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Luminescent dendritic cyclometalated iridium(III) polypyridine complexes: synthesis, emission behavior, and biological properties.
5432 Inorg. Chem. 2010, 49, 5432–5443
DOI: 10.1021/ic902443e
Luminescent Dendritic Cyclometalated Iridium(III) Polypyridine Complexes:
Synthesis, Emission Behavior, and Biological Properties
Kenneth Yin Zhang, Hua-Wei Liu, Tommy Tsz-Him Fong, Xian-Guang Chen, and Kenneth Kam-Wing Lo*
Department of Biology and Chemistry, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong,
People’s Republic of China
Received December 9, 2009
Luminescent dendritic cyclometalated iridium(III) polypyridine complexes [{Ir(N∧C)2}n(bpy-n)](PF6)n (HN∧C =
2-phenylpyridine, Hppy, n = 8 (ppy-8), 4 (ppy-4), 3 (ppy-3); HN∧C = 2-phenylquinoline, Hpq, n = 8 (pq-8), 4 (pq-4),
3 (pq-3)) have been designed and synthesized. The properties of these dendrimers have been compared to those of
their monomeric counterparts [Ir(N∧C)2(bpy-1)](PF6) (HN∧C = Hppy (ppy-1), Hpq (pq-1)). Cyclic voltammetric
studies revealed that the iridium(IV/III) oxidation and bpy-based reduction occurred at about þ1.24 to þ1.29 V and
-1.21 to -1.27 V versus SCE, respectively, for all the complexes. The molar absorptivity of the dendritic iridium(III)
complexes is approximately proportional to the number of [Ir(N∧C)2(N∧N)] moieties in one complex molecule.
However, the emission lifetimes and quantum yields are relatively independent of the number of [Ir(N∧C)2(N∧N)]
units, suggesting negligible electronic communications between these units. Upon photoexcitation, the complexes
displayed triplet metal-to-ligand charge-transfer (3MLCT) (dπ(Ir) fπ*(bpy-n)) emission. The interaction of these
complexes with plasmid DNA has been investigated by agarose gel retardation assays. The results showed that the
dendritic iridium(III) complexes, unlike their monomeric counterparts, bound to the plasmid, and the interaction was
electrostatic in nature. The lipophilicity of all the complexes has been determined by reversed-phase high-performance
liquid chromatography (HPLC). Additionally, the cellular uptake of the complexes by the human cervix epithelioid
carcinoma (HeLa) cell line has been examined by inductively coupled plasma mass spectrometry (ICP-MS), laserscanning confocal microscopy, and flow cytometry. Upon internalization, all the complexes were localized in the
perinuclear region, forming very sharp luminescent rings surrounding the nuclei. Interestingly, in addition to these
rings, HeLa cells treated with the dendritic iridium(III) complexes showed specific labeled compartments, which have
been identified to be the Golgi apparatus. Furthermore, the cytotoxicity of these iridium(III) complexes has been
evaluated by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay.
Introduction
The role of dendrimers in biomedical applications has
attracted increasing interest.1-4 They have been used as
transfection agents for gene therapy (GT),5,6 contrast agents
*To whom correspondence should be addressed. E-mail: bhkenlo@cityu.
edu.hk. Phone: (852) 2788 7231. Fax: (852) 2788 7406.
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pubs.acs.org/IC
Published on Web 05/21/2010
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r 2010 American Chemical Society
Article
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5433
work has not been explored.24 In view of the interesting emissive
behavior of iridium(III) polypyridine complexes,25-43 and our
recent work on the mononuclear complexes of this type in
biological studies,43 we believe that it will be interesting to
investigate the potential applications of polynuclear complexes or dendrimers modified with luminescent cyclometalated iridium(III) polypyridine complexes as new probes for
biological molecules and live cells. The objectives of this work
are based on the following considerations: (1) an increasing
number of luminescent units in a macromolecular probe may
alter its photophysical properties which could offer new
biological sensing possibilities; (2) an increase of formal
charge of a dendrimer because of the addition of cationic
[Ir(N∧C)2(N∧N)]þ units is expected to change the biological
behavior of the resultant adduct; (3) while a change of the
ligand and hence the lipophilicity of the mononuclear complex [Ir(N∧C)2(N∧N)]þ has been found to significantly perturb its biomolecular binding, cellular uptake, and cytotoxic
properties,43a,e,g,h,j-l it will be interesting to investigate
whether these effects will still be observed when the complexes
are linked together by a dendritic skeleton; and (4) most
importantly, while the biological and cellular uptake properties of mononuclear cyclometalated iridium(III) polypyridine complexes modified with various biologically relevant
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5434 Inorganic Chemistry, Vol. 49, No. 12, 2010
Chart 1. Structures of Polypyridine Ligands
Zhang et al.
(ppy-4), 3 (ppy-3); HN∧C = 2-phenylquinoline, Hpq, n =8
(pq-8), 4 (pq-4), 3 (pq-3)). The structures of the polypyridine
ligands are shown in Chart 1. The properties of these dendrimers have been compared to those of their monomeric counterparts [Ir(N∧C)2(bpy-1)](PF6) (HN∧C = Hppy (ppy-1), Hpq
(pq-1)).43g The interaction of these complexes with plasmid
DNA has been investigated by agarose gel retardation assays.
The lipophilicity of all the complexes has been determined
by reversed-phase high-performance liquid chromatography
(HPLC). Additionally, the cellular uptake of the complexes by
the human cervix epithelioid carcinoma (HeLa) cell line has
been examined by inductively coupled plasma mass spectrometry (ICP-MS), laser-scanning confocal microscopy, and flow
cytometry. Furthermore, the cytotoxicity of these iridium(III)
complexes has been evaluated by the 3-(4,5-dimethyl-2thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay.
The most important results are that the dendritic complexes
showed interesting DNA-binding capabilities and a cellular
uptake pathway that is different to their mononuclear counterparts. Also, these dendritic complexes bound to specific
cellular compartments in HeLa cells, which has not been
observed in the cellular uptake studies of related luminescent
inorganic and organometallic transition metal complexes.
Results and Discussion
units are well documented, related studies on multinuclear
complexes have not been explored. All these reasons have
prompted us to design new luminescent dendritic cyclometalated iridium(III) polypyridine complexes and investigate their
photophysical and biological properties. In particular, the
effects of the lipophilicity, formal charge, and molecular size
on the biological and cellular uptake properties of these
complexes, and a comparison with their mononuclear counterparts are of much interest.
In this work, we report the synthesis and characterization of a series of luminescent dendritic cyclometalated
iridium(III) polypyridine complexes [{Ir(N∧C)2}n(bpy-n)](PF6)n (HN∧C = 2-phenylpyridine, Hppy, n = 8 (ppy-8), 4
Synthesis. The bpy-terminated dendritic ligands bpy-n
(n = 8, 4, 3) (Chart 1) were obtained from the reaction of
amine-terminated dendrimers with bpy-NHS (1.5 equiv
per terminal primary amine moiety) and triethylamine
(1.5 equiv per terminal primary amine moiety) in dried
N,N-dimethylformamide (DMF) at room temperature
for 18 h. These ligands were purified by silica gel column
chromatography and characterized by 1H NMR spectroscopy and positive-ion electrospray ionization mass
spectrometry (ESI-MS). The preparation of the dendritic
iridium(III) complexes ppy-n and pq-n (n = 8, 4, 3) was
accomplished by reacting the bpy-terminated dendritic ligands bpy-n (n = 8, 4, 3) with [Ir2(ppy)4Cl2] or [Ir2(pq)4Cl2]
(0.5 equiv per terminal bpy moiety) in refluxing CH2Cl2/
MeOH, followed by metathesis with KPF6. The complexes were purified by silica gel column chromatography
and recrystallized from CH2Cl2/diethyl ether to yield yellow
to orange crystals. All the iridium(III) complexes were characterized by 1H NMR spectroscopy, positive-ion ESI-MS, IR
spectroscopy, and elemental analysis. Positive-ion ESI-MS
was very useful in confirming the structures of these complexes. Consecutive loss of PF6- units and addition of Hþ were
observed in all the spectra of the dendritic iridium(III) complexes. For example, the spectrum of complex pq-4 was
dominated by four peaks corresponding to {M þ Hþ PF6-}2þ, {M - 2 PF6-}2þ, {M þ Hþ - 2 PF6-}3þ,
and {M - 3 PF6-}3þ ions. All the dendritic iridium(III)
complexes were very soluble in common organic solvents such
as CH2Cl2 and CH3CN, and aqueous DMSO solution. However, they were only sparingly soluble in aqueous buffers.
Electrochemical Properties. The electrochemical properties of all the complexes have been studied by cyclic voltammetry. The electrochemical data are listed in Table 1.
Similar to the monomeric complexes ppy-1 and pq-1 that
showed a reversible iridium(IV/III) oxidation couple at
about þ1.27 V and a bipyridine-based reduction couple at
about -1.28 V versus SCE, the dendritic complexes ppy-n
Article
Inorganic Chemistry, Vol. 49, No. 12, 2010
and pq-n (n = 8, 4, 3) exhibited reversible couples at
about þ1.24 to þ1.29 V and -1.21 to -1.27 V. These data
indicate that there are negligible electronic communications between the [Ir(N∧C)2(N∧N)] moieties in the same
dendritic molecule. The reduction waves at potentials more
negative than -1.7 V are quasi-reversible or highly irreversible in nature, and have been assigned to the reduction
of the bipyridine and the cyclometalating ligands.
Electronic Absorption and Luminescence Properties.
The electronic absorption spectral data of all the complexes
are listed in Table 2. The electronic absorption spectra of
complexes pq-n (n = 8, 4, 3, 1) in CH3CN at 298 K are
shown in Figure 1. All the complexes displayed intense
absorption bands and shoulders at about 253-344 nm
(ε on the order of 104 to 105 dm3 mol-1 cm-1), which have
been assigned to spin-allowed intraligand (1IL) (π f π*)
(bpy-n and N∧C) transitions. The less intense shoulders
at > about 350 nm have been assigned to spin-allowed
metal-to-ligand charge-transfer (1MLCT) (dπ(Ir) f π*(bpy-n and N∧C)) transitions. The weak absorption tailing
at about 475-550 nm has been assigned to spin-forbidden
3
MLCT (dπ(Ir) f π*(bpy-n and N∧C)) transitions. As
expected, the molar absorptivity of the dendritic iridium(III) complexes is approximately proportional to the number of [Ir(N∧C)2(N∧N)] moieties in the complex molecule.
The absorption profiles of the complexes containing the
same N∧C ligand are highly similar, further indicating
that there are no electronic communications between the
[Ir(N∧C)2(N∧N)] moieties in the same dendritic complex
molecule.
5435
Upon photoexcitation, all the complexes displayed
intense and long-lived greenish-yellow to orange luminescence in fluid solutions under ambient conditions and in
low-temperature glass. The photophysical data are summarized in Table 3. The emission spectra of complexes
ppy-8 and pq-8 in CH3CN at 298 K are shown in Figure 2.
For the ppy complexes, on going from relatively nonpolar
CH2Cl2 to polar MeOH, they exhibited longer emission
wavelengths, shorter emission lifetimes, and lower luminescence quantum yields (Table 3). Thus, the emission
has been tentatively assigned to an excited state of
3
MLCT (dπ(Ir) f π*(bpy-n)) character. Their emission
maxima also displayed significant blue-shifts upon cooling the samples from room temperature to 77 K (Table 3),
which is a common feature for cyclometalated iridium(III)
polypyridine 3MLCT emitters. Interestingly, in aqueous
buffer at 298 K, the dendritic complexes ppy-n (n =
8, 4, 3) showed an unexpected shorter emission wavelength (λem = 588 to 594 nm) which is in contrast to the
mononuclear complex ppy-1 (λem = 625 nm). We have
ascribed this to a more nonpolar local environment for
the polynuclear complexes offered by the dendritic cores.
The emission quantum yields in buffer followed the order:
ppy-1 < ppy-3 ≈ ppy-8<ppy-4, which is in line with the
trend of the emission wavelengths (Table 3). The more
highly branched dendritic skeleton of complex ppy-8
increases the flexibility of the complex, which may account
for a lower quantum yield compared to that of complex
Table 1. Electrochemical Data of the Iridium(III) Complexesa
complex
oxidation, E1/2/V
reduction, E1/2 or Ec/V
ppy-8
ppy-4
ppy-3
ppy-1d
pq-8
pq-4
pq-3
pq-1d
þ1.24
þ1.28
þ1.26
þ1.26
þ1.25
þ1.28
þ1.29
þ1.28
-1.27, -1.87,b -2.25,c -2.47c
-1.26, -1.83,b -2.25,c -2.48c
-1.27, -1.77,b -2.24,c -2.51c
-1.28, -1.84,b -2.27,b -2.48b
-1.22, -1.79,b -1.92,c -2.21c
-1.21, -1.72,b -1.90,c -2.21c
-1.22, -1.77,b -1.97,c -2.22c
-1.28, -1.72,b -1.93,b -2.22b
a
In CH3CN (0.1 mol dm-3 nBu4NPF6) at 298 K, glassy carbon
electrode, sweep rate 100 mV s-1, all potentials versus SCE. b Quasireversible couples. c Irreversible waves. d From reference 43g.
Figure 1. Electronic absorption spectra of complexes pq-8 (red), pq-4
(orange), pq-3 (green), and pq-1 (blue) in CH3CN at 298 K.
Table 2. Electronic Absorption Spectral Data of the Iridium(III) Complexes at 298 K
complex
solvent
λabs/nm (ε/dm3 mol-1 cm-1)
ppy-8
CH2Cl2
CH3CN
CH2Cl2
CH3CN
CH2Cl2
CH3CN
CH2Cl2
CH3CN
CH2Cl2
CH3CN
CH2Cl2
CH3CN
CH2Cl2
CH3CN
CH2Cl2
CH3CN
257 (388,410), 267 sh (368,135), 356 sh (68,030), 382 sh (59,890), 467 (8,150)
255 (347,240), 267 sh (323,130), 360 sh (57,210), 381 sh (49,860), 466 (6,840)
257 (197,740), 267 sh (185,170), 358 sh (33,705), 382 sh (30,345), 468 (3,975)
255 (174,720), 267 sh (160,105), 360 sh (29,365), 381 sh (25,930), 466 (3,460)
257 (146,095), 268 sh (136,135), 355 sh (35,865), 382 sh (31,930), 468 (4,150)
255 (123,735), 264 sh (115,075), 350 sh (22,665), 381 sh (18,585), 466 (2,490)
256 (66,075), 271 sh (58,420), 320 sh (21,640), 339 sh (12,130), 361 sh (10,105), 384 (9,240), 413 sh (5,990), 464 sh (1,205)
253 (57,730), 268 sh (52,710), 318 sh (20,440), 336 sh (11,505), 358 sh (9,610), 381 (8,190), 416 sh (3,670), 476 sh (1,215)
263 (336,940), 267 sh (335,585), 282 sh (334,480), 313 sh (160,780), 342 sh (150,970), 438 (35,230)
263 (277,455), 267 sh (275,025), 278 sh (273,875), 317 sh (126,135), 340 sh (119,150), 436 (25,910)
263 (175,165), 267 sh (174,455), 278 sh (174,035), 313 sh (78,190), 342 sh (54,190), 438 (18,250)
263 (126,440), 267 sh (125,600), 278 sh (125,300), 317 sh (59,135), 339 sh (55,880), 434 (12,835)
263 (139,185), 267 sh (138,715), 281 sh (139,100), 313 sh (67,030), 342 sh (63,200), 438 (14,465)
263 (99,750), 267 sh (99,285), 281 sh (97,215), 313 sh (47,425), 339 sh (44,155), 435 (10,050)
260 (56,110), 281 (54,440), 318 (24,090), 343 (23,300), 394 (5,700), 437 (5,510), 464 sh (3,620)
259 (46,610), 281 (45,490), 314 sh (21,680), 344 (19,470), 393 sh (4,860), 435 (4,800), 455 sh (3,600)
ppy-4
ppy-3
ppy-1a
pq-8
pq-4
pq-3
pq-1a
a
From reference 43g.
5436 Inorganic Chemistry, Vol. 49, No. 12, 2010
Zhang et al.
Table 3. Photophysical Data of the Iridium(III) Complexes
complex
medium (T/K)
λem/nm
τo/μs
Φem
ppy-8
CH2Cl2 (298)
CH3CN (298)
MeOH (298)
buffera (298)
glassb (77)
CH2Cl2 (298)
CH3CN (298)
MeOH (298)
buffera (298)
glassb (77)
CH2Cl2 (298)
CH3CN (298)
MeOH (298)
buffera (298)
glassb (77)
CH2Cl2c (298)
CH3CNc (298)
MeOH (298)
buffera (298)
glassb,c (77)
CH2Cl2 (298)
CH3CN (298)
MeOH (298)
buffera (298)
glassb (77)
CH2Cl2 (298)
CH3CN (298)
MeOH (298)
buffera (298)
glassb (77)
CH2Cl2 (298)
CH3CN (298)
MeOH (298)
buffera (298)
glassb (77)
CH2Cl2c (298)
CH3CNc (298)
MeOH (298)
buffera (298)
glassb,c (77)
599
613
623
592
541, 580 sh
603
614
620
588
540, 576 sh
597
611
620
594
541, 572 sh
609
613
619
625
541, 569 sh
554 sh, 587
562, 592 sh
557 sh, 608
572, 601 sh
543 (max), 587
560 sh, 587
560 sh, 587
551 sh, 603
567 sh, 597
543 (max), 583
557, 599 sh
557, 600 sh
557, 599 sh
563 sh, 597
542 (max), 583
557 sh, 592
566, 606 sh
570, 600 sh
574, 596 sh
543 (max), 585
0.33
0.18
0.065
0.14
4.53
0.26
0.14
0.061
0.16
4.69
0.34
0.20
0.066
0.13
4.75
0.40
0.23
0.10
0.34
4.80
0.45
0.40
0.11
0.11
4.27
0.46
0.34
0.11
0.10
4.88
0.83
0.46
0.12
0.13
4.76
0.67
0.54
0.22
0.17
4.69
0.10
0.036
0.011
0.0067
ppy-4
ppy-3
ppy-1
pq-8
pq-4
pq-3
pq-1
0.081
0.037
0.023
0.011
0.10
0.036
0.015
0.0061
0.13
0.058
0.015
0.0023
0.11
0.077
0.019
0.011
0.11
0.096
0.018
0.0056
0.16
0.078
0.030
0.0034
0.18
0.14
0.026
0.013
a
b
50 mM potassium phosphate buffer pH 7.4/MeOH (9:1, v/v).
EtOH/MeOH (4:1, v/v). c From reference 43g.
ppy-4. The pq complexes showed two emission features at
about 551-574 and 587-608 nm, respectively, in fluid
solutions at 298 K (Table 3). Their emission maxima
underwent a much smaller blue-shift upon cooling to 77
K compared to the ppy complexes, which is possibly a
result of the mixing of some 3IL (π f π*) (pq) character
into their emissive state.43a,e,g,h,j,l In general, the dendritic
complexes ppy-n and pq-n (n = 8, 4, 3) showed rather similar
emission energies, quantum yields, and emission lifetimes at
room temperature or 77 K compared to their monomeric counterparts ppy-1 and pq-1, respectively, further supporting that the
[Ir(C∧N)2(N∧N)] units exhibit negligible electronic communications.
The emission quantum yields of these complexes in
aqueous buffer were only moderate (Table 3), which is a
typical feature for cyclometalated iridium(III) bipyridinebased 3MLCT emitters.43e,g-i,l However, this is not considered a disadvantage as the most important requirement for luminescent probes in biological recognition and
sensing applications is that they show strongly environment-sensitive emission. For example, the weak emission
of ruthenium(II) and iridium(III) dppz and dpq com(44) (a) Puckett, C. A.; Barton, J. K. J. Am. Chem. Soc. 2007, 129, 46–47.
(b) Puckett, C. A.; Barton, J. K. Biochemistry 2008, 47, 11711–11716.
Figure 2. Emission spectra of complexes ppy-8 (solid) and pq-8 (dashed)
in CH3CN at 298 K.
plexes in aqueous solution does not hamper their interesting DNA and cellular sensing capabilities.43c,j,44 In this
work, there is a large difference between the emission
quantum yields of the complexes in solvents of different
polarity. Indeed, intracellular location of the complexes
can be readily detected by laser-scanning confocal microscopy (see below), and thus, the low emission quantum
yields in aqueous buffer are not a limitation for these
iridium(III) complexes.
DNA Interaction. Cationic dendrimers usually show
good DNA condensation ability, and the association of
these dendrimers with DNA results in the formation of
dendriplexes.45-47 We have studied the possible interaction
of the iridium(III) complexes with DNA. Although addition of double-stranded calf thymus DNA to a solution of
the complex in Tris-Cl buffer (50 mM, pH 7.4) did not cause
any change to the absorption and emission spectra, the
condensation of plasmid DNA (pDNA) by the complexes
has been demonstrated by agarose gel retardation assays
(Figure 3). It can be seen that complexes ppy-8 and pq-8
effectively formed dendriplexes with pDNA (1 μg) at [Ir]
(concentration with respect to iridium) being as low as
12.5 nM whereas complexes ppy-4, pq-4 and ppy-3, pq-3
were slightly less potent and could only inhibit the migration of pDNA at [Ir] g 25 and 100 nM, respectively. This
implies that the interaction of these dendritic complexes
with pDNA originates from electrostatic attraction. Another possible reason is the interaction of the dendritic
motifs of the complexes with the biopolymer. In contrast,
for the monomeric complexes ppy-1 and pq-1, no obvious
pDNA condensation was observed even at [Ir]=400 nM,
which is probably due to their lower cationic charge and the
lack of dendritic skeleton.
Lipophilicity. The lipophilicity of a compound refers to
its ability to dissolve in fats, oils, lipids, and nonpolar
solvents, and is highly related to its efficiency to permeate
biological membranes.48 It is commonly estimated by the
partition coefficient (Po/w) in n-octanol/water.49 The
lipophilicity (log Po/w) of the complexes in this work has
(45) Navarro, G.; de ILarduya, C. T. Nanomedicine 2009, 5, 287–297.
(46) Mintzer, M. A.; Merkel, O. M.; Kissel, T.; Simanek, E. E. New J.
Chem. 2009, 33, 1918–1925.
(47) Merkel, O. M.; Mintzer, M. A.; Sitterberg, J.; Bakowsky, U.;
Simanek, E. E.; Kissel, T. Bioconjugate Chem. 2009, 20, 1799–1806.
(48) VanBrocklin, H. F.; Liu, A.; Welch, M. J.; O’Neil, J. P.; Katzenellenbogen,
J. A. Steroids 1994, 59, 34–45.
(49) Minick, D. J.; Frenz, J. H.; Patrick, M. A.; Brent, D. A. J. Med.
Chem. 1988, 31, 1923–1933.
Article
Inorganic Chemistry, Vol. 49, No. 12, 2010
5437
Figure 3. Results of agarose gel retardation assays of the iridium(III) complexes and plasmid DNA in PBS/DMSO (10 μL, 7:3, v/v). Lane 1: DNA (1 μg)
only. Lane 2: the iridium(III) complex only. Lanes 3-8: DNA (1 μg) and the iridium(III) complex ([Ir] = 12.5, 25, 50, 100, 200, and 400 nM, respectively).
Table 4. Lipophilicity (log Po/w) of the Iridium(III) Complexes
a
complex
log Po/w
ppy-8
ppy-4
ppy-3
ppy-1a
pq-8
pq-4
pq-3
pq-1a
1.66
2.06
1.87
0.44
2.61
3.40
2.93
2.01
From reference 43g.
been determined by reversed-phase HPLC, and the results are listed in Table 4. The log Po/w values of the pq
complexes are larger than those of their corresponding
ppy counterparts, resulting from the additional fused
phenyl ring of the pq ligand. Interestingly, the lipophilicity of the dendritic complexes ppy-n (n=8, 4, 3) (log Po/w=
1.66-2.06) and pq-n (n=8, 4, 3) (log Po/w=2.61-3.40) is
substantially higher than that of the monomeric complexes ppy-1 (log Po/w =0.44) and pq-1 (log Po/w =2.01),
respectively, reflecting the hydrophobic nature of the
dendritic skeleton moiety. This is in accordance with the
observation that the tetranuclear complexes ppy-4 and
pq-4 showed larger log Po/w values (2.06 and 3.40,
respectively) than the corresponding trinuclear complexes ppy-3 and pq-3 (log Po/w = 1.87 and 2.93, respectively). However, complexes ppy-8 and pq-8 exhi-
Table 5. Numbers of Moles and Concentrations of Iridium Associated with
an Average HeLa Cell upon Incubation with the Iridium(III) Complexes ([Ir] =
2 μM) at 37 °C for 2 h as Determined by ICP-MS
complex
number of mol/fmol
[Ir]/mM
ppy-8
ppy-4
ppy-3
ppy-1
pq-8
pq-4
pq-3
pq-1
0.65
0.95
0.98
3.8
0.66
0.94
0.88
7.1
0.19
0.28
0.29
1.1
0.19
0.26
0.28
2.1
bited the lowest lipophilicity (log Po/w = 1.66 and 2.61,
respectively) among all the dendritic complexes, which
may be a consequence of the polar amine and amide
groups and the þ8 formal charge.
Cellular Uptake. The cellular uptake of the complexes
has been studied by ICP-MS measurements. The amounts
and concentrations of iridium associated with HeLa cells
incubated with the complexes ([Ir] = 2 M) at 37 °C for 2 h
are listed in Table 5. An average cell (mean volume of
3.4 pL) contained 0.65 to 7.1 fmol of iridium, which
is comparable to those reported in the cellular uptake
studies of iridium43i,l and other inorganic complexes.50-53
(50) Brunner, J.; Barton, J. K. Biochemistry 2006, 45, 12295–12302.
(51) Yu, J.; Parker, D.; Pal, R.; Poole, R. A.; Cann, M. J. J. Am. Chem.
Soc. 2006, 128, 2294–2299.
5438 Inorganic Chemistry, Vol. 49, No. 12, 2010
Zhang et al.
Figure 4. Laser-scanning confocal microscopy images of HeLa cells incubated with the iridium(III) complexes ([Ir] = 2 μM) at 37 °C for 2 h.
The dendritic complexes exhibited a lower cellular uptake
efficiency (approximately one order of magnitude lower
in terms of [Ir]) compared to the monomeric complexes,
probably because of their higher cationic charge and much
larger molecular size. The monomeric pq complex pq-1
displayed almost a double cellular uptake efficiency ([Ir]=
2.1 mM) compared to that of complex ppy-1 ([Ir] = 1.1
mM), which can be ascribed to the higher lipophilicity of
the former complex (Table 4). However, the dendritic pq
complexes pq-n (n=3, 4, 8), despite their higher lipophilicity, showed similar cellular uptake efficiencies compared to their corresponding ppy counterparts ppy-n
(n=3, 4, 8). This illustrates that, regarding cellular uptake
(52) Chauvin, A.-S.; Comby, S.; Song, B.; Vandevyver, C. D. B.; B€unzli,
J.-C. G. Chem.;Eur. J. 2008, 14, 1726–1739.
(53) Louie, M.-W.; Liu, H.-W.; Lam, M. H.-C.; Lau, T.-C.; Lo, K. K.-W.
Organometallics 2009, 28, 4297–4307.
efficiency of the dendritic complexes, the branched skeletons play a more important role compared to the cyclometalating ligands. It is important to point out that the [Ir]
of all the complexes (0.19-2.1 mM, Table 5) are much
higher than that of the free complexes in the medium
before the uptake (2 μM), indicating that the complexes
were concentrated within the cells. However, the measured concentrations indicate iridium associated with the
cells, which is not necessary all being in the interior. Thus,
the data can only serve as an approximate indicator for
cellular uptake efficiencies.
Confocal Imaging and Flow Cytometry. The internalization and intracellular localization of all the complexes
have been investigated using laser-scanning confocal
microscopy. The fluorescence microscopy images of
HeLa cells treated with the complexes ([Ir]=2 μM) for
2 h are illustrated in Figure 4. We found that the
monomeric complexes ppy-1 and pq-1 were localized in
Article
Inorganic Chemistry, Vol. 49, No. 12, 2010
5439
Figure 5. Laser-scanning confocal microscopy images of HeLa cells treated successively with complexes ppy-8 or pq-8 ([Ir] = 2 M) at 37 °C for 2 h, PBS
containing 3% paraformaldehyde, anti-golgin-97 (human) mouse IgG1 (1 μg/mL, 1 h), and Alexa 635 goat anti-mouse IgG (HþL) (10 μg/mL, 30 min).
Table 6. Mean Emission Intensities of HeLa Cells Incubated with Blank Medium
and the Iridium(III) Complexes at 37 and 4 °C for 2 h as Determined by Flow
Cytometry
emission intensity after incubation
complex
at 37 °C
at 4 °C
ppy-8
ppy-4
ppy-3
ppy-1
pq-8
pq-4
pq-3
pq-1
blank medium
18.5
19.1
18.1
33.5
15.9
31.6
53.7
1641.3
3.0
3.0
3.2
3.7
4.8
3.0
3.4
3.9
625.3
3.0
the perinuclear region, forming very sharp luminescent
rings surrounding the nuclei. Z-scans of the cells confirmed that the complexes were indeed located inside the
cells (Supporting Information, Figure S1). Similar intracellular distributions are commonly observed for other
iridium(III) complexes.38b,43g-i,k,l However, in addition to
the perinuclear region, the dendritic complexes also bound
to specific compartments of the cells, which are likely to be
the Golgi apparatus (Figure 4). This has been confirmed
by co-staining experiments using complexes ppy-8 and
pq-8 as examples. Specifically, HeLa cells treated with
complexes ppy-8 and pq-8, respectively, were fixed and
incubated with anti-golgin-97 (human) mouse IgG1 (1 μg/
mL, 1 h), and subsequently stained by Alexa 635 goat antimouse IgG (HþL) (10 μg/mL, 30 min). The dye Alexa 635
was used because its spectral properties do not interfere
with those of the iridium(III) complexes. It can be seen in
Figure 5 that the Golgi apparatus has been co-stained by
the complexes and the fluorescent antibody.
The cellular uptake has been investigated by temperaturedependence experiments and analyzed by flow cytometry.
Figure 6. Flow cytometric results of HeLa cells incubated with blank
medium (black) and complexes pq-8 (blue), pq-4 (green), pq-3 (orange),
and pq-1 (red) at (a) 37 °C and (b) 4 °C for 2 h.
The mean emission intensities of HeLa cells incubated with
all the complexes and blank medium at 37 and 4 °C are
presented in Table 6. All the cell samples incubated with the
complexes at 37 °C displayed emission intensities that are
higher than the autofluorescence of untreated HeLa cells,
reflecting efficient cellular uptake of the complexes. The
emission intensities of HeLa cells treated with the dendritic
ppy complexes ppy-n, (n = 8, 4, 3) at 37 °C are similar to
each other and lower than those incubated with their
monomeric counterpart ppy-1 (Table 6). For the pq complexes at 37 °C, the emission intensities increased with a
decreasing number of [Ir(pq)2(N∧N)] moieties and followed
5440 Inorganic Chemistry, Vol. 49, No. 12, 2010
Zhang et al.
Figure 7. Laser-scanning confocal microscopy images of HeLa cells incubated with complexes ppy-8, ppy-1, pq-8, and pq-1 ([Ir] = 2 μM) at 4 °C for 2 h.
the order: pq-8<pq-4<pq-3 , pq-1 (Table 6 and Figure 6a).
Upon lowering the incubation temperature to 4 °C, the
emission intensities of HeLa cells treated with all the
complexes were reduced (Table 6). The cells treated with
the dendritic complexes ppy-n and pq-n (n = 8, 4) became
hardly emissive, whereas the cells loaded with complex ppy-1
showed a small but noticeable emission intensity and those
incubated with complex pq-1 still displayed rather strong
luminescence (Table 6 and Figure 6b). These results are
consistent with the confocal microscopy images of HeLa
cells treated with the dendritic complexes ppy-8 and pq-8 at
4 °C, which did not reveal luminescence (Figure 7). In
contrast, cells loaded with the monomeric complexes ppy-1
and pq-1 at 4 °C were emissive, with the latter complex
giving very strong emission intensity (Figure 7 and Supporting Information, Figure S1). All these results indicate
that the dendritic complexes entered the cells by an energydependent process (which is likely to be endocytosis),
whereas an energy-independent diffusion-like mechanism
also occurred for the monomeric complexes, especially for
complex pq-1. The coexistence of these cellular internalization pathways has been observed for other cyclometalated
iridium(III) complexes.43i It is conceivable that the different internalization pathways and localizations between the
dendritic and mononuclear complexes originate from the
different formal charge and molecular size of the complexes, and the dendritic skeletons also play a role in the
cellular uptake properties.54
Cytotoxicity. The cytotoxicity of the complexes toward
the HeLa cell line has been studied by the MTT assay.55
The dose dependence of surviving cells after exposure to
the complexes for 48 h has been evaluated, and the IC50
values are shown in Table 7. The iridium(III) complexes
showed higher or comparable cytotoxicity (IC50 =1.4 to
(54) The possibility of aggregation of the dendritic complexes in aqueous
solutions has been eliminated by light-scattering experiments, and we did not
have evidence that the lack of passive diffusion of these complexes was due to
aggregation.
(55) Mosmann, T. J. Immunol. Methods 1983, 65, 55–63.
Table 7. Cytotoxicity (IC50, 48 h) of the Iridium(III) Complexes and Cisplatin
Toward the HeLa Cell Line
complex
IC50 [complex]/μM
IC50 [Ir]/μM
ppy-8
ppy-4
ppy-3
ppy-1
pq-8
pq-4
pq-3
pq-1
cisplatin
2.1 ( 0.2
3.3 ( 0.1
5.2 ( 0.3
26.4 ( 3.4
1.4 ( 0.1
2.1 ( 0.1
2.7 ( 0.1
5.5 ( 0.7
26.4 ( 2.0
16.8 ( 1.6
13.2 ( 0.4
15.6 ( 0.9
26.4 ( 3.4
11.2 ( 0.8
8.4 ( 0.4
8.1 ( 0.3
5.5 ( 0.7
N.A.
26.4 μM) compared to cisplatin (IC50 = 26.4 μM) under
the same experimental conditions. In general, from our
previous work, we found that there is a correlation
between the lipophilicity and cytotoxicity of this type of
complexes, with higher lipophilicity resulting in higher
cytotoxicity.43g-l This relationship has also been observed in this work: the IC50 values of the pq complexes
are smaller than those of their corresponding ppy counterparts, indicative of higher cytotoxicity related to the
more hydrophobic pq ligand (Table 7). To illustrate the
effects of the dendritic structure of the complexes on their
cytotoxicity, the IC50 values are also presented in terms of
[Ir] (Table 7). The dendritic ppy complex ppy-8 (IC50 =
16.8 μM) is slightly more cytotoxic than its monomeric
counterpart ppy-1 (IC50=26.4 μM). However, the reverse
was observed for the pq analogues: the cytotoxicity of
complex pq-8 (IC50 = 11.2 μM) is apparently lower than
that of complex pq-1 (IC50 =5.5 μM).
For the monomeric complexes, complex pq-1 is about
5 times more cytotoxic than complex ppy-1 (Table 7). We
believe that the observed cytotoxicity is associated with
the lipophilicity of the complex because pq-1 is much
more lipophilic (log Po/w = 2.01) than ppy-1 (log Po/w =
0.44, Table 4). This is also in agreement with much
higher membrane-permeability and hence higher cellular
uptake efficiency of the former complex by passive diffusion (Table 5), for which the lipophilicity plays a very
Article
important role. The IC50 values of the dendritic complexes
ppy-8 and pq-8 are similar (16.8 and 11.2 μM, respectively,
Table 7). Interestingly, their cellular uptake efficiencies
are also very similar, and the intracellular [Ir] are lower
than those of their monomeric counterparts by one order
of magnitude (Table 5). We have attributed the different
cytotoxicity of the monomeric complexes and their dendritic counterparts to a shift of the cellular uptake mechanism from essentially a passive-diffusion mode for the
monomers to an energy-requiring endocytosis pathway
for the dendrimers (see above). It is likely that these
findings are a consequence of an increase of the formal
charge, molecular size, and molecular weight of the dendritic complexes. In summary, we believe that the cytotoxicity of the complexes in this work originates primarily
from their binding to organelles, in particular, the Golgi
apparatus. Thus, higher cellular uptake efficiency results
in higher cytotoxicity. While the uptake efficiency (and
hence cytotoxicity) is strongly dependent on the lipophilicity of the mononuclear complexes which are internalized via essentially a passive diffusion pathway, it is less
important to the polynuclear complexes because the
dendritic cores play a more important role on the properties of the complexes and the uptake mechanism is energyrequiring endocytosis which is less dependent on the
lipophilicity of the complexes.
Conclusion
In this work, dendrimers containing terminal luminescent
cyclometalated iridium(III) polypyridine complexes have
been synthesized and characterized. Their electrochemical
and photophysical properties have been investigated. The
results revealed that there are no electronic communications
between the [Ir(N∧C)2(N∧N)] moieties in the same dendritic
molecule. Agarose gel retardation assays showed that all the
dendritic iridium(III) complexes, in contrast to their mononuclear counterparts, exhibited DNA condensation ability.
Additionally, the lipophilicity, cellular uptake properties, and
cytotoxicity of the complexes have been examined. Treatment
of HeLa cells with the complexes resulted in localization in the
perinuclear regions. Interstingly, cells loaded with the dendritic complexes also displayed Golgi staining, which is a
novel finding. While the dendritic complexes are internalized
essentially by an energy-dependent process, the mononuclear
complexes exhibit an additional passive-diffusion pathway.
These differences are most likely due to factors such as formal
charge and molecular size. In conclusion, the luminescent
dendritic cyclometalated iridium(III) polypyridine complexes
in this work not only show favorable photophysical characteristics that are common to iridium(III) complexes but also
exhibit interesting DNA condensation properties and intracellular localization. Studies of related dendrimers of higher
generations containing luminescent inorganic and organometallic complexes are underway.
Experimental Section
Materials and Synthesis. All solvents were of analytical
reagent grade and purified according to standard procedures.56
(56) Perrin, D. D.; Armarego, W. L. F. Purification of Laboratory
Chemicals; Pergamon: Oxford, 1997.
(57) Telser, J.; Cruickshank, K. A.; Schanze, K. S.; Netzel, T. L. J. Am.
Chem. Soc. 1989, 111, 7221–7226.
Inorganic Chemistry, Vol. 49, No. 12, 2010
5441
All buffer components were of biological grade and used as
received. IrCl3 3 3H2O, Hppy, Hpq, 4,40 -dimethyl-2,20 -bipyridine,
tris(2-aminoethyl)amine, PAMAM dendrimers (generations 0
and 1), n-octanol, 4-methoxyaniline, 4-methoxyphenol, phenol,
acetophenone, naphthalene, tert-butylbenzene, anthracene, and
pyrene were purchased from Aldrich. N-Hydroxysuccinimide,
N,N0 -dicyclohexylcarbodiimide, triethylamine, KPF6, cisplatin,
and paraformaldehyde were purchased from Acros. MTT was
purchased from Sigma. Double-stranded calf thymus DNA
was obtained from Calbiochem. PmCherry-C1 vector (4722 bp)
was procured from Clontech. 4-Succinimidyl-carboxy-40 -methyl2,20 -bipyridine,57 the precursor complexes [Ir2(ppy)4Cl2]25a
and [Ir2(pq)4Cl2],25a and the monomeric complexes ppy-1 and
pq-143g were prepared according to reported procedures. Tetra-nbutylammonium hexafluorophosphate (TBAP) was obtained
from Aldrich and was recrystallized from hot ethanol and dried
in vacuo at 110 °C before use. 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, penicillin/streptomycin, anti-golgin97 (human), mouse IgG1, and Alexa 635 goat anti-mouse IgG
(HþL) were purchased from Invitrogen. The growth medium for
cell culture contained DMEM with 10% FBS and 1% penicillin/
streptomycin.
bpy-8. A 20 wt % MeOH solution of PAMAM dendrimer,
generation 1 (306 μL, 0.035 mmol) was evaporated to dryness
under vacuum for 2 h. The resulting residue was dissolved in a
mixture of DMF (3 mL) and triethylamine (163 μL, 1.16 mmol).
To that solution was added 4-succinimidyl-carboxy-40 -methyl2,20 -bipyridine (120 mg, 0.38 mmol) in DMF (2 mL). The
reaction mixture was stirred at room temperature for 48 h, and
then the DMF was removed under vacuum. The residue was
purified by column chromatography on silica gel. The desired
product was eluted with CH2Cl2/MeOH (6:1, v/v). The ligand
bpy-8 was subsequently isolated as a colorless oil. Yield: 80 mg
(76%). 1H NMR (300 MHz, CDCl3, 298 K, TMS): δ 8.72 (br,
8H, CONH), 8.62-8.57 (m, 16H, H3 and H6 of bpy), 8.34 (d,
8H, J = 4.5 Hz, H60 of bpy), 8.27 (br, 8H, CONH), 8.20-8.04
(m, 12H, CONH and H30 of bpy), 7.64 (d, 8H, J = 4.5 Hz, H5 of
bpy), 7.02 (d, 8H, J=4.5 Hz, H50 of bpy), 3.53-3.44 (m, 32H,
CONHCH2), 3.20-2.60 (m, 44H, CONHCH2 and NCH2),
2.54-2.33 (m, 48H, CH2CO and CH3 on C40 of bpy).
bpy-4. A 20 wt % MeOH solution of PAMAM dendrimer,
generation 0 (141 μL, 0.047 mmol) was evaporated to dryness
under vacuum for 2 h. The resulting residue was dissolved in a
mixture of DMF (5 mL) and triethylamine (108 μL, 0.77 mmol).
To that solution was added 4-succinimidyl-carboxy-40 -methyl2,20 -bipyridine (0.26 mmol, 80 mg) in DMF (2 mL). The reaction
mixture was stirred at room temperature for 48 h, and then
DMF was removed under vacuum. The residue was purified by
column chromatography on silica gel. The desired product was
eluted with CH2Cl2/MeOH (6:1, v/v). The ligand bpy-4 was
subsequently isolated as a colorless oil. Yield: 50 mg (82%). 1H
NMR (300 MHz, CDCl3, 298 K, TMS): δ 8.66-8.57 (m, 12H,
CONH, H3 and H6 of bpy), 8.36-8.34 (m, 8H, CONH, H60 of
bpy), 8.07 (s, 4H, H30 of bpy), 7.62 (d, 4H, J = 5.1 Hz, H5 of
bpy), 7.06 (d, 4H, J = 5.1 Hz, H50 of bpy), 3.53 (br, 8H,
CONHCH2), 3.43 (br, 8H, CH2NHCO), 2.76 (br, 8H, NCH2),
2.40-2.36 (m, 24H, NCH2, CH2CO and CH3 on C40 of bpy).
Positive-ion ESI-MS ion cluster at m/z 1302 {M þ Hþ}þ.
bpy-3. A mixture of 4-succinimidyl-carboxy-40 -methyl-2,20 bipyridine (0.23 mmol, 70 mg,), tris(2-aminoethyl)amine (8.4 μL,
0.056 mmol), and triethylamine (95 μL, 0.68 mmol) in DMF
(5 mL) was stirred at room temperature for 12 h. The colorless
solution was then evaporated to dryness under vacuum. The
residue was purified by column chromatography on silica gel. The
desired product was eluted with CH2Cl2/MeOH (6:1, v/v).
The ligand bpy-3 was subsequently isolated as a colorless oil.
Yield: 40 mg (93%). 1H NMR (300 MHz, CD3OD, 298 K, TMS):
5442 Inorganic Chemistry, Vol. 49, No. 12, 2010
δ 8.30-8.29 (m, 6H, H3 and H6 of bpy), 8.22 (d, 3H, J = 4.8 Hz,
H60 of bpy), 7.81 (s, 3H, H30 of bpy), 7.37 (d, 3H, J = 5.1 Hz, H5
of bpy), 7.03 (d, 3H, J = 4.8 Hz, H50 of bpy), 3.44 (t, 6H, J=5.7
Hz, CONHCH2), 2.70 (t, 6H, J = 5.7 Hz, NCH2), 2.24 (s, 9H,
CH3 on C40 of bpy). Positive-ion ESI-MS ion cluster at m/z 736
{M þ Hþ}þ.
[{Ir(ppy)2}8(bpy-8)](PF6)8 (ppy-8). A mixture of [Ir2(ppy)4Cl2]
(139 mg, 0.11 mmol) and the ligand bpy-8 (80 mg, 0.027 mmol) in
CH2Cl2/MeOH (20 mL, 1:1, v/v) was refluxed under nitrogen
for 4 h. The solution was then cooled to room temperature,
and KPF6 (79 mg, 0.43 mmol) was added to the solution. The
mixture was then evaporated to dryness. The solid was dissolved
in CH2Cl2/MeOH and purified by column chromatography on
silica gel. The product was eluted with CH2Cl2/MeOH (10:1,
v/v) and subsequently recrystallized from a mixture of CH2Cl2
and diethyl ether as yellow crystals. Yield: 47 mg (22%). 1H
NMR (300 MHz, (CD3)2CO, 298 K, TMS): δ 9.34 (br, 8H, H3 of
bpy), 8.97 (br, 8H, H30 of bpy), 8.17-8.02 (m, 36H, CONH, H6
of bpy, and H3 of pyridyl ring of ppy), 7.85-7.66 (m, 72 H, H5
and H60 of bpy, H3 of phenyl ring and H4 and H6 of pyridyl ring
of ppy, and CONH), 7.46 (br, 8H, H50 of bpy), 7.11-6.83 (m,
48H, H5 of pyridyl ring of ppy, H4 and H5 of phenyl ring of
ppy), 6.29 (br, 16H, H6 of phenyl ring of ppy), 3.41-3.29 (m,
40H, CONHCH2, CH2NHCO, CONHCH2), 2.62-2.49 (m,
36H, NCH2, CH2N), 2.40-2.08 (m, 48H, CH2CO, CH3 on C40
of bpy). Positive-ion ESI-MS ion cluster at m/z 2722 {M þ
3 Hþ}3þ, 2674 {M þ 2 Hþ - PF6-}3þ, 2625 {M þ Hþ 2 PF6-}3þ, 2576 {M - 3 PF6-}3þ, 2043 {M þ 4 Hþ}4þ, 1999
{M þ 3 Hþ - PF6-}4þ, 1970 {M þ 2 Hþ - 2 PF6-}4þ, 1933
{M þ Hþ - 3 PF6-}4þ, 1896 {M - 4 PF6-}4þ. IR (KBr)
ν/cm-1: 3425 (s, br, N-H), 1655 (s, CdO), 845 (s, PF6-). Anal.
Calcd for Ir8C334H320N58O20P8F48 3 2CH2Cl2 3 2H2O: C, 48.22; H,
3.95; N, 9.71. Found: C, 48.16; H, 4.23; N, 9.92.
[{Ir(ppy)2}4(bpy-4)](PF6)4 (ppy-4). The procedure was similar
to that for the preparation of complex ppy-8, except that bpy-4
was used instead of bpy-8. Subsequent recrystallization from
CH2Cl2/diethyl ether afforded complex ppy-4 as yellow crystals.
Yield: 40%. 1H NMR (300 MHz, (CD3)2CO, 298 K, TMS): δ
9.02 (s, 4H, H3 of bpy), 8.69 (s, 4H, H30 of bpy), 8.37 (br, 4H,
CONH), 8.21-8.13 (m, 12H, H6 of bpy and H3 of pyridyl ring
of ppy), 7.89-7.78 (m, 32H, H5 and H60 of bpy and H3 of
phenyl ring and H4 and H6 of pyridyl ring of ppy), 7.65 (br, 4H,
CONH), 7.49 (d, 4H, J = 5.7 Hz, H50 of bpy), 7.15-6.83 (m,
24H, H5 of pyridyl ring and H4 and H5 of phenyl ring of ppy),
6.31 (t, 8H, J = 5.7 Hz, H6 of phenyl ring of ppy), 3.43 (br, 8H,
CONHCH2), 3.37 (br, 8H, CH2NHCO), 3.10 (br, 8H, NCH2),
2.64-2.60 (m, 12H, CH2N and CH2CO), 2.52 (s, 12H, CH3 on
C40 of bpy). Positive-ion ESI-MS ion cluster at m/z 1797 {M 2 PF6-}2þ, 1150 {M - 3 PF6-}3þ, 826 {M - 4 PF6-}4þ. IR
(KBr) ν/cm-1: 3424 (s, br, N-H), 1656 (s, CdO), 845 (s, PF6-).
Anal. Calcd for Ir4C158H144N26O8P4F24 3 4CH2Cl2: C, 46.07; H,
3.63; N, 8.62. Found: C, 46.00; H, 3.76; N, 8.83.
[{Ir(ppy)2}3(bpy-3)](PF6)3 (ppy-3). The procedure was similar
to that for the preparation of complex ppy-8, except that bpy-3
was used instead of bpy-8. Subsequent recrystallization from
CH2Cl2/diethyl ether afforded complex ppy-3 as yellow crystals.
Yield: 43%. 1H NMR (300 MHz, (CD3)2CO, 298 K, TMS): δ
9.11 (s, 3H, H3 of bpy), 8.72 (s, 3H, H30 of bpy), 8.42 (br, 3H,
CONH), 8.23-8.18 (m, 6H, H3 of pyridyl ring of ppy), 8.08 (d,
3H, J = 5.7 Hz, H6 of bpy), 7.93-7.76 (m, 24H, H5 and H60 of
bpy and H3 of phenyl ring and H4 and H6 of pyridyl ring of
ppy), 7.47 (d, 3H, J=5.7 Hz, H50 of bpy), 7.12-6.83 (m, 18H,
H5 of pyridyl ring and H4 and H5 of phenyl ring of ppy), 6.31 (t,
6H, J = 5.7 Hz, H6 of phenyl ring of ppy), 3.55 (br, 6H,
CONHCH2), 2.84 (br, 6H, NCH2), 2.42 (s, 9H, CH3 on C40 of
bpy). Positive-ion ESI-MS ion cluster at m/z 2527 {M - PF6-}þ,
1191 {M - 2 PF6-}2þ. IR (KBr) ν/cm-1: 3427 (s, br, N-H), 1661
(s, CdO), 845 (s, PF6-). Anal. Calcd for Ir3C108H90N16O3P3F18 3
H2O: C, 48.23; H, 3.45; N, 8.33. Found: C, 47.93; H, 3.61; N, 8.63.
Zhang et al.
[{Ir(pq)2}8(bpy-8)](PF6)8 (pq-8). The procedure was similar to
that for the preparation of complex ppy-8, except that
[Ir2(pq)4Cl2] was used instead of [Ir2(ppy)4Cl2]. Subsequent
recrystallization from CH2Cl2/diethyl ether afforded complex
pq-8 as orange crystals. Yield: 24%. 1H NMR (300 MHz,
(CD3)2CO, 298 K, TMS): δ 8.75 (br, 12H, CONH), 8.53-8.41
(m, 56H, H3, H30 , and H6 of bpy and H3 of quinoline and H3 of
phenyl ring of pq), 8.23 (d, 8H, J=8.1 Hz, H5 of bpy), 8.178.12 (m, 16H, H4 of quinoline of pq), 8.03-8.01 (m, 8H, H60 of
bpy), 7.84-7.77 (m, 24H, H8 of quinoline of pq, CONH),
7.51-7.49 (m, 8H, H50 of bpy), 7.39-7.28 (m, 32H, H5, H7 of
quinoline of pq), 7.18-7.03 (m, 32H, H4 of phenyl ring and H6
of quinoline of pq), 6.76-6.68 (m, 8H, H5 of phenyl ring of pq),
6.67 (br, 8H, H5 of phenyl ring of pq), 6.50-6.48 (m, 16H, H6 of
phenyl ring of pq), 3.41-3.29 (m, 40H, CONHCH2, CH2NHCO,
and CONHCH2), 2.58-2.46 (m, 36H, NCH2 and CH2N), 2.312.21 (m, 48H, CH2CO and CH3 on C40 of bpy). Positive-ion
ESI-MS ion cluster at m/z 2892 {M þ Hþ - 2 PF6-}3þ, 2844
{M - 3 PF6-}3þ, 2242 {M þ 4 Hþ}4þ, 2207 {M þ 3 Hþ PF6-}4þ, 2171 {M þ 2 Hþ - 2 PF6-}4þ, 2133 {M þ Hþ - 3
PF6-}4þ, 2097 {M - 4 PF6-}4þ, 1766 {M þ 4 Hþ - PF6-}5þ,
1735 {M þ 3 Hþ - 2 PF6-}5þ, 1707 {M þ 2 Hþ - 3
PF6-}5þ, 1677 {M þ Hþ - 4 PF6-}5þ, 1649 {M - 5 PF6-}5þ.
IR (KBr) ν/cm-1: 3424 (s, br, N-H), 1657 (s, CdO), 847 (s, PF6-).
Anal. Calcd for Ir8C398H352N58O20P8F48 3 6H2O: C, 53.87; H, 4.41;
N, 8.63. Found: C, 54.09; H, 4.66; N, 8.80.
[{Ir(pq)2}4(bpy-4)](PF6)4 (pq-4). The procedure was similar to
that for the preparation of complex ppy-4, except that [Ir2(pq)4Cl2] was used instead of [Ir2(ppy)4Cl2]. Subsequent recrystallization from CH2Cl2/diethyl ether afforded complex pq-4 as orange
crystals. Yield: 40%. 1H NMR (300 MHz, (CD3)2CO, 298 K,
TMS): δ 8.65 (br, 4H, CONH), 8.54-8.41 (m, 20H, H3 of bpy
and H3 of phenyl ring and H3 of quinoline of pq), 8.31-8.17 (m,
20H, H30 , H6, and H60 of bpy and H4 of quinoline of pq), 7.96
(d, 4H, J = 6.0 Hz, H5 of bpy), 7.86-7.82 (m, 8H, H8 of
quinoline of pq), 7.62 (br, 4H, CONH), 7.54 (d, 4H, J = 5.4 Hz,
H50 of bpy), 7.42-7.31 (m, 16H, H5 and H7 of quinoline of pq),
7.19-7.05 (m, 16H, H4 of phenyl ring and H6 of quinoline of
pq), 6.84-6.74 (m, 8H, H5 of phenyl ring of pq), 6.54-6.49 (m,
8H, H6 of phenyl ring of pq), 3.47-3.26 (m, 16H, CONHCH2
and CH2NHCO), 3.07 (br, 8H, NCH2), 2.58-2.46 (m, 12H,
CH2N and CH2CO), 2.39 (s, 12H, CH3 on C40 of bpy). Positiveion ESI-MS ion cluster at m/z 2070 {M þ Hþ - PF6-}2þ, 1998
{M - 2 PF6-}2þ, 1331 {M þ Hþ - 2 PF6-}3þ, 1283 {M 3 PF6-}3þ. IR (KBr) ν/cm-1: 3422 (s, br, N-H), 1660 (s,
CdO), 847 (s, PF6-). Anal. Calcd for Ir4C190H160N26O8P4F24 3
4CH2Cl2: C, 50.39; H, 3.66; N, 7.88. Found: C, 50.18; H, 3.74;
N, 8.08.
[{Ir(pq)2}3(bpy-3)](PF6)3 (pq-3). The procedure was similar
to that for the preparation of complex ppy-3, except that
[Ir2(pq)4Cl2] was used instead of [Ir2(ppy)4Cl2]. Subsequent
recrystallization from CH2Cl2/diethyl ether afforded complex
pq-3 as orange crystals. Yield: 42%. 1H NMR (300 MHz,
(CD3)2CO, 298 K, TMS): δ 8.67 (br, 3H, CONH), 8.55-8.36
(m, 15H, H3 of bpy and H3 of phenyl ring and H3 of quinoline
of pq), 8.26-8.16 (m, 15H, H30 , H5, and H6 of bpy and H4
of quinoline of pq), 7.90-7.83 (m, 9H, H60 of bpy and H8 of
quinoline of pq), 7.55 (d, 3H, J = 4.8 Hz, H50 of bpy), 7.40-7.24
(m, 12H, H5 and H7 of quinoline of pq), 7.20-7.09 (m, 6H, H4
of phenyl ring of pq), 7.06-6.91 (m, 6H, H6 of quinoline of pq),
6.85-6.73 (m, 6H, H5 of phenyl ring of pq), 6.56-6.50 (m, 6H,
H6 of phenyl ring of pq), 3.36 (br, 6H, CONHCH2), 2.68 (br,
6H, NCH2), 2.30 (s, 9H, CH3 on C40 of bpy). Positive-ion
ESI-MS ion cluster at m/z 2827 {M - PF6-}þ, 1341 {M - 2
PF6-}2þ. IR (KBr) ν/cm-1: 3421 (s, br, N-H), 1666 (s, CdO),
846 (s, PF6-). Anal. Calcd for Ir3C132H102N16O3P3F18 3 2H2O:
C, 52.71; H, 3.55; N, 7.45. Found: C, 52.57; H, 3.64; N, 7.50.
Physical Measurements and Instrumentation. Equipment for
the characterization, photophysical, electrochemical, lipophilicity,
Article
and cytotoxicity measurements, has been described previously.43i
Luminescence quantum yields were measured by the optically
dilute method58 using an aerated aqueous solution of [Ru(bpy)3]Cl2 (Φ = 0.028) as the standard solution.59 The lipophilicity and
cytotoxicity have been determined with the reported methods.43i
Agarose Gel Retardation Assays. Agarose gel retardation
assays were used to study the DNA condensation ability of the
complexes. Plasmid DNA (1 μg) was mixed with the iridium(III)
complex with [Ir] ranging from 12.5 to 400 nM in PBS/DMSO
(10 μL, 7:3, v/v). The mixture was incubated at room temperature
for 30 min and then electrophoresced (Power-Pac, Bio-Rad,
100 V, 40 min) on a 0.9% (w/v) agarose gel containing ethidium
bromide (0.5 μg/mL) in Tris-acetate-EDTA (TAE) buffer. The
agarose gel was examined by the Bio-Rad Gel Doc imager.
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 removed and replaced with
medium/DMSO (99:1, v/v) containing the complex at [Ir] =
2 μM. After incubation for 3 h, the medium was removed, and
the cell layer was washed gently with PBS (1 mL 3). After that,
the cell layer was trypsinized, digested in 65% HNO3 (2 mL) at
70 °C for 2 h, and then diluted in Milli-Q water to the final
volume of 10 mL for ICP-MS (PerkinElmer SCIEX, ELAN
DRC Plus) analysis.
Confocal Microscopy. HeLa cells were grown on a sterile glass
coverslip in a 35 mm tissue culture dish. The incubation procedure was similar to that of the ICP-MS. After washing with
PBS, the coverslip was mounted onto slides for measurements.
(58) Demas, J. N.; Crosby, G. A. J. Phys. Chem. 1971, 75, 991–1024.
(59) Nakamura, K. Bull. Chem. Soc. Jpn. 1982, 55, 2697–2705.
Inorganic Chemistry, Vol. 49, No. 12, 2010
5443
Imaging was performed using a confocal microscope (Leica
TCS SPE) with an excitation wavelength at 405 nm. The
emission was measured using a long-pass filter at 532 nm. In
the 4 °C imaging experiments, HeLa cells were preincubated at
4 °C for 1 h before treatment with complexes. In the co-staining
experiments, after treated with complexes, the HeLa cells were
fixed by PBS containing 3% paraformaldehyde, and then
incubated with anti-golgin-97 (human) mouse IgG1 (1 μg/mL,
1 h), washed with PBS (1 mL 3), then incubated with Alexa
635 goat anti-mouse IgG (HþL) (10 μg/mL, 30 min), and finally
washed with PBS (1 mL 3).
Flow Cytometry. The incubation procedure was similar to
that for the ICP-MS measurements. After washing with PBS,
the cell layer was trypsinized and added up to a final volume of
2 mL with PBS. The samples were analyzed by a FACSCalibur
flow cytometer (Becton, Dickinson and Co., Franklin Lakes,
NJ, U.S.A.) with excitation at 488 nm. The emission was
measured using a long-pass filter at 505 nm. The number of
cells analyzed for each sample was between 9,000 and 10,000.
Acknowledgment. We thank The Hong Kong Research
Grants Council (Project Nos. CityU 101606 and 101908) for
financial support. K.Y.Z. acknowledges the receipt of a Postgraduate Studentship, a Research Tuition Scholarship, and an
Outstanding Academic Performance Award, all of which were
administered by the City University of Hong Kong.
Supporting Information Available: Laser-scanning confocal
microscopy images (z stacks) of HeLa cells incubated with
complex pq-1. This material is available free of charge via the
Internet at http://pubs.acs.org.