity due to their limited uptake. Remarkably, most of the complexes displayed distinctive organelleꢀ
staining; for example, the ppyꢀCOOMe and pqꢀCOOMe complexes 1a and 2a targeted endosomes and mitochondria, respectively,
−
whereas the ppyꢀCOO complex 1b was located on the surface of plasma membrane. Also, the membrane impermeability of
complex 1b enabled the imaging of circulation system of zebrafish larvae. Related work on phosphorescent inorganic and
organometallic iridium(III) complexes with a focus on live cell and animal imaging is in progress.
Experimental
General considerations
1
9
All solvents were of analytical reagent grade and purified according to published procedures. All buffer components were of
biological grade and used as received. Autoclaved MilliꢀQ water was used for the preparation for the aqueous solutions. Hpqꢀ
COOH and MgSO were obtained from Acros. 4ꢀ(2ꢀPyridyl)benzaldehyde (Hpba), AgNO , LiOH⋅H O, methylene blue, DPBF
4
3
2
and IrCl3ꢁnH O were obtained from SigmaꢀAldrich. BSA was purchased from Calbiochem and used as received. CCCP was
2
purchased from Acros. MTT and tricaine were purchased from SigmaꢀAldrich. Dulbecco’s modified Eagle’s medium (DMEM),
glucoseꢀfree DMEM, fetal bovine serum (FBS), PBS at pH 7.4, trypsinꢀEDTA, penicillin/streptomycin, Alexa Fluorꢀ633
conjugated transferrin, MitoTracker Green FM and CellMask Deep Red Plasma Membrane Stain were purchased from Invitrogen.
Unless specified, the growth medium for cell culture contained DMEM with 10% FBS and 1% penicillin/streptomycin. HeLa
cells were obtained from American Type Culture Collection. Mature zebrafish (Danio rerio) was purchased from Chong Hing
2
0
Aquarium, Hong Kong, China and maintained as described by Westerfield.
photoinduced spawning over green plants and then cultured at 28.5 C in filtered tap water with Instant Ocean (60 ng mL ), which
was purchased from Aquatic System. All experiments involving living zebrafish embryos and larvae have been approved by
The zebrafish embryos were obtained by
−
1
°
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Department of Health, the Government of the Hong Kong Special Administrative Region and the Animal Research Ethics Subꢀ
Committee of City University of Hong Kong. The instruments for the characterisation and photophysical measurements have been
2
1
22
described previously.
Emission quantum yields were measured using the optically dilute method with an aerated aqueous
2
3
solution of [Ru(bpy) ]Cl (Φem = 0.028) as the standard solution.
3
2
Synthesis
Preparation of methyl 4-(2-pyridyl)benzoate (Hppy-COOMe). Aliquots of NaOH (1 M, 60 mL) were added dropwise to a mixture of
Hpba (1.99 g, 10.89 mmol) and AgNO (2.81 g, 16.54 mmol) in 80% EtOH(aq) (100 mL) and then stirred at ambient temperature for 24 h.
3
The mixture was concentrated under vacuum, filtered and then acidified with concentrated HCl to precipitate HppyꢀCOOH as a white solid.
The solid was collected by filtration, washed with H O and Et O and then air dried (1.84 mg, 85%). A solution of HppyꢀCOOH (850 mg, 4.3
2
2
mmol), concentrated H SO (2 mL) and MeOH (30 mL) was refluxed under an inert atmosphere of nitrogen for 24 h. The solution was
2
4
evaporated to dryness under vacuum, leaving a pale yellow oil. The crude product was dissolved in H O (100 mL) and then neutralised with
2
a saturated Na CO solution. The aqueous phase was extracted with Et O. The combined organic phase was washed with H O, brine and
2
3
2
2
1
dried over MgSO . The solvent was removed under vacuum to yield the product as a white solid. Yield: 815 mg (90%). H NMR (300
4
MHz, chloroformꢀd, 298 K, relative to TMS):
δ 8.74 − 8.72 (m, 1H, H6 of pyridyl ring), 8.16 − 8.05 (m, 4H, H2, H3, H5 and H6 of phenyl
ring), 7.82 − 7.77 (m, 2H, H3 and H4 of pyridyl ring), 7.32 − 7.27 (m, 1H, H5 of pyridyl ring), 3.95 (s, 3H, CH ). Positiveꢀion ESIꢀMS (m/z):
3
+
+
2
15 [M + H ] .
Preparation of methyl 2-phenyl-4-quinolinecarboxylate (Hpq-COOMe). The synthetic procedure was similar to that for HppyꢀCOOMe,
1
except that HpqꢀCOOH (1.50 g, 6.02 mmol) was used instead of HppyꢀCOOH. Yield: 1.44 g (91%). H NMR (300 MHz, chloroformꢀd,
2
98 K, relative to TMS):
δ 8.76 (d, 1H, J = 8.7 Hz, H5 of quinoline ring), 8.42 (s, 1H, H3 of quinoline ring), 8.25 − 8.20 (m, 3H, H8 of
quinoline ring, H2 and H6 of phenyl ring), 7.77 (t, 1H, J = 7.7 Hz, H7 of quinoline ring), 7.63 (t, 1H, J = 7.8 Hz, H6 of quinoline ring), 7.61
−
+
+
7.47 (m, 3H, H3, H4 and H5 of phenyl ring), 4.08 (s, 3H, CH ). Positiveꢀion ESIꢀMS (m/z): 264 [M + H ] .
3
Preparation of [Ir(ppy-COOMe) (en)](Cl) (1a). A mixture of [Ir (ppyꢀCOOMe) Cl ] (500 mg, 0.38 mmol) and en (50 ꢀL, 0.76 mmol) in
2
2
4
2
CH Cl /MeOH (30 mL, 1:1, v/v) was refluxed under an inert atmosphere of nitrogen in the dark for 24 h. The solution was evaporated to
2
2
dryness yielding an orange solid, which was purified by column chromatography on silica gel. The desired product was eluted with
CH Cl /MeOH (2:1, v/v). The solvent was removed under vacuum to yield an orange solid. Subsequent recrystallisation from MeOH/Et O
2
2
2
1
afforded complex 1a as orange crystals. Yield: 340 mg (63%). H NMR (400 MHz, methanolꢀd , 298 K, relative to TMS):
5.6 Hz, H6 of pyridyl ring), 8.19 (d, 2H, J = 8.0 Hz, H4 of phenyl ring), 8.05 (t, 2H, J = 7.2 Hz, H4 of pyridyl ring), 7.72 (d, 2H, J = 8.0 Hz,
δ
8.88 (d, 2H, J
4
=
H3 of pyridyl ring), 7.54 (t, 2H, J = 7.2 Hz, H5 of pyridyl ring), 7.42 (d, 2H, J = 8.0 Hz, H3 of phenyl ring), 6.91 (s, 2H, H6 of phenyl ring),
−
1
4
.83 (br s, 2H, NH ), 4.07 (br s, 2H, NH ), 3.67 (s, 6H, OCH ), 2.98 (br s, 2H, CH ), 2.79 (br s, 2H, CH ). IR (KBr) ν/cm : 1701 (C=O).
2 2 3 2 2
+
−
Positiveꢀion ESIꢀMS (m/z): 678 [M − Cl ] . Anal. calcd (%) for IrC H N O Cl⋅3H O⋅CH Cl : C, 40.92; H, 4.26; N, 6.58; found: C, 40.87;
28
28
4
4
2
2
2
H, 4.46; N, 6.85.
Preparation of [Ir(pq-COOMe) (en)](Cl) (2a). A mixture of [Ir (pqꢀCOOMe) Cl ] (400 mg, 0.27 mmol) and en (40 ꢀL, 0.55 mmol) in
2
2
4
2
CH Cl (20 mL) was refluxed under an inert atmosphere of nitrogen in the dark for 8 h. The solution was evaporated to dryness yielding a
2
2
dark brown residue, which was purified by column chromatography on silica gel. The desired product was eluted with CH Cl /MeOH (8:1,
2
2
v/v). The solvent was removed under vacuum to yield a brown solid. Subsequent recrystallisation from MeOH/Et O afforded complex 2a as
2
1
brown crystals. Yield: 240 mg (54%). H NMR (400 MHz, methanolꢀd , 298 K, relative to TMS):
δ
8.80 (dd, 2H, J = 8.4, 2.4 Hz, H5 of
quinoline ring), 8.72 (s, 2H, H3 of quinoline ring), 8.09 (d, 2H, J = 8.8 Hz, H3 of phenyl ring), 8.02 (d, 2H, J = 7.6 Hz, H8 of quinoline ring),
4
7
6
2
.75 – 7.65 (m, 4H, H6 and H7 of quinoline ring), 7.03 (t, 2H, J = 7.0 Hz, H4 of phenyl ring), 6.73 (t, 2H, J = 7.0 Hz, H5 of phenyl ring),
.60 (d, 2H, J = 7.6 Hz, H6 of phenyl ring), 4.81 (br s, 2H, NH ), 4.18 (s, 6H, OCH ), 3.26 (br s, 2H, NH ), 2.57 (br s, 2H, CH ), 1.91 (br s,
2
3
− +
2
2
−1
H, CH ). IR (KBr) ν/cm : 1720 (C=O). Positiveꢀion ESIꢀMS (m/z): 777 [M – Cl ] . Anal. calcd (%) for IrC H N O Cl⋅2H O: C, 50.97;
2
36 32
4
4
2
H, 4.28; N, 6.60; found: C, 50.84; H, 4.48; N, 6.91.
Preparation of [Ir(ppy-COO) (en)](Li) (1b). A mixture of complex 1a (53 mg, 0.08 mmol) and LiOH⋅H O (34 mg, 0.8 mmol) in 80%
2
2
aqueous MeOH (5 mL) was stirred at ambient temperature for 48 h. The solution was evaporated to dryness yielding a yellow residue.
1
Subsequent recrystallisation from MeOH/Et O afforded complex 1b as yellow crystals. Yield: 18 mg (34%). H NMR (400 MHz,
2
methanolꢀd , 298 K, relative to TMS):
J = 8.0 Hz, H4 of pyridyl ring), 7.66 (d, 2H, J = 8.0 Hz, H3 of pyridyl ring), 7.39 – 7.35 (m, 4H, H3, H5 of pyridyl ring), 6.92 (s, 2H, H6 of
δ
8.77 (d, 2H, J = 5.2 Hz, H6 of pyridyl ring), 8.13 (d, 2H, J = 8.4 Hz, H4 of phenyl ring), 7.94 (t, 2H,
4
−1
phenyl ring), 4.80 (br s, 2H, NH ), 3.77 (br s, 2H, NH ), 2.97 (br s, 2H, CH ), 2.74 (br s, 2H, CH ). IR (KBr) ν/cm : 1608 (C=O).
2
2
2
2
+
−
Negativeꢀion ESIꢀMS (m/z): 647 [M − Li ] . Anal. calcd (%) for IrC H N O Li⋅5H O: C, 41.69; H, 4.00; N, 6.95; found: C, 41.70; H, 4.33;
N, 7.16.
26
22
4
4
2
Preparation of [Ir(pq-COO) (en)](Li) (2b). A mixture of complex 2a (49 mg, 0.06 mmol) and LiOH⋅H O (5.1 mg, 0.12 mmol) in 80%
2
2
aqueous THF (5 mL) was stirred at ambient temperature for 24 h. The solution was evaporated to dryness yielding a red residue.
1
Subsequent recrystallisation from MeOH/Et O afforded complex 2b as red crystals. Yield: 35 mg (77%). H NMR (400 MHz, methanolꢀd4,
2
2
98 K, relative to TMS): δ 8.42 (dd, 2H, J = 6.8, 3.2 Hz, H5 of quinoline ring), 8.27 (s, 2H, H3 of quinoline ring), 8.00 (dd, 2H, J = 6.8, 3.6
Hz, H3 of phenyl ring), 7.94 (d, 2H, J = 7.2 Hz, H8 of quinoline ring), 7.64 – 7.60 (m, 4H, H6 and H7 of quinoline ring), 6.97 (t, 2H, J = 7.2
Hz, H4 of phenyl ring), 6.66 (t, 2H, J = 8.0 Hz, H5 of phenyl ring), 6.59 (d, 2H, J = 7.6 Hz, H6 of phenyl ring), 4.77 (br s, 2H, NH ), 3.01 (br
2
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−1
+ −
s, 2H, NH ), 2.60 (br s, 2H, CH ), 2.00 (br s, 2H, CH ). IR (KBr) ν/cm : 1586 (C=O). Negativeꢀion ESIꢀMS (m/z): 747 [M – Li ] . Anal.
2
2
2
calcd (%) for IrC H N O Li⋅6H O: C, 47.38; H, 4.44; N, 6.50; found: C, 47.36; H, 4.80; N, 6.70.
34
26
4
4
2
pH-dependence studies
pH titrations were conducted with a stock solution of complex 1b or 2b (50
ꢀ
M, 20 mL) in an aerated 10 mM KOH(aq) and 100
mM KCl(aq)/DMSO (9:1, v/v) solution. The pH value of the buffer solution was decreased gradually by addition of appropriate
amounts of 5.00, 2.50, 1.25, 0.65, 0.30, 0.15, 0.08, 0.04 or 0.02 N HCl(aq). After a desired pH was achieved, a portion of the
solution (2 mL) was transferred to a quartz cuvette for electronic absorption, emission and lifetime measurements. The overall
increase in solution volume due to pH adjustment was kept below 2%. The pKa values were obtained from the Hendersonꢀ
2
4
Hasselbalch type equation: pH = pKa + log [(Imax
− I)/(I − Imin)], where I is the emission intensity of the solution upon excitation
at 350 nm and Imax and Imin are the maximum and minimum intensities, respectively.
Protein-binding studies
Complexes 1a, 1b and 2b (10 ꢀM) in 2 mL potassium phosphate buffer (50 mM, pH 7.4) and 2a (10 ꢀM) in 2 mL potassium
phosphate buffer (50 mM, pH 7.4) containing 0.5% DMSO, respectively, were mixed with BSA in a series of [BSA]:[Ir] ratios
ranging from 0 to 2.5. The mixtures were incubated in the dark at room temperature for 24 h. The emission spectra and lifetime
of the solutions were then recorded.
Determination of lipophilicity
The lipophilicity (log Po/w values) of the complexes was measured using the shakeꢀflask method. An aliquot of stock solution of
the complex in PBS solution (pH 7.4 and saturated with nꢀoctanol) was added to an equal volume of nꢀoctanol (saturated with PBS
buffer) and shaken vigorously for 4 h. The mixture was then centrifuged at 40 rpm for 30 min for phase separation. The two
immiscible layers were carefully isolated and the concentrations of the complexes were determined by emission spectroscopy.
Dark cytotoxicity assays
−
1
HeLa cells were seeded in a 96ꢀwell flatꢀbottomed microplate (10 000 cells well ) in growth medium (100
ꢀL) and incubated at
2
5
3
7 °C under a 5% CO atmosphere for 24 h. A detailed procedure for the MTT assay has been described previously. The
2
absorbance of the solutions at 570 nm was measured with a SPECTRAmax 340 microplate reader (Molecular Devices Corp.,
Sunnyvale, CA). The IC50 values of the complexes were determined from the dose dependence of surviving cells after exposure to
the complexes at concentrations ranging from 3.13 to 200 ꢀM for 6 and 24 h.
Live cell confocal imaging
HeLa cells in growth medium were seeded on a sterilised glass coverslip in a 35ꢀmm tissue culture dish and grown at 37 °C under
a 5% CO atmosphere for 48 h. The culture medium was removed and replaced with medium/DMSO (99:1, v/v) containing the
2
complexes at various concentrations. At different incubation time points, the medium was removed and the cell layer was washed
with PBS (1 mL
microscope.
× 3). The coverslip was mounted onto a sterilised glass slide and then imaged using a Leica TCS SPE confocal
Cellular uptake studies
In the temperatureꢀdependence experiments, HeLa cells were incubated with complex 1a (100 ꢀM) or 2a (10 ꢀM) for 1.5 h at 37
or 4
°
C. In the inhibition experiments, HeLa cells pretreated with CCCP (10
ꢀ
M, 30 min) in a glucoseꢀfree medium were
incubated with complex 1a (100
used as the control.
ꢀ
M) or 2a (10
ꢀ
M) for 1.5 h at 37
°
C. Cells grown at 37 C without treatment of CCCP were
°
Costaining studies
−
1
In the endosomal imaging experiments, HeLa cells pretreated with Alexa Fluor 633ꢀconjugated transferrin (50
medium for 1.5 h were incubated with complex 1a (50 M) for 6 h and then washed with PBS. In the mitochondrial imaging
experiments, HeLa cells were incubated with complex 2a (10 M) in medium for 1.5 h and then with MitoTracker Green FM (300
nM) in a FBSꢀfree medium for 15 min and then washed with PBS. In the cell membrane imaging experiments, HeLa cells were
ꢀg mL ) in
ꢀ
ꢀ
–
1
incubated with complex 1b (50 ꢀM) for 6 h and then with CellMask Deep Red Plasma Membrane Stain (12.5 ꢀg mL ) for 10 min
and then washed with PBS. The colocalisation coefficients were determined by the program ImageJ (Version 1.4.3.67).
ICP-MS
HeLa cells were grown to 50% confluency in a 60ꢀmm tissue culture dish. The cells were incubated with the complexes (10 ꢀM)
in medium/DMSO (99:1, v/v) at 37
thoroughly with PBS (1 mL 3). The cells were typsinised by trypsinꢀEDTA (500
further washed with PBS (250 2) and the PBS portions were collected. The harvested cells, together with the collected PBS,
were digested with 65% HNO (1 mL) at 60 C. The concentration of iridium in the filtrate was measured using an ELan 6100
°C under a 5% CO atmosphere for 6 h. The medium was then removed and washed
2
×
ꢀL) and harvested. The culture dish was
ꢀL ×
°
3
DRCꢀICPꢀMS system (PerkinElmer SCIEX Instruments, USA).
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1
Determination of O quantum efficiency
2
1
26
The quantum yields for photosensitised O (Φꢁ) were determined according to a literature procedure.
An airꢀequilibrated
2
DMSO solution (2 mL) containing the complexes and DPBF (10
ꢀM) was introduced to a quartz cuvette of 1ꢀcm path length and
1
irradiated at 365 nm with a 6 W UVꢀA lamp (Spectroline, USA). Methylene blue was used as a reference for O sensitisation (Φꢁ
2
=
0.52). The absorbance of the complexes and methylene blue at 365 nm was ca. 0.15. The absorbance of DPBF at 417 nm was
monitored every 6 s. A DMSO solution of DPBF without the complexes was examined to determine its photostability under
identical irradiation conditions. The Φꢁ of the complex was determined by comparing Φꢁ of iridium(III)ꢀsensitised DPBF
photooxidation to Φꢁ of methylene blueꢀsensitised DPBF photooxidation (as reference) and calculated by the following equation:
unk
MB
(
(
m
m
× F
× F
)
)
unk
Φꢁ
MB
= Φꢁ
×
MB
unk
where
m
is the slope of a linear fit of the change in absorbance of DPBF at 417 nm against the irradiation time and
F
is the
−
AL
absorption correlation factor, which is given as
F
= 1 – 10
(A = absorbance at 365 nm and L = pathlength of the cuvette).
Photoinduced cytotoxicity assays
−
1
HeLa cells were seeded in two 96ꢀwell flatꢀbottomed microplates (10 000 cells well ) in growth medium (100
ꢀ
L) and incubated
L, 99:1, v/v)
M) and grown in the dark for 6 h. The culture medium was
L) was added to each well. One of the microplates was irradiated
at 37 °C under a 5% CO atmosphere for 24 h. The cells were replenished with fresh medium/DMSO (100
ꢀ
2
containing the complexes at various concentrations (0.09
− 200 ꢀ
removed, the cells were washed twice with PBS and PBS (100
ꢀ
at 365 nm with a 6 W UVꢀA lamp (Spectroline, USA) for 1 h while the other one was kept in the dark for 1 h. The cells were
replenished with fresh growth medium and further incubated in the dark for 24 h. The cell viability was assessed using the MTT
2
5
assay as described previously.
Live zebrafish imaging
A selected 48 hpf zebrafish embryo (Danio rerio) was manually dechorionated using forceps under a dissecting stereomicroscope.
The zebrafish larva was then anaesthetised in 0.016 M tricaine and mounted in 0.3% agarose gel. A PBS (pH 7.4) solution (40 nL)
of complex 1b (2 mM) was microinjected into the blood vessels at the heart region of the larva. The larva was imaged at three
postmicroinjection time points (5 min, 24 h and 48 h) using a fluorescence microscope (Olympus SZX 12) equipped with a
mercury lamp (EXFO Xꢀcite 120Q) as the excitation source. Subsequent to the imaging experiments, the larva was released from
the agarose tray and then returned to the water tank.
Acknowledgements
We thank The Hong Kong Research Grants Council (Project Nos. CityU 7008174 and 9667090) for financial support. T. S.ꢀM.
Tang acknowledges the receipt of a Postgraduate Studentship and an outstanding academic performance award administrated by
City University of Hong Kong.
Notes and references
a
Centre for Functional Photonics and Department of Biology and Chemistry, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong,
People’s Republic of China. Eꢀmail: bhkenlo@cityu.edu.hk (K. K.ꢀW. Lo); Fax: (852) 3442ꢀ0522.
b
Centre for Functional Photonics and Department of Biomedical Sciences, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong,
People’s Republic of China.
†
Electronic Supplementary Information (ESI) available: electronic absorption and photophysical data, pHꢀtitration and proteinꢀbinding results,
fluorescence and Zꢀscan images of HeLa cells, singlet oxygen determination data and luminescence images of zebrafish larve. See
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Graphical abstract
Four new phosphorescent biscyclometallated iridium(III) ethylenediamine complexes functionalised with polar ester or carboxylate groups
have been found to achieve organelleꢀselective staining in live cells and bright visualisation of physiological structures in zebrafish.
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Dalton Discussion XX | 17