Luminescent cyclometallated iridium(iii) bis(quinolylbenzaldehyde) diimine complexes - Synthesis, photophysics, electrochemistry, protein cross-linking properties, cytotoxicity and cellular uptake

Article abstract of DOI:10.1039/c0dt00501k

Four new luminescent cyclometallated iridium(iii) bis(quinolylbenzaldehyde) diimine complexes [Ir(qba)₂(NN)](PF₆) (Hqba = 4-(2-quinolyl)benzaldehyde, NN = 2,2′-bipyridine, bpy (1); 1,10-phenanthroline, phen (2); 3,4,7,8-tetramethyl-1,10-phenanthroline, Me ₄-phen (3); 4,7-diphenyl-1,10-phenanthroline, Ph₂-phen (4)) have been synthesised and characterised, and their electronic absorption, emission and electrochemical properties investigated. The X-ray crystal structures of complexes 1 and 2 have been determined. Upon irradiation, complexes 1-4 exhibited intense and long-lived orange-yellow emission in fluid solutions at 298 K and in alcohol glass at 77 K. The emission has been assigned to a triplet intra-ligand (³IL) excited state associated with the qba ligand, probably with mixing of some triplet metal-to-ligand charge-transfer (³MLCT) (dπ(Ir) → π*(qba)) character. Reductive amination reactions of complexes 1-4 with the protein bovine serum albumin (BSA) afforded the bioconjugates 1-BSA-4-BSA, respectively. Upon photoexcitation, these bioconjugates displayed intense and long-lived ³MLCT (dπ(Ir) → π*(NC)) emission in aqueous buffer at 298 K. The cross-linked nature of the Ir-BSA bioconjugates has been verified by SDS-PAGE. Additionally, the cytotoxicity of the complexes towards human cervix epithelioid carcinoma (HeLa) cells has been examined by 3-(4,5-dimethyl-2-thiazolyl)-2,5- diphenyltetrazolium bromide (MTT) assays, and the cellular uptake of complex 4 has been investigated by laser-scanning confocal microscopy and flow cytometry.

Full text of DOI:10.1039/c0dt00501k

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Dalton
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An international journal of inorganic chemistry
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Volume 40 | Number 10 | 14 March 2011 | Pages 2077–2392
Themed issue: New Talent: Asia
ISSN 1477-9226
COVER ARTICLE
Lo et al.
Luminescent cyclometallated
iridium(III) bis(quinolylbenzaldehyde)
diimine complexes
1477-9226(2011)40:10;1-0

PAPER
www.rsc.org/dalton | Dalton Transactions
Luminescent cyclometallated iridium(III) bis(quinolylbenzaldehyde) diimine
complexes—synthesis, photophysics, electrochemistry, protein cross-linking
properties, cytotoxicity and cellular uptake†
Pui-Kei Lee, Hua-Wei Liu, Shek-Man Yiu, Man-Wai Louie and Kenneth Kam-Wing Lo*
Received 19th May 2010, Accepted 28th July 2010
DOI: 10.1039/c0dt00501k
Four new luminescent cyclometallated iridium(III) bis(quinolylbenzaldehyde) diimine complexes
[Ir(qba)2(N∧N)](PF6) (Hqba = 4-(2-quinolyl)benzaldehyde, N∧N = 2,2¢-bipyridine, bpy (1);
1,10-phenanthroline, phen (2); 3,4,7,8-tetramethyl-1,10-phenanthroline, Me4-phen (3);
4,7-diphenyl-1,10-phenanthroline, Ph2-phen (4)) have been synthesised and characterised, and their
electronic absorption, emission and electrochemical properties investigated. The X-ray crystal
structures of complexes 1 and 2 have been determined. Upon irradiation, complexes 1–4 exhibited
intense and long-lived orange-yellow emission in fluid solutions at 298 K and in alcohol glass at
77 K. The emission has been assigned to a triplet intra-ligand (3IL) excited state associated with the qba
ligand, probably with mixing of some triplet metal-to-ligand charge-transfer (3MLCT) (dp(Ir) →
p*(qba)) character. Reductive amination reactions of complexes 1–4 with the protein bovine serum
albumin (BSA) afforded the bioconjugates 1-BSA–4-BSA, respectively. Upon photoexcitation, these
bioconjugates displayed intense and long-lived 3MLCT (dp(Ir) → p*(N∧C)) emission in aqueous buffer
at 298 K. The cross-linked nature of the Ir–BSA bioconjugates has been verified by
SDS-PAGE. Additionally, the cytotoxicity of the complexes towards human cervix epithelioid
carcinoma (HeLa) cells has been examined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium
bromide (MTT) assays, and the cellular uptake of complex 4 has been investigated by laser-scanning
confocal microscopy and flow cytometry.
the harmful effects of the excitation source and background
Introduction
fluorescence in biological systems, respectively. Additionally, a
The rich photophysical and photochemical properties of many
reactive group such as an aldehyde will render these reagents
transition metal complexes have been well documented. In view
active towards various amine-containing biological entities. While
of the advantages of these systems such as variable molecular
the first goal can be achieved by introducing an extended planar
structures, intense absorption and tunable emission in the vis-
ible region with high luminescence quantum yields and long
cyclometallating ligand with lower-lying p* orbitals, the latter
target can be accomplished by the incorporation of an aldehyde
emission lifetimes, there is an expanding interest in applying
group into the same ligand. We envisage that new complexes
them in biological studies. Recently, the cellular uptake properties
with these characteristics would exhibit desirable absorption and
and cytotoxicity of polypyridine complexes of platinum(II),1,2
emission in aqueous solutions under ambient conditions owing
ruthenium(II),3–7 rhenium(I),8–10 rhodium(III)11 and gold(III)12 have
to significant participation of intraligand (IL) character in the
low-lying emissive states,20c potential cross-linking properties20a as
well as unique emission characteristics upon reductive amination
via the aldehyde groups.20f Herein we describe the synthesis,
characterisation, photophysics and electrochemistry of four lu-
minescent iridium(III) bis(quinolylbenzaldehyde) diimine com-
plexes [Ir(qba)2(N∧N)](PF6) (Hqba = 4-(2-quinolyl)benzaldehyde,
N∧N = 2,2¢-bipyridine, bpy (1); 1,10-phenanthroline, phen
(2); 3,4,7,8-tetramethyl-1,10-phenanthroline, Me4-phen (3);
4,7-diphenyl-1,10-phenanthroline, Ph2-phen (4)) (Chart 1). The
bioconjugation products of the protein bovine serum albumin
(BSA) modified with these complexes have been studied by
emission spectroscopy and SDS-PAGE. Additionally, the cy-
totoxicity of the complexes towards human cervix epithelioid
carcinoma (HeLa) cells has been examined by 3-(4,5-dimethyl-
2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assays.
Furthermore, the cellular uptake of complex 4 has been in-
vestigated by laser-scanning confocal microscopy and flow
cytometry.
been widely reported. Since many cyclometallated iridium(III)
polypyridine complexes are known to be excellent environment-
sensitive emitters,13–20 this family of complexes are attractive
choices for biomolecular and cellular probes.21,22 Following our
interest in the utilisation of cyclometallated iridium(III) polypyri-
dine complexes in biological labelling studies, we anticipate that
functionalisation of these luminescent compounds with reactive
groups will lead to useful cellular labels and probes.
The main objective of this work is to design new cyclometallated
iridium(III) diimine complexes [Ir(N∧C)2(N∧N)]+ with biological
reactivity and desirable photophysical properties for bio-imaging.
In this context, compounds that feature both low-energy absorp-
tion and emission are attractive candidates as this can minimise
Department of Biology and Chemistry, City University of Hong Kong, Tat
Chee Avenue, Kowloon, Hong Kong, People’s Republic of China. E-mail:
bhkenlo@cityu.edu.hk; Fax: (+852) 3442 0522; Tel: (+852) 3442 7231
† CCDC reference numbers 775620 and 775621. For crystallographic data
in CIF or other electronic format see DOI: 10.1039/c0dt00501k
2180 | Dalton Trans., 2011, 40, 2180–2189
This journal is
The Royal Society of Chemistry 2011
©

to give a reddish brown solid. Subsequent recrystallisation from
diffusion of diethyl ether into a CH2Cl2 solution of [Ir2(qba)4Cl2]
afforded reddish brown crystals. The complex was used without
further purification. Yield: 379 mg (62%). Positive-ion ESI-MS
+
(m/z): 656 {Ir(qba)2} .
[Ir(qba)2(N∧N)](PF6). A mixture of [Ir2(qba)4Cl2] (83 mg,
0.06 mmol) and N∧N (0.26 mmol) in 25 ml of CH2Cl2–MeOH–
H2O (2 : 2 : 1 v/v) was heated at reflux under nitrogen for 4 h.
The red solution was then cooled to room temperature, and solid
KPF6 (35 mg, 0.19 mmol) was added to the solution. The mixture
was then evaporated to dryness, and the solid was dissolved in
CH2Cl2 and purified by column chromatography on silica gel. The
desired product was eluted with CH2Cl2–acetone (9 : 1 v/v) and
subsequently recrystallised from a mixture of acetone and diethyl
ether.
[Ir(qba)2(bpy)](PF6) (1). Complex 1 was isolated as red crys-
tals. Yield: 84 mg (80%). 1H NMR (400 MHz, acetone-d6, 298 K,
relative to TMS): d 9.66 (s, 2H, CHO), 8.72–8.66 (m, 4H, H3
and H4 of quinolyl ring of qba), 8.52 (d, J = 8.1 Hz, 2H, H5 of
phenyl ring of qba), 8.47–8.42 (m, 4H, H5, H5¢, H6 and H6¢ of
bpy), 8.17 (dt, J = 8.1 and 1.4 Hz, 2H, H4 of bpy), 8.14 (dd, J =
8.1 and 1.3 Hz, 2H, H5 of quinolyl ring of qba), 7.75–7.72 (m,
4H, H3 and H3¢ of bpy and H8 of quinolyl ring of qba), 7.54–
7.48 (m, 4H, H6 of phenyl ring and H7 of quinolyl ring of qba),
7.20–7.15 (m, 2H, H6 of quinolyl ring of qba). Anal. Calc. for
C42H28N4O2PF6Ir·1/2H2O: C, 52.17; H, 3.02; N, 5.79. Found: C,
52.47; H, 3.32; N, 5.54%. IR (KBr) n/cm-1: 1685 (s, CHO), 839 (s,
Chart 1 Structures of the iridium(III) complexes.
Experimental
General considerations
All solvents were of analytical reagent grade and purified
according to published procedures.23 All buffer components
were of biological grade and used as received. 2-
Hydroxyquinoline (Aldrich), N-bromosuccinimide (Aldrich),
triphenylphosphine (Acros), 4-formylphenylboronic acid (Acros),
-
- +
PF6 ). Positive-ion ESI-MS (m/z): 813 {M – PF6 } .
cis-dichlorobis(triphenylphosphine)palladium(II)
(Aldrich),
[Ir(qba)2(phen)](PF6) (2). Complex 2 was isolated as orange-
red crystals. Yield: 148 mg (72%). 1H NMR (400 MHz, acetone-d6,
298 K, relative to TMS): d 9.70 (s, 2H, CHO), 8.82 (dd, J = 5.1 and
1.4 Hz, 2H, H2 and H9 of phen), 8.78 (dd, J = 8.3 and 1.4 Hz, 2H,
H4 and H7 of phen), 8.73 (d, J = 8.9 Hz, 2H, H4 of quinolyl ring of
qba), 8.62 (d, J = 8.9 Hz, 2H, H3 of quinolyl ring of qba), 8.58 (d,
J = 8.2 Hz, 2H, H5 of phenyl ring of qba), 8.13 (s, 2H, H5 and H6
of phen), 8.10 (dd, J = 8.3 and 5.1 Hz, 2H, H3 and H8 of phen),
7.88 (dd, J = 8.2 and 1.4 Hz, 2H, H5 of quinolyl ring of qba), 7.79
(dd, J = 8.1 and 1.6 Hz, 2H, H8 of quinolyl ring of qba), 7.41 (d,
J = 9.0 Hz, 2H, H6 of phenyl ring of qba), 7.37–7.33 (m, 2H, H7
of quinolyl ring of qba), 7.30 (d, J = 1.4 Hz, 2H, H2 of phenyl
ring of qba), 6.95–6.91 (m, 2H, H6 of quinolyl ring of qba). Anal.
Calc. for C44H28N4O2PF6Ir: C, 53.82; H, 2.88; N, 5.71. Found: C,
53.66; H, 2.94; N, 5.76%. IR (KBr) n/cm-1: 1683 (s, CHO), 845 (s,
K2CO3 (Acros), IrCl3·3H2O (Aldrich), bpy (Aldrich), phen
(Acros), Me4-phen (Acros), Ph2-phen (Acros) and KPF6 (Acros)
were used without further purification. Hqba was prepared as
described previously.24 BSA was obtained from Calbiochem
and used as received. NAP-10 size-exclusion columns and
YM-30 microcons were purchased from Pharmacia and Amicon,
respectively. HeLa cells were obtained from American Type
Culture Collection. Dulbecco’s modified Eagle’s medium
(DMEM), fetal bovine serum (FBS), phosphate buffered
saline (PBS), trypsin–EDTA and penicillin/streptomycin were
purchased from Invitrogen. The growth medium for cell culture
contained DMEM with 10% FBS and 1% penicillin/streptomycin.
Sugar-free growth medium (Invitrogen), 2-deoxyglucose (Acros),
carbonyl cyanide 3-chlorophenylhydrazone (CCCP) (Acros),
potassium nitrate (Acros), sodium azide (Acros), sodium
orthovanadate (Acros) and sodium molybdate(VI) dihydrate
(Acros) were used without purification.
-
- +
PF6 ). Positive-ion ESI-MS (m/z): 837 {M – PF6 } .
[Ir(qba)2(Me4-phen)](PF6) (3). Complex 3 was isolated as
1
orange-red crystals. Yield: 60.3 mg (65%). H NMR (400 MHz,
acetone-d6, 298 K, relative to TMS): d 9.68 (s, 2H, CHO), 8.74 (d,
J = 8.8 Hz, 2H, H4 of quinolyl ring of qba), 8.61 (d, J = 8.6 Hz,
2H, H3 of quinolyl ring of qba), 8.58 (d, J = 8.2 Hz, 2H, H5 of
phenyl ring of qba), 8.45 (s, 2H, H2 and H9 of Me4-phen), 8.23
(s, 2H, H5 and H6 of Me4-phen), 7.88 (dd, J = 8.1 and 5.4 Hz,
2H, H5 of quinolyl ring of qba), 7.77 (dd, J = 8.1 and 1.6 Hz,
2H, H8 of quinolyl ring of qba), 7.44 (d, J = 8.9 Hz, 2H, H6
of phenyl ring of qba), 7.36–7.32 (m, 2H, H7 of quinolyl ring of
qba), 7.23 (d, J = 1.5 Hz, 2H, H2 of phenyl ring of qba), 6.93–6.91
(m, 2H, H6 of quinolyl ring of qba), 2.78 (s, 6H, CH3 on C4 and
C7 of Me4-phen), 2.45 (s, 6H, CH3 on C3 and C8 of Me4-phen).
Synthesis
[Ir2(qba)4Cl2]. The synthetic procedure was similar to that
reported for related dinuclear iridium(III) complexes.20a A mixture
of IrCl3·3H2O (311 mg, 0.88 mmol) and Hqba (548 mg, 2.26 mmol)
in 50 ml of 2-ethoxyethanol–water (3 : 1 v/v) was refluxed for 24 h
in the dark under an inert atmosphere of nitrogen. The reddish
brown precipitate formed was collected by centrifugation and
washed with 50 ml of ethanol–water (95 : 5 v/v) and air dried.
The solid was then dissolved in CH2Cl2 (100 ml) and the solution
was filtered with Celite. The filtrate was then evaporated to dryness
This journal is
The Royal Society of Chemistry 2011
Dalton Trans., 2011, 40, 2180–2189 | 2181
©

Table 1 Crystal data and summary of data collection and refinement for
complexes 1 and 2
Anal. Calc. for C48H36N4O2PF6Ir·H2O: C, 54.59; H, 3.63; N, 5.31.
Found: C, 54.73; H, 3.87; N, 5.44%. IR (KBr) n/cm-1: 1668 (s,
-
CHO), 845 (s, PF6 ). Positive-ion ESI-MS ion cluster (m/z): 893
1
2
-
+
{M – PF6 } .
Empirical formula
C42H28F6IrN4O2P·
C2H5O0.50
C44H28F6IrN4O2P·
[Ir(qba)2(Ph2-phen)](PF6) (4). Complex 4 was isolated as or-
ange crystals. Yield: 54 mg (42%). 1H NMR (400 MHz, acetone-
d6, 298 K, relative to TMS): d 9.71 (s, 2H, CHO), 8.89 (d, J = 5.
4 Hz, 2H, H2 and H9 of Ph2-phen), 8.76 (d, J = 4.4 Hz, 2H, H4
of quinolyl ring of qba), 8.66 (d, J = 8.7 Hz, 2H, H3 of quinolyl
ring of qba), 8.60 (d, J = 8.2 Hz, 2H, H5 of phenyl ring of qba),
8.07 (d, J = 5.4 Hz, 2H, H3 and H8 of Ph2-phen), 8.03 (s, 2H,
H5 and H6 of Ph2-phen), 7.93 (d, J = 8.3 Hz, 2H, H5 of quinolyl
ring of qba), 7.80 (d, J = 8.3 Hz, 2H, H8 of quinolyl ring of qba),
7.63–7.51 (m, C6H5 on C4 and C7 of Ph2-phen and H6 of phenyl
ring of qba), 7.42–7.38 (m, 2H, H7 of quinolyl ring of qba), 7.30
(d, J = 1.4 Hz, 2H, H2 of phenyl ring of qba), 7.09–7.05 (m, 2H,
H6 of quinolyl ring of qba). Anal. Calc. for C54H48N4O2PF6Ir: C,
57.80; H, 4.31; N, 4.99. Found: C, 57.76; H, 4.52; N, 5.10%. IR
C
6.50H14O2
Mr
994.91
0.58 ¥ 0.15 ¥ 0.12
133
1106.05
0.64 ¥ 0.28 ¥ 0.16
173
Crystal size/mm
T/K
Crystal system
Triclinic
Triclinic
¯
¯
Space group
P1
P1
˚
a/A
10.6285(3)
11.1167(3)
˚
b/A
13.1359(3)
14.5805(3)
1895.12(7)
2
1.744
3.64
982
3.6–32.2
12.1327(4)
16.8271(5)
2202.76(12)
2
1.668
3.15
1102
3.4–27.5
˚
c/A
3
˚
V/A
Z
rc/g cm-3
m/mm-1
F/000
q range/◦
Oscillation/◦
Distance/mm
Exposure time/s
Index ranges
1
60
20
1
55
10
(KBr) n/cm-1: 1687 (s, CHO), 845 (s, PF6 ). Positive-ion ESI-MS
-
-12 £ h £ 12; -15 £ k
£ 15; -17 £ l £ 17
0.025
-13 £ h £ 11; -14 £ k
£ 14; -19 £ l £ 20
0.019
-
+
(m/z): 977 {M – PF6 } .
a
Rint
No. of unique
data/restraints/
parameters
6659/4/523
7729/70/646
Physical measurements and instrumentation
Instruments for the characterisation and photophysical and
electrochemical measurements have been described previously.22
Luminescence quantum yields were measured using the optically
dilute method25 with an aerated aqueous solution of [Ru(bpy)3]Cl2
(Uem = 0.028) as the standard solution.26
GOF on F2
1.07
0.0170, 0.0447
0.98
0.0209, 0.0469
b
R1, wR2 (I >
2s(I))c
R1, wR2 (all data)
0.0193, 0.0452
1.12, -0.46
0.0258, 0.0476
1.11, -0.69
Largest diff. peak,
-3
˚
hole/e A
a Rint = R|Fo - Fo (mean)|/R[Fo2]. b GOF = {R[w(Fo - Fc2)2]/(n -
2
2
2
Crystal structure determination
p)}1/2, where n is the number of reflections and p is the total number of
2
parameters refined. The weighting scheme is w = 1/[s (Fo2) + (0.0321P)2 +
X-Ray structural analysis of complex 1. Single crystals of com-
plex 1 suitable for X-ray crystallographic studies were obtained
by slow diffusion of diethyl ether vapour into a concentrated
acetone solution of the complex. A crystal of dimensions 0.58 ¥
0.15 ¥ 0.12 mm coated with paratone-N and mounted on a nylon
cryoloop was used for X-ray diffraction analysis. X-ray diffraction
data were collected on an Oxford Diffraction Gemini S Ultra
4-circle kappa diffractometer with a 92 mm diagonal Sapphire
CCD detector using graphite monochromatised Mo-Ka radiation
at 133 K. Data collection was made with a 1◦ oscillation step
of w, a 20 s exposure time and a detector distance of 60 mm.
A total of 1117 frames in 17 runs were collected. The images
were interpreted and intensities integrated using the programme
CrysAlis.27 The structure was solved and refined using full-matrix
least-squares based on F2 with the programmes SHELXS-9728 and
SHELXL-9728 within WinGX29 on a PC. Iridium and many non-
hydrogen atoms were located according to direct methods. The
positions of other non-hydrogen atoms were found after successful
refinement by full-matrix least-squares using SHELXL-97. One
crystallographic asymmetric unit consists of one Ir formula unit,
one PF6 anion and half a diethyl ether solvent molecule. In the
final stage of least-squares refinement, non-hydrogen atoms on
the disordered diethyl ether solvent molecule and the disordered
formyl group on one of the qba ligands were refined isotropically
and the other non-hydrogen atoms were refined anisotropically.
Hydrogen atoms were generated by SHELXL-97. The positions
of the hydrogen atoms were calculated on the basis of riding mode
with thermal parameters equal to 1.2 times that of the associated
2
0.1169P] for complex 1 and w = 1/[s (Fo2) + (0.0028P)2] for complex 2,
where P is (Fo2 + 2Fc2)]/3. c R1 = RꢀFo| - |Fcꢀ/R|Fo|, wR2 = {R[w(Fo
-
2
1/2
Fc2)2]/R[w(Fo2)]}
.
C atoms, and participated in the calculation of final R-indices.
Crystal data and a summary of data collection and refinement
details are given in Table 1.
X-Ray structural analysis of complex 2. Single crystals of com-
plex 2 suitable for X-ray crystallographic studies were obtained
by slow diffusion of diethyl ether vapour into a concentrated
acetone solution of the complex. A crystal of dimensions 0.64 ¥
0.28 ¥ 0.16 mm coated with paratone-N and mounted on a nylon
cryoloop was used for X-ray diffraction analysis. X-ray diffraction
data were collected on an Oxford Diffraction Gemini S Ultra
4-circle kappa diffractometer with a 92 mm diagonal Sapphire
CCD detector using graphite monochromatised Mo-Ka radiation
at 173 K. Data collection was made with a 1◦ oscillation step
of w, a 10 s exposure time and a detector distance at 55 mm.
A total of 422 frames in 5 runs were collected. The images
were interpreted and intensities integrated using the programme
CrysAlis. The structure was solved and refined using full-matrix
least-squares based on F2 with the programmes SHELXS-97 and
SHELXL-97 within WinGX. Iridium and many non-hydrogen
atoms were located according to direct methods. The positions of
other non-hydrogen atoms were found after successful refinement
by full-matrix least-squares using SHELXL-97 on a PC. One
2182 | Dalton Trans., 2011, 40, 2180–2189
This journal is
The Royal Society of Chemistry 2011
©

crystallographic asymmetric unit consists of one Ir formula unit,
one PF6 anion, one acetone solvent molecule and half an acetone
and half a diethyl ether solvent molecule occupying the same
void. In the final stage of least-squares refinement, all non-
hydrogen atoms were refined anisotropically. Hydrogen atoms
were generated by the programme SHELXL-97. The positions
of the hydrogen atoms were calculated on the basis of riding mode
with thermal parameters equal to 1.2 or 1.5 times that of the
associated C atoms, and participated in the calculation of final
R-indices. Crystal data and a summary of data collection and
refinement details are given in Table 1.
Flow cytometry
HeLa cells in growth medium (100,000 cells ml-1)◦were seeded in
a 35 mm tissue culture dish and incubated at 37 C under a 5%
CO2 atmosphere for 48 h. The culture medium was removed and
replaced with medium–DMSO (99 : 1 v/v) containing the irid-
ium(III) complexes at a concentration of 5 mM. After incubation
for 1 h, the medium was removed, and the cell layer was washed
gently with PBS (1 ml ¥ 3). The cell layer was then trypsinised and
made up to a final volume of 1 ml with PBS. The samples were
analysed by a FACSCalibur flow cytometer (Becton, Dickinson
and Co., Franklin Lakes, NJ, USA) with excitation at 488 nm.
The number of cells analysed for each sample was between ca.
9,000 and 10,000.
Conjugation of BSA with complexes 1–4
In a typical procedure, the iridium(III) complex (1 mg) in anhy-
drous DMSO (20 ml) was added to BSA (5 mg) dissolved in 50 mM
carbonate buffer at pH 10.0 (180 ml). After the mixture was gently
stirred in the dark for 6 h, NaCNBH3 (0.63 mg, 10 mmol) in 2 ml
of 2 M NaOH was added, and the mixture was stirred gently
in the dark for 12 h at room temperature. Then, the mixture was
diluted with 50 mM phosphate buffer at pH 7.4 and the precipitate
was removed by centrifugation. The filtrate was loaded to a PD-
10 size-exclusion column that had been equilibrated with 50 mM
phosphate buffer at pH 7.4. The same buffer was used as the mobile
phase, and the first band with strong orange-yellow emission was
collected, concentrated with a YM-30 microcon and stored at 4 ◦C
before use.
Live-cell confocal imaging
HeLa cells were grown on sterile glass coverslips in a 35 mm
tissue culture dish. The sample preparation procedure was similar
to that of the flow cytometry. After washing with PBS, the
coverslips were mounted onto slides for measurements. Imaging
was performed using a confocal microscope (Carl Zeiss, LSM510)
with an excitation wavelength at 488 nm. The emission intensity
was measured between 505 and 800 nm.
Fixed-cell confocal imaging
The incubation procedure was similar to that of live-cell confocal
imaging except that after washing with PBS, the coverslips were
incubated in (1) MeOH–PBS (1 : 1 v/v) for 4 min and then in
MeOH for 4 min, or (2) 10% paraformaldehyde for 10 min
and then washed successively with MeOH. The coverslips were
finally washed with PBS before being mounted onto the slides for
measurements.
SDS-PAGE
The Ir-BSA bioconjugate ([BSA] = ca. 10 mg ml-1; 10 ml) was mixed
with a sample buffer (25% glycerol, 5% b-mercaptoethanol, 2%
SDS and 0.01% bromophenol blue in 0.06 M Tris-HCl at pH 6.8;
10 ml). The mixture was denatured at 95 ◦C for 5 min before being
loaded into a 6% polyacrylamide gel for SDS-PAGE analysis (Bio-
Rad electrophoresis accessories). The gel was directly imaged using
a Fujifilm LAS-4000 luminescent image analyser with excitation
at 460 nm.
ICP-MS
HeLa cells were grown in a 60 mm tissue culture dish and
incubated at 37 ◦C under a 5% CO2 atmosphere for 48 h. The
culture medium was then removed and replaced with medium–
DMSO (99 : 1 v/v) containing the iridium(III) complexes at a
concentration of 5 mM. After incubating for 1 h, the medium was
removed, and the cell layer was washed gently with PBS (1 ml ¥ 3).
The cell layer was then trypsinised and made up to a final volume of
1 ml with PBS. The harvested cells were digested with 65% HNO3
(2 ml) at 60 ◦C for 1 h, and then diluted in Milli-Q water to a
final volume of 10 ml. The concentration of iridium was measured
using an Elan 6100 DRC-ICP-MS system (PerkinElmer SCIEX
Instruments, USA) equipped with a peristaltic pump, Meinhard
quartz nebuliser, cyclonic spray chamber, nickel skimmer, and
sample cones.
Cytotoxicity assays
HeLa cells were seeded in a 96-well flat-bottomed microplate
(10,00◦0 cells well-1) in growth medium (100 ml) and incubated
at 37 C under a 5% CO2 atmosphere for 24 h. The iridium(III)
complexes and cisplatin (positive control) were then added,
respectively, to the wells with concentrations ranging from ca.
10-7 to 10-4 M in a mixture of growth medium–DMSO (99 : 1 v/v).
Wells containing growth medium without cells were used as blank
controls. The microplate was incubated at 37 ◦C under a 5% CO2
atmosphere for 48 h. Then, 10 mL of MTT in PBS (5 mg ml-1)
◦
was added to each well. The microplate was incubated at 37 C
Results and discussion
under a 5% CO2 atmosphere for another 3 h. The medium in each
well was then removed, and 100 ml of isopropanol was added to
each well and the microplate was further incubated for 5 min.
The absorbance of the solutions at 570 nm was measured with a
SPECTRAmax 340 microplate reader (Molecular Devices Corp.,
Sunnyvale, CA, USA). The IC50 values of the complexes were
determined from dose dependence of surviving cells after exposure
to the complexes.
Synthesis
The dimeric precursor compound [Ir2(qba)4Cl2] was synthesised,
in a moderate yield, from the reaction of IrCl3·3H2O and excess
Hqba ligand. The complex was isolated as reddish brown crystals
after recrystallisation from a mixture of CH2Cl2 and diethyl ether.
The mononuclear iridium(III) complexes 1–4 were obtained from
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The Royal Society of Chemistry 2011
Dalton Trans., 2011, 40, 2180–2189 | 2183
©

the reactions of the dinuclear complex with two equivalents of
the corresponding diimine ligands. These four complexes were
isolated as air-stable orange-red crystals in good yields. They
1
were characterised by H NMR spectroscopy, positive-ion ESI-
MS and IR spectroscopy, and gave satisfactory microanalysis.
The aldehyde moieties of complexes 1–4 were characterised by
a singlet at ca. d = 9.7 ppm in the 1H NMR spectra and an intense
IR absorption band at ca. 1685 cm-1. The crystal structures of
complexes 1 and 2 have also been studied by X-ray crystallography.
X-Ray crystal structure determination
The perspective views of the cations of complexes 1 and 2 are
depicted in Fig. 1 and 2, respectively. Selected bond lengths and
angles are listed in Table 2. In both complexes, the iridium(III)
centre adopted a distorted octahedral geometry, and the angles
of the trans ligands at the metal centre ranged from 168.59(8) to
˚
˚
170.19(8) A for complex 1 and from 168.17(9) to 169.71(10) A
for complex 2. The coordination geometry of the qba ligands
around the iridium(III) centre was such that the metal–carbon
bonds were in a cis orientation.16,20a,b,22d The trans influence of
Fig. 1 Perspective view of the cation of complex 1. Thermal ellipsoids are
set at the 50% probability level. Hydrogen atoms are omitted for clarity.
the carbon donors rendered slightly longer Ir–N bond lengths in
∧
˚
the N N ligands (Ir1–N3 and Ir1–N4; 2.173(2) and 2.1657(19) A
˚
for complex 1 and Ir1–N3 and Ir–N4; 2.197(2) and 2.172(2) A
for complex 2) than those in the qba ligands (Ir1–N1 and Ir1–
˚
N2; 2.096(2) and 2.091(2) A for complex 1 and Ir1–N1 and
˚
Ir1–N2; 2.091(2) A for complex 2). The bite angles of the qba
ligands (79.17(9) and 79.61(9)◦ for complex 1 and 79.67(10) and
79.56(10)◦ for complex 2) were larger than those of the N∧N
ligands (74.99(8)◦ for complex 1 and 75.88(9)◦ for complex 2).
These observations have been made in related cyclometallated
iridium(III) polypyridine systems [Ir(N∧C)2(N∧N)]+.16,20a,b,22d The
distance between the oxygen atoms of the two aldehyde groups
˚
˚
was ca. 9.7 A for complex 1 and 9.6 A for complex 2, respectively.
◦
˚
Table 2 Selected bond lengths (A) and angles ( ) for complexes 1 and 2
Complex 1
Ir1–C21
Ir1–C6
Ir1–N2
1.993(2)
1.998(2)
2.091(2)
Ir1–N1
Ir1–N4
Ir1–N3
2.096(2)
2.1657(19)
2.173(2)
C21–Ir1–C6
C21–Ir1–N2
C6–Ir1–N2
C21–Ir1–N1
C6–Ir1–N1
N2–Ir1–N1
C21–Ir1–N4
C6–Ir1–N4
92.27(9)
79.61(9)
92.44(9)
94.42(9)
79.17(9)
169.57(7)
95.77(8)
170.19(8)
N2–Ir1–N4
N1–Ir1–N4
C21–Ir1–N3
C6–Ir1–N3
N2–Ir1–N3
N1–Ir1–N3
N4–Ir1–N3
83.52(7)
105.72(8)
168.59(8)
97.63(9)
105.50(8)
81.96(8)
74.99(8)
Fig. 2 Perspective view of the cation of complex 2. Thermal ellipsoids are
set at the 50% probability level. Hydrogen atoms are omitted for clarity.
Electronic absorption and luminescence properties
The electronic absorption spectral data of complexes 1–4 in
CH2Cl2 and CH3CN at 298 K are listed in Table 3. The electronic
absorption spectra of complexes 1 and 4 in CH3CN at 298 K are
displayed in Fig. 3. Complexes 1–3 showed intense absorption
features in the UV region (e on the order of 104 dm3 mol-1 cm-1)
and moderate absorption bands in the visible region. With
reference to the photophysical studies of related cyclometallated
iridium(III) polypyridine systems,14–17,19,20a–e,22 the high-energy ab-
sorption bands at ca. 256–320 nm have been assigned to spin-
allowed 1IL transitions (p → p*) (qba and diimine), whereas
the low-energy absorption shoulders at ca. 388–478 nm have
been assigned to spin-allowed metal-to-ligand charge-transfer
Complex 2
Ir1–C17
Ir1–C1
1.987(3)
1.996(3)
2.091(2)
Ir1–N2
Ir1–N4
Ir1–N3
2.091(2)
2.172(2)
2.197(2)
Ir1–N1
C17–Ir1–C1
C17–Ir1–N1
C1–Ir1–N1
C17–Ir1–N2
C1–Ir1–N2
N1–Ir1–N2
C17–Ir1–N4
C1–Ir1–N4
93.50(11)
93.62(10)
79.67(10)
79.56(10)
92.38(10)
169.26(8)
95.73(10)
169.71(10)
N1–Ir1–N4
N2–Ir1–N4
C17–Ir1–N3
C1–Ir1–N3
N1–Ir1–N3
N2–Ir1–N3
N4–Ir1–N3
104.22(9)
84.85(8)
168.17(9)
95.61(10)
80.64(8)
107.53(9)
75.88(9)
2184 | Dalton Trans., 2011, 40, 2180–2189
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©

Table 3 Electronic absorption spectral data of the iridium(III) complexes
at 298 K
Table 4 Photophysical data of the iridium(III) complexes
Complex
1
Medium (T/K)
l
em/nm
to/ms
U
Complex Medium
l
abs /nm (e/dm3 mol-1 cm-1)
CH2Cl2 (298)
CH3CN (298)
Glassa (77)
CH2Cl2 (298)
CH3CN (298)
Glassa (77)
CH2Cl2 (298)
CH3CN (298)
Glassa (77)
CH2Cl2 (298)
CH3CN (298)
Glassa (77)
573, 621 sh
577, 622 sh
565 (max), 615, 670 sh
575, 621 sh
577, 623 sh
566 (max), 614, 669 sh
577, 621 sh
581, 623 sh
568 (max), 617, 671 sh
575, 621 sh
577, 624 sh
2.67
2.56
4.40
2.58
2.49
4.41
2.43
2.50
4.65
2.52
2.49
4.45
0.36
0.34
1
2
3
4
CH2Cl2 250 (38805), 273 sh (51760), 284 (60040), 293 sh
(56225), 308 sh (32225), 342 (31290), 358 sh
(27185), 451 (5660), 488 sh (3080), 551 (300)
CH3CN 246 sh (38120), 282 (59310), 292 sh (54270), 339
(29900), 354 sh (26515), 391 sh (5935), 450 (5225),
484 sh (2880), 550 (280)
CH2Cl2 252 sh (40510), 279 (73850), 292 sh (62255), 322 sh
(26780), 341 (32015), 356 (31125), 451 (6485), 487
sh (3220), 551 (325)
CH3CN 231 sh (46430), 278 (65195), 292 sh (51625), 341
(28135), 355 sh (26740), 389 sh (6870), 448 (5460),
483 sh (2815), 550 (310)
CH2Cl2 271 sh (56545), 282 (71625), 295 sh (53430), 342
(29065), 357 sh (26325), 388 sh (7505), 414 sh
(4805), 454 (4870), 486 sh (2865), 551 (330)
CH3CN 235 sh (49095), 282 (78485), 339 (31055), 355 sh
(27485), 391 sh (7270), 453 (5040), 488 sh (2670),
553 (315)
2
3
4
0.43
0.29
0.38
0.29
0.38
0.27
566 (max), 613, 672 sh
a EtOH–MeOH (4 : 1 v/v).
CH2Cl2 272 sh (53845), 285 (72085), 293 sh (67795), 341 sh
(31945), 356 (28290), 386 sh (11890), 451 (5580),
487 sh (2810), 551 (290)
CH3CN 225 sh (59180), 284 (83390), 341 sh (34570), 355 sh
(31200), 389 sh (10550), 448 (5990), 486 sh (2770),
550 (305)
Fig. 4 Emission spectra of complex 4 in CH2Cl2 (—) and in CH3CN (---)
at 298 K and in EtOH–MeOH (4 : 1 v/v) at 77 K ( ◊ ◊ ◊ ).
independence of the emission energy on the identity of the diimine
ligands, together with the observation of structured emission
features, long emission lifetimes and high luminescence quantum
yields, suggest that the emission of complexes 1–4 in fluid solutions
is derived from an 3IL (p → p*) (qba) excited state, perhaps with
mixing of some 3MLCT (dp(Ir) → p*(qba)) character.14,16–18,19b,20,22
This is supported by the red-shifted emission energy of complexes
1–4 compared to their pba (Hpba = 4-(2-pyridyl)benzaldehyde)
counterparts [Ir(pba)2(N∧N)](PF6) (N∧N = bpy, phen, Me4-phen,
Ph2-phen)20a owing to the more p-conjugated cyclometallating
ligand qba. In alcohol glass at 77 K, the structural features in the
emission spectra of complexes 1–4 became more evident. Again,
the emission decays followed first-order kinetics, with a lifetime
of ca. 4.4–4.7 ms. The blue shifts of complexes 1–4 were relatively
small, and the emission energy was independent of the nature of
the diimine ligands. Thus, it is likely that the low-temperature
glass emission of complexes 1–4, similar to their emission in
Fig. 3 Electronic absorption spectra of complexes 1 (—) and 4 (---) in
CH3CN at 298 K.
(1MLCT) transitions (dp(Ir) → p*(qba and diimine)). Although
similar assignments have been made for complex 4, the absorption
shoulders of complex 4 have substantial 1IL character due to
the lower-lying p* orbitals of Ph2-phen.14–17,19,20a–e,22 The spectra of
all four complexes exhibited weaker absorption tails towards the
lower energy region, which may be attributed to spin-forbidden
3MLCT (dp(Ir) → p*(qba and diimine)) transitions.14–17,19,20a–e,22
Photoexcitation of complexes 1–4 resulted in strong and
long-lived orange-yellow luminescence in fluid solutions under
ambient conditions and in low-temperature alcohol glass. The
photophysical data are summarised in Table 4. The emission
spectra of complex 4 are shown in Fig. 4. In fluid solutions at
room temperature, all four complexes displayed structured and
indistinguishable emission spectra with an emission maximum at
ca. 573–581 nm and a shoulder at ca. 621–624 nm. The emission
lifetimes of complexes 1–4 were single-exponential (ca. 2.4 to
2.7 ms) and about one order of magnitude longer than those of
common cyclometallated iridium(III) polypyridine systems. The
3
solutions, originated from an excited state of predominant IL
(p → p*) (qba) character,14,16–18,19b,20,22 although the existence of
some 3MLCT (dp(Ir) → p*(qba)) character cannot be ruled out.
Electrochemical properties
The electrochemical properties of the cyclometallated iridium(III)
polypyridine complexes have been studied by cyclic voltam-
metry. The electrochemical data are listed in Table 5. The
cyclic voltammograms of complex 1 are displayed in Fig. 5.
All the complexes showed a reversible couple at ca. +1.41 to
+1.46 V vs. SCE, which has been assigned to the iridium(IV/III)
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Dalton Trans., 2011, 40, 2180–2189 | 2185
©

Table 5 Electrochemical data of the iridium(III) complexesa
Table 6 Photophysical data of the bioconjugates in 50 mM potassium
phosphate buffer pH 7.4 at 298 K
Complex
Oxidation E1/2/V
Reduction E1/2 or Ec/V
Conjugates
l
em/nm
to/ms
U
1
2
3
4
+1.46
+1.46
+1.41
+1.45
-1.25, -1.43, -1.68, -1.85,b -2.06b
-1.24, -1.42, -1.66, -1.87,b -2.06b
-1.27, -1.47, -1.75 -1.90,b, -2.07b
-1.24, -1.41, -1.59, -1.80b, -2.02b
1-BSA
2-BSA
3-BSA
4-BSA
586
587
593
589
0.42 (16%), 1.28 (84%)
0.44 (16%), 1.25 (84%)
0.29 (15%), 1.06 (85%)
0.41 (16%), 1.23 (84%)
0.10
0.086
0.039
0.098
a In CH3CN (0.1 M TBAP) at 298 K (glassy carbon working electrode,
sweep rate = 100 mV s-1, all potentials vs. SCE). b Irreversible waves.
ca. 0.3–0.9. Upon photoexcitation, the bioconjugates 1-BSA–4-
BSA exhibited moderately intense long-lived yellow emission in
50 mM potassium phosphate buffer pH 7.4 at 298 K (Table 6).
The emission spectra of these labelled proteins in aqueous buffer
displayed structureless bands ca. 586–593 nm, which were at lower
energies compared to those of the free complexes in organic
solvents. The emission has been tentatively assigned to a 3MLCT
(dp(Ir) → p*(N∧C)) excited state mixed with a 3IL (p → p*)
(N∧C) character. The emission lifetimes of the bioconjugates were
biexponential (ca. 0.29–1.28 ms), which were not uncommon for
proteins labelled with transition metal complexes. Additionally,
the luminescent bioconjugates have been analyzed by SDS-
PAGE. The results of the analysis are shown in Fig. 6. Upon
photoexcitation, all the Ir–BSA bioconjugates displayed several
luminescent bands on the gel. The brightest bands corresponded
to a molecular weight close to that of BSA (MW = 66 kDa),
indicating that the molecular weight of the labelled protein was not
altered much by the iridium(III) complex. Additionally, the other
bands have been ascribed to the labelled dimer (MW = ca. 132
kDa) as well as other oligomers (Fig. 6). The two amine-reactive
aldehyde moieties of each molecule of the iridium(III) complexes
rendered them useful luminescent cross-linkers for biomolecules
such as the model protein BSA in this study.
Fig. 5 Cyclic voltammograms of complex 1 in CH3CN at 298 K.
oxidation couple.13–19,20a–e,22a–f With reference to previous electro-
chemical studies of related cyclometallated iridium(III) polypyri-
dine complexes,14,16,18,19b,20a–e,22a–f the first reversible reduction couple
of all the complexes at ca. -1.24 to -1.27 V has been assigned to the
reduction of one of the qba ligands, which is in agreement with the
photophysical data that the LUMOs of the complexes should be
the qba ligands. Additionally, all the complexes displayed more
reduction waves at more negative potentials, which have been
assigned to the reduction of the diimine and cyclometallating
ligands.14,16,18,19b,20a–e,22a–f
Fig. 6 Results of SDS-PAGE of the Ir–BSA bioconjugates. Lanes A–D
are 1-BSA, 2-BSA, 3-BSA and 4-BSA, respectively.
Cytotoxicity
Conjugation of BSA
We have examined the cytotoxic effect of the cyclometallated
iridium(III) polypyridine complexes towards HeLa cells using the
MTT assay.30 The IC50 values have been determined from the dose-
dependence of surviving cells after exposure to the complexes for
48 h. The IC50 values of the iridium(III) complexes ranged from
4.0 to 9.8 mM (Table 7), which are significantly smaller than that
of cisplatin (23.5 mM) under the same experimental conditions.
In general, these complexes are similarly cytotoxic to related
iridium(III) pq (Hpq = 2-phenylquinoline) complexes.22a,b,d,f It is
interesting to note that there is a slight decrease in the IC50 values of
complexes 3 and 4, probably due to an increase of the lipophilicity
Since the aldehyde group can react readily with a primary amine
group to form a secondary amine after reductive amination,
complexes 1–4 are considered as amine-specific biological labelling
reagents. We have conjugated the protein BSA with complexes 1–4.
The electronic absorption spectra of the bioconjugates 1-BSA–4-
BSA revealed intense absorption at 280 nm, attributable to both
the protein and complex absorption, and other absorption features
in the visible region mainly due to the iridium(III) complexes.
The dye-to-protein ratios (D/P) determined from the electronic
absorption spectra of the free complexes and bioconjugates are
2186 | Dalton Trans., 2011, 40, 2180–2189
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©

Table 7 IC50 values of the iridium(III) complexes
Table 8 Flow cytometric results of HeLa cells incubated with complex 4
under various conditions for 1 h
Complex
IC50/mM
Condition/inhibitor
Blank
Relative intensity (A. U.)
1
2
3
4
9.6 0.5
9.8 0.7
6.0 0.9
4.0 0.2
23.5 3.3
3.5
100.0
71.2
37 ◦
C
C
4 ◦
Cisplatin
Nitrate (50 mM)
91.6
Vanadate (50 mM)
Molybdate (250 mM)
2-Deoxyglucose (50 mM)
Azide (3 mM)
103.5
113.0
115.5
109.6
56.1
of the diimines. Although cytotoxicity is often a concern in cellular
imaging studies, this drawback can be compensated by employing
a low dose of the complex with a short incubation time with the
cells. It is likely that further structural modification of this class
of complexes such as the attachment of a poly(ethylene glycol)
(PEG) pendant,22g can alleviate the cytotoxicity associated with
the parent skeleton, while the desirable photophysical features of
the complexes can be retained.
CCCP (20 mM)
Cellular-uptake studies
HeLa cells loaded with the cyclometallated iridium(III) polypyri-
dine complexes (5 mM) at 37 ◦C for 1 h have been analysed
using laser-scanning confocal microscopy. The results for complex
4 are shown in Fig. 7. Upon excitation, the cells incubated
with the complex displayed intense emission intensity, indicating
efficient cellular internalisation of the complex molecules. The
complex distribution in the cytoplasm was not even but localised
in the perinuclear region, forming very sharp luminescent rings
surrounding the nuclei. The nuclei showed much weaker emission,
indicative of negligible nuclear uptake of the complex. From the
image (Fig. 7), it is likely that the complex binds to hydrophobic
organelles such as Golgi apparatus, endoplasmic reticulum and
mitochondria.31◦Interestingly, incubation of the HeLa cells with
complex 4 at 4 C resulted in reduced cellular uptake efficiency
as revealed by confocal microscopy (Fig. 8) and flow cytometry
(Fig. 9). This suggests that an energy-requiring process should
play a part in the cellular uptake process. The cellular uptake of
complex 4 has been further investigated using different reagents
Fig. 9 Flow cytometric results of HeLa cells incubated with blank
medium (black) and complex 4 for 1 h at 37 ◦C (red), 4 ◦C (green) and
after pretreatment with CCCP at 37 ◦C (blue), respectively.
including metabolic- and ATPase-inhibitors. The flow cytometric
results are listed in Table 8. While most of the pretreatments
did not result in any observable changes in the cellular uptake
efficiency, incubation of HeLa cells with the metabolic inhibitor
CCCP (20 mM) at 37 ◦C in a glucose-free medium before treatment
with complex 4 led to a moderate decrease of cellular uptake
efficiency (Table 8 and Fig. 9). Since CCCP inhibits oxidative
phosphorylation, resulting in decreased ATP production and
hence a lowered metabolic rate, the observed decrease of cellular
uptake is in accordance with the argument that internalisation
Fig. 7 Fluorescence (left), brightfield (middle), and overlaid (right) images of HeLa cells incubated with complex 4 (5 mM) at 37 ◦C for 1 h.
Fig. 8 Fluorescence (left), brightfield (middle), and overlaid (right) images of HeLa cells incubated with complex 4 (5 mM) at 4 ◦C for 1 h.
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Fig. 10 Fluorescence images of HeLa cells incubated with complex 4 (5 mM) at 37 ◦C for 1 h (a) before and (b) after subsequent fixation with MeOH,
or (c) fixation with paraformaldehyde, followed by extensive washing with MeOH; (d)–(f): fluorescence images of HeLa cells treated and fixed with the
same procedures except that [Ir(pq)2(Ph2-phen)](PF6) was used as the staining reagent.
of complex 4 occurred via an energy-requiring pathway such as
endocytosis.32
two complexes require more detailed investigations, since the
only difference between the two complexes is the two aldehyde
groups of complex 4, it is conceivable that the amine-specific
reactivity and protein cross-linking properties of the complex
should play a key role in the cellular staining. It is possible that this
complex, unlike its aldehyde-free counterpart, became anchored
onto cellular structures (e.g., via proteins on organelles) and was
still retained even after the cells were extensively washed with
MeOH. In summary, functionalisation of luminescent complexes
with an aldehyde unit is useful for cellular staining experiments
as it possibly allows covalent attachment of the imaging tools to
the amine groups of various biomolecules as well as intracellular
structures.
The effects of the aldehyde groups of complex 4 on cellular
labelling have been examined by fixed-cell imaging studies. HeLa
cells were treated with complex 4 (at 37 ◦C for 1 h) and then
fixed with MeOH. The confocal images showed that complex
4 was localised in the perinuclear region before (Fig. 10a) and
after (Fig. 10b) the cells were fixed. Also, during the fixation, the
complex diffused across the compromised nuclear envelop and
stained the nuclear components including what appeared to be
nucleoli. Remarkably, although many cellular reagents suffer from
severe loss of staining ability after the cells are fixed with organic
solvents, the emission intensity of the stained cells was retained
after treatment with MeOH. This finding has been compared to
the cell-staining properties of the aldehyde-free counterpart of
complex 4, [Ir(pq)2(Ph2-phen)](PF6).33 HeLa cells incubated with
this pq complex at 37 ◦C for 1 h exhibited similar intracellular
staining with an emission intensity that was similar to that of
cells treated with complex 4 (Fig. 10d). This is in accordance
with the results from ICP-MS analysis that revealed similar
cellular uptake efficiencies for both complexes (the intracellular
iridium contents of complex 4 and the pq complex were 0.38
and 0.39 fmol in an average cell, respectively). However, in sharp
contrast to complex 4, after the pq-complex-treated cells were
fixed with MeOH, the emission intensity decreased significantly
(Fig. 10e). The cellular staining properties of the two complexes
have been further studied using paraformaldehye as the fixing
reagent. HeLa cells were first stained by the complexes, then fixed
by paraformaldehyde and extensively washed with MeOH. The
confocal images showed that while the staining patterns of HeLa
cells were similar to those under normal incubation of the two
complexes, this fixation and washing treatment led to a significant
decrease of emission intensity for the control complex (Fig. 10f).
Remarkably, HeLa cells incubated with complex 4 with subsequent
fixation and washing still displayed very intense emission intensity
(Fig. 10c). Although the reasons for the drastic difference between
the emission intensities of the fixed cells pretreated with the
Conclusion
We report herein the synthesis, characterisation and photophysical
and electrochemical properties of four luminescent cyclometal-
lated iridium(III) bis(quinolylbenzaldehyde) diimine complexes.
Flow cytometry and laser-scanning confocal microscopy stud-
ies revealed efficient uptake of complex 4 by HeLa cells and
subsequent localisation in the perinuclear region. Results from
temperature-dependence and inhibition experiments indicated
that the internalisation mechanism was likely to be via endocytosis.
The highly efficient internalisation of the complex at a low
concentration in a relatively short incubation time and the
fixed-cell confocal experiments suggests that these luminescent
cyclometallated iridium(III) polypyridine complexes represent a
new class of bio-imaging reagents.
Acknowledgements
We thank The Hong Kong Research Grants Council (Project
No. CityU 102109) and City University of Hong Kong (Project
No. 7002575) for financial support. The work described in this
paper was also supported by a Special Equipment Grant from
the Hong Kong University Grants Committee (SEG_CityU02).
2188 | Dalton Trans., 2011, 40, 2180–2189
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The Royal Society of Chemistry 2011
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P.-K. L. and M.-W. L acknowledge the receipt of a Postgraduate
Studentship, a Research Tuition Scholarship and an Outstanding
Academic Performance Award, all administered by City Univer-
sity of Hong Kong. We thank Mr. Kenneth King-Kwan Lau and
Mr. Michael Wai-Lun Chiang for their assistance on the cellular
experiments.
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The Royal Society of Chemistry 2011
Dalton Trans., 2011, 40, 2180–2189 | 2189
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