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Modification of luminescent iridium(III) polypyridine complexes with discrete poly(ethylene glycol) (PEG) pendants: synthesis, emissive behavior, intracellular uptake, and PEGylation properties.
FULL PAPER
DOI: 10.1002/chem.201000474
Modification of Luminescent IridiumACHTUNGRE(III) Polypyridine Complexes with
Discrete Poly(ethylene glycol) (PEG) Pendants: Synthesis, Emissive
Behavior, Intracellular Uptake, and PEGylation Properties
Steve Po-Yam Li, Hua-Wei Liu, Kenneth Yin Zhang, and Kenneth Kam-Wing Lo*[a]
Abstract: We report the synthesis, characterization, and photophysical properties of a new class of luminescent cyclometalated iridiumACHTUNGRE(III) polypyridine
poly(ethylene glycol) (PEG) complexes
[IrACHTUNGRE(N^C)2ACHTUNGRE(N^N)]ACHTUNGRE(PF6) (HN^C = Hppy
(2-phenylpyridine),
N^N = bpy
CONHPEG1 (bpy = 2,2’-bipyridine;
1 a),
bpyCONHPEG3
(1 b);
HN^C = Hpq
(2-phenylquinoline),
N^N = bpyCONHPEG1 (2 a), bpy
CONHPEG3 (2 b); HN^C = Hpba (4(2-pyridyl)benzaldehyde), N^N = bpy
CONHPEG1 (3)) and their PEG-free
counterparts (N^N = bpyCONHEt,
HN^C = Hppy (1 c); HN^C = Hpq
(2 c)). The cytotoxicity and cellular
uptake of these complexes have been
investigated by the MTT assay, ICPMS,
laser-scanning confocal microscopy,
and flow cytometry. The results showed
that the complexes supported by the
water-soluble PEG can act as biological
probes and labels with considerably reKeywords: DNA · iridium · luminescence · PEGylation probes
Introduction
PEGylation is the process of covalent modification of proteins, peptides, antibody fragments, and drug molecules with
poly(ethylene glycol) (PEG). Because this derivatization
procedure can significantly reduce the toxicity of the molecules without sacrificing their specific biological or therapeutic properties, PEGylation reagents with 1) a wide range
of molecular weights (MWs), 2) various shapes such as
linear and branched structures, and 3) different reactive
functional groups for modification have been developed.[1]
Many of these compounds are already commercially available. Those with fluorescence properties are particularly
useful because they not only confer the aforementioned
properties but also enable detection and quantization of the
targets by fluorescence methods.[2] In addition to the work
[a] S. P.-Y. Li, Dr. H.-W. Liu, K. Y. Zhang, Dr. K. K.-W. Lo
Department of Biology and Chemistry
City University of Hong Kong, Tat Chee Avenue
Kowloon, Hong Kong (P.R. China)
Fax: (+ 852) 2788-7406
E-mail: bhkenlo@cityu.edu.hk
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201000474.
Chem. Eur. J. 2010, 16, 8329 – 8339
duced cytotoxicity. Because the aldehyde groups of complex 3 are reactive
toward primary amines, the complex
has been utilized as the first luminescent PEGylation reagent. Bovine
serum albumin (BSA) and poly(ethACHTUNGREylACHTUNGREeneimine) (PEI) have been PEGylated
with this complex, and the resulting
conjugates have been isolated, purified,
and their photophysical properties
studied. The DNA-binding and genedelivery properties of the luminescent
PEI conjugate 3-PEI have also been investigated.
on the modification of cisplatin with PEG and the incorporation of related anticancer drugs into micelles composed of
PEG,[3] there is an emerging interest in the use of transitionmetal PEG complexes in biological studies.[4] Despite these
reports, applications of luminescent transition-metal PEG
complexes in biological studies are still unexplored.
The use of transition-metal complexes as cellular probes
has attracted much attention recently.[5–13] In our previous
bioconjugation and cellular studies of luminescent inorganic
and organometallic transition-metal complexes,[14–18] we
found that the solubility of the complexes in aqueous media
might not be sufficiently high to fully utilize their potential
in biological applications. Another observation is that most
of the complexes are considerably cytotoxic to eukaryotic
cell lines.[14h,i, 15d, 17h–m] Although this may be an advantage in
the development of anticancer drugs, the high cytotoxicity
may limit their use as probes in live-cell imaging studies. We
anticipate that the attachment of PEG pendants to the
metal complexes will circumvent these problems. To the
best of our knowledge, there has been no report about the
development of PEGylation reagents based on luminescent
transition-metal complexes.
Herein, we report the synthesis, characterization, and
photophysical and biological properties of a new class of lu-
2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
8329
minescent cyclometalated iridiumACHTUNGRE(III) polypyridine PEG
complexes [IrACHTUNGRE(N^C)2ACHTUNGRE(N^N)]ACHTUNGRE(PF6) (HN^C = 2-phenylpyridine (Hppy), N^N = 4-(N-(4,7,10,13,16,19,22,25,28,31,ACHTUNGRE34,ACHTUNGRE37,ACHTUNGRE40,43,46,49,52,55,58,61,64,67,70,73-tetracosaoxapentaheptacontyl)aminocarbonyl)-4’-methyl-2,2’-bipyridine
(bpy
CONHPEG1; 1 a), 4-(tris(N-(2,5,8,11,14,17,20,23,26,29,ACHTUNGRE32,ACHTUNGRE35-dodecaoxaheptatriacontyl)aminocarbonyl-(2-oxabutyl))methylamino-N-(3,6,9,12-tetraoxapentadecanoyl)aminocarbonyl)-4’-methyl-2,2’-bipyridine (bpyCONHPEG3; 1 b);
HN^C = 2-phenylquinoline (Hpq), N^N = bpyCONH
PEG1 (2 a), bpyCONHPEG3 (2 b); HN^C = 4-(2-pyridyl)benzaldehyde (Hpba), N^N = bpyCONHPEG1 (3))
and their PEG-free counterparts (N^N = 4-(N-ethylaminocarbonyl)-4’-methyl-2,2’-bipyridine
(bpyCONHEt),
HN^C = Hppy (1 c); HN^C = Hpq (2 c); Scheme 1). The cytotoxicity and cellular uptake of these iridiumACHTUNGRE(III) PEG
complexes have been investigated by the MTT assay,
ICPMS, laser-scanning confocal microscopy, and flow cytometry. The results showed that these complexes that are supported by the water-soluble PEG can act as biological
probes and labels with considerably reduced cytotoxicity.
Because the aldehyde groups of complex 3 are reactive
toward primary amines, this complex has been utilized as
the first luminescent PEGylation reagent. Bovine serum albumin (BSA) and poly(ethyleneimine) (PEI) have been
PEGACHTUNGREylated with this complex, and the resulting conjugates
have been isolated, purified, and their photophysical properties studied. The DNA-binding and gene-delivery properties
of the luminescent PEI conjugate 3-PEI have also been investigated.
Results and Discussion
Synthesis: Discrete PEG units have been selected for this
work because these molecules have defined chain lengths,
MWs, and molecular structures, which will result in homogeneous compounds that can be readily characterized.[19] Additionally, PEGylation reagents that are functionalized with
discrete PEG units are expected to offer higher consistency
in bioconjugation reactions. The diimine ligands bpy
CONHPEG1, bpyCONHPEG3, and bpyCONHEt
were synthesized from the reaction of 4-(N-hydroxysuccinimidyl)-4’-methyl-2,2’-bipyridine (bpyNHS) with the amines
4,7,10,13,16,19,22,25,28,31,34,37,40,43,46,49,52,55,ACHTUNGRE58,ACHTUNGRE61,ACHTUNGRE64,
ACHTUNGRE67,ACHTUNGRE70,ACHTUNGRE73-tetracosaoxapentaheptacontane
(m-dPEG24
amine), tris((37-N-amido-2,5,8,11,14,17,20,23,26,29,32,35-dodecaoxaheptatriacontane)-4-oxapentanoyl)-(15-amino-4,ACHTUNGRE7,ACHTUNGRE10,ACHTUNGRE13-tetACHTUNGREraACHTUNGREoxapentadecanoylamido)methane
(amino
dPEG4ACHTUNGRE(m-dPEG12)3), and ethylamine, respectively, in
CH2Cl2 in the presence of triethylamine. The 1H NMR spectra of the ligACHTUNGREands revealed that the aromatic amide proton
resonated at 7.50 ppm. All the iridiumACHTUNGRE(III) complexes 1 a–
c, 2 a–c, and 3 were prepared from the reaction of [Ir2ACHTUNGRE(N^C)4Cl2] (HN^C = Hppy, Hpq, Hpba) with the diimine ligands in a mixture of CH2Cl2 and MeOH (1:1, v/v) under
reflux conditions, followed by anion exchange with KPF6
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Scheme 1. Structures of the iridiumACHTUNGRE(III) complexes.
and purification by column chromatography. Unfortunately,
the iridiumACHTUNGRE(III) PEG complexes 1 a,b, 2 a,b, and 3 were hygroscopic, rendering it impossible to check the purity of the
samples by elemental analysis. However, because discrete
PEG molecules have been used, the PEG complexes can be
readily characterized by high-resolution (HR) ESI-TOF MS
and 1H NMR spectroscopy. The HR mass spectra showed
the relevant isotopic patterns and MWs of all the complexes,
which confirmed their identities. In contrast to common cyclometalated iridiumACHTUNGRE(III) polypyridine complexes, all the
PEG complexes in this work are soluble in aqueous buffers.
The water solubility was determined to be > 0.3 m, which is
sufficiently high for common biological applications.
Electronic absorption and emission properties: The electronic absorption spectral data of all the complexes are
listed in Table 1. The intense absorption bands and should-
2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2010, 16, 8329 – 8339
Luminescent IrIII Polypyridine Complexes
FULL PAPER
is in line with the appearance of a similar HE emission band
ers at 253–352 nm have been assigned to spin-allowed intrafor a related iridium nondiscrete PEG complex [IrACHTUNGREliACHTUNGREgand (1IL) (p!p*) (N^N and N^C) transitions.[17, 20–23, 24a, 25]
ACHTUNGRE(ppy)2(bpyCONHPEG)]ACHTUNGRE(PF6) (bpyCONHPEG = 4-(NThe less intense absorption bands and shoulders at > 352 nm
and the weak absorption tail beyond 450 nm have been as{2-[w-methoxypoly(1-oxaACHTUNGREproACHTUNGREpyl)]ACHTUNGREethACHTUNGREyl}ACHTUNGREaminocarbonyl)-4’signed to spin-allowed and spin-forbidden metal-to-ligand
methyl-2,2’-bipyridine, MWPEG = 5000 Da, PDIPEG = 1.08)
charge-transfer (MLCT) (dp(Ir)!p*(N^N and N^C)) tran(lem = 539 and 615 nm (sh); to = 0.30 ms; Fem = 0.0050). This
sitions, respectively.[17, 20–23, 24a, 25] Upon photoexcitation, all
argument is supported by the observation of a less intense
HE band and a dominant LE emission band for complexes
the complexes displayed intense and long-lived orange to
1 a and 1 b in water (Figure S1 and S2 in the Supporting Ingreenish-yellow emission. The photophysical data are sumformation) because the degree of PEG–complex interaction
marized in Table 2 and the emission spectra of the PEG
should be lower in water than in more polar 50 mm phoscomplexes 1 a,b, 2 a,b, and 3 are shown in Figure 1. In generphate buffer. Although the pq complexes 2 a–c displayed
al, the emission maxima of ppy complexes 1 a–c occurred at
typical 3MLCT (dp(Ir)!p*ACHTUNGRE(N^N)) emission properties, the
601–625 nm in fluid solutions with a small red-shift upon
increasing the polarity of the
solvent. We have tentatively assigned the emission to an excit- Table 1. Electronic absorption spectral data of the cyclometalated iridiumACHTUNGRE(III) PEG complexes at 298 K.
ed state of 3MLCT (dp(Ir)!p*- Complex
Solvent
labs [nm] (e [dm3 mol1 cm1])
[17, 20a, 22, 24–26]
ACHTUNGRE(N^N))
character.
1a
CH2Cl2
253 (22 375), 323 sh (6390), 386 sh (3140), 500 sh (470)
The
electron-withdrawing
253 (21 205), 323 sh (5360), 389 sh (2380), 472 sh (655)
CH3CN
254 (24 260), 321 sh (7500), 387 sh (3630), 501 sh (600)
CH2Cl2
amide substituent stabilizes the 1 b
253 (28 865), 323 sh (7365), 386 sh (3220), 480 sh (475)
CH3CN
p* orbitals of the diimine lig2a
CH2Cl2
260 (29 505), 284 sh (25 235), 350 sh (10 495), 440 sh (2840), 530 sh (365)
ACHTUNGREands and renders the emission
258 (29 240), 282 sh (25 545), 349 sh (9865), 440 sh (2630)
CH3CN
of these complexes to occur at 2 b
261 (38 065), 285 sh (30 875), 352 sh (12 930), 444 sh (3950), 530 sh (500)
CH2Cl2
259 (43 185), 284 sh (34 780), 349 sh (14 195), 440 sh (3985)
CH3CN
lower energy than their unsub262 (35 930), 302 sh (25 500), 318 sh (20 620), 371 sh (5670), 452 sh (2740)
CH2Cl2
stituted 2,2’-bipyridine counter- 3
264 (33 330), 297 sh (23 515), 317 sh (18 140), 366 sh (4990), 444 sh (2475)
CH3CN
[17d]
parts.
Interestingly, the emission spectrum of complex 1 a in
degassed buffer showed an unexpected high-energy (HE) Table 2. Photophysical data of the cyclometalated iridiumACHTUNGRE(III) complexes and the conjugates 3-BSA and 3band at 536 nm and a low- PEI.
to [ms][a]
Fem
Medium (T [K])
lem [nm]
energy (LE) shoulder at Complex
615 nm
(Figure 1
and 1 a
CH2Cl2 (298)
601
0.36
0.051
607
0.22
0.038
CH3CN (298)
Table 2). For complex 1 b, this
536, 615 sh
0.31
0.0053
buffer (298)[b]
special HE emission feature ap477, 513 sh, 549 (max)
4.87 (54 %), 2.42 (46 %)
–
glass (77)[c]
peared as a shoulder at
603
0.28
0.062
1b
CH2Cl2 (298)
518 nm whereas the expected
611
0.20
0.038
CH3CN (298)
LE band occurred at 624 nm
518 sh, 624
0.31
0.0023
buffer (298)[b]
489 sh, 554
4.50 (57 %), 2.82 (43 %)
–
glass (77)[c]
(Figure 1 and Table 2). The lifeCH2Cl2 (298)
609
0.40
0.13
times of these HE features 1 c
613
0.23
0.058
CH3CN (298)
were indistinguishable from
625
0.34
0.0023
buffer (298)[d]
those of the LE ones. Although
541, 560 sh
4.80
–
glass (77)[c]
562, 586 sh
3.03 (20 %), 0.67 (80 %)
0.19
CH2Cl2 (298)
PEG is commonly considered 2 a
562, 595 sh
5.66 (38 %), 0.57 (62 %)
0.10
CH3CN (298)
as a polar molecule, its poly518, 545 (max), 596 sh
11.82 (61 %), 3.19 (39 %)
0.0031
buffer (298)[b]
ether nature is less polar com505, 539 (max), 580, 631 sh
20.61 (2 %), 4.56 (98 %)
–
glass (77)[c]
pared with aqueous buffer. Be- 2 b
561 sh, 583
2.79 (18 %), 0.58 (82 %)
0.16
CH2Cl2 (298)
cause the MLCT emission of
564, 597 sh
5.43 (22 %), 0.49 (78 %)
0.087
CH3CN (298)
517, 546 (max), 597 sh
12.70 (62 %), 3.35 (38 %)
0.0048
buffer (298)[b]
this type of cycloACHTUNGREmetACHTUNGREalated
502, 545 (max), 583
15.84 (5 %), 3.79 (95 %)
–
glass (77)[c]
iridiumACHTUNGRE(III) complex occurs at
595
0.67
0.18
2c
CH2Cl2 (298)
higher energy in less polar sol587
0.54
0.14
CH3CN (298)
vents, it is likely that in 50 mm
574, 596 sh
1.63 (8 %), 0.17 (92 %)
0.013
buffer (298)[d]
543 (max), 585
4.69
–
glass (77)[c]
phosphate buffer, the long PEG
532, 566 sh
3.82
0.26
CH2Cl2 (298)
pendants are in close proximity 3
536, 566 sh
2.84
0.20
CH3CN (298)
to the relatively hydrophobic
521 (max), 567, 615 sh
6.99
–
glass (77)[c]
complex molecules, giving rise 3-BSA
buffer (298)[b]
490 (max), 520
2.61
0.069
to a comparatively nonpolar 3-PEI
504, 525 sh
2.45 (56 %), 0.87 (44 %)
0.074
buffer (298)[b]
local environment and hence [a] The lifetimes were measured at the emission maxima. [b] Potassium phosphate buffer (50 mm, pH 7.4).
the HE emission features. This [c] EtOH/MeOH (4:1, v/v). [d] Potassium phosphate buffer (50 mm, pH 7.4) containing 10 % methanol.
Chem. Eur. J. 2010, 16, 8329 – 8339
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K. K.-W. Lo et al.
Figure 1. Emission spectra of complexes 1 a,b, 2 a,b, and 3 in degassed CH2Cl2 (c), CH3CN (b), and potassium phosphate buffer (g) at 298 K.
PEG complexes 2 a and 2 b showed biphasic emission decay
with lifetimes (to 3–6 and 0.5–0.7 ms), which are noticeably
longer than those of their ppy counterparts (Table 2). We
have tentatively assigned the long- and short-lived components to excited states of predominant 3IL (p!p*) (pq) and
3
MLCT (dp(Ir)!p*(N^N or pq)) character, respectively.[17c,d,f,h,i,k,m] Again, both complexes 2 a and 2 b exhibited a
HE emission feature with rich vibronic structures at
518 nm in aqueous phosphate buffer. Lifetime measurements also revealed biphasic decay with longer lifetimes (to
12–13 and 3–5 ms). Very similar emission spectra and lifetimes are observed for a related nondiscrete PEG complex
[Ir(pq)2(bpyCONHPEG)]ACHTUNGRE(PF6) (lem = 518 (sh), 550 and
596 nm (sh); to = 12.36 (37 %) and 1.46 (63 %) ms; Fem =
0.0045). On the basis of the very long lifetime and structural
band shapes, it is conceivACHTUNGREable that the HE feature originated
from a 3IL state associated with the pq ligand. Unlike the
ppy complexes, the emission spectra of complexes 2 a and
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2 b in water (Figure S3 and S4 in the Supporting Information) are very similar to those in buffer. Probably the interactions of the PEG pendants with these complexes are similarly strong in both water and buffer, which is not unreasonable because of the higher hydrophobicity of the pq complexes compared with their ppy counterparts. Note that addition of free PEG molecules to a buffer solution of the
control bpyCONHEt complexes 1 c and 2 c did not result
in the production of such HE emission bands/shoulders.
These findings resemble the interesting emission properties
of related inorganic complexes incorporated with aliphatic
chains.[27] All these results strongly indicate that the covalently attached PEG1 and PEG3 pendants significantly perturb the emission properties of the complexes in aqueous
buffer. The pba complex 3 showed a vibronically structured
emission band (Figure 1) with very long emission lifetimes in
fluid solutions at 298 K and in low-temperature alcohol glass
( 3–7 ms), typical of 3IL (p!p*) (pba) emission.[17b,l, 18a,b]
2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Luminescent IrIII Polypyridine Complexes
FULL PAPER
Cytotoxicity: The cytotoxicity of all the complexes toward
HeLa cells has been examined by the MTT assay and the results are listed in Table 3. The most important finding is that
Table 3. Cytotoxicity (IC50, 48 h) of the cyclometalated iridiumACHTUNGRE(III) complexes and cisplatin towards HeLa cells.
Complex
IC50 [mm]
1a
1b
1c
2a
2b
2c
3
cisplatin
830.4 54.5
1180.0 70.5
14.6 1.5
565.9 49.4
286.5 35.2
4.1 0.4
1050.0 64.9
10.3 0.7
the IC50 values of the iridiumACHTUNGRE(III) PEG complexes 1 a,b,
2 a,b, and 3 (ranging from 286.5 to 1180.0 mm) are significantly higher than those of cisplatin (10.3 mm) and the control bpyCONHEt complexes 1 c (14.6 mm) and 2 c
(4.1 mm). We have ascribed the exceptionally low cytotoxicity of the PEG complexes to the long PEG chains which protect the complexes from 1) interacting nonspecifically with
the extracellular proteins and 2) triggering immunogenicity
and antigenicity inside the cells.[28, 29] The ppy complexes 1 a
and 1 b revealed lower cytotoxicity than their pq counterparts 2 a and 2 b, which can be accounted for by the higher
lipophilicity of the latter complexes due to the more nonpolar pq ligands. A similar dependence of cytotoxicity on lipophilicity has been commonly observed for related cycloACHTUNGREmetACHTUNGREalated iridiumACHTUNGRE(III) polypyridine complexes.[17h–m]
Cellular uptake studies: The cellular uptake of all the complexes has been studied by ICPMS measurements and the
results are listed in Table 4. The amounts of iridium taken
up by the HeLa cells are of the same order of magnitude
among all the complexes. Specifically, an average cell (mean
volume of 3.4 pL) contained 1.38 to 4.45 fmol of iridium,
which is comparable to those reported in the cellular uptake
studies of other iridium[17j,k,m] and inorganic complexes.[6a, 14i]
Note that the intracellular iridium concentrations are in the
submolar range, which is much higher than that of the free
complexes in the medium before the uptake (5 mm), indicating that the complexes were concentrated within the cells.
The difference of uptake efficiencies between the complexes
is too small for meaningful comparison (Table 4). Thus, the
similar intracellular iridium concentrations indicate that the
much larger IC50 values of the PEG complexes 1 a,b, 2 a,b,
and 3 compared with those of the control bpyCONHEt
complexes 1 c and 2 c (Table 3) are due to the favorable effects of the PEG pendants.
Laser-scanning confocal microscopy: The cellular uptake of
the complexes has been investigated by laser-scanning confocal microscopy. Related cyclometalated iridiumACHTUNGRE(III) polypyridine complexes that we have designed previously are
quite cytotoxic and thus the complex concentration was limited to a few micromolar. However, because the current
PEG complexes are almost noncytotoxic to HeLa cells, complex 2 a at higher concentrations has been used in the experiments. Incubation of the cells with the complex at 10 mm
at 37 8C for 1 h resulted in efficient cellular uptake and the
microscopy images revealed obvious punctate staining in the
cytoplasm (Figure 2). It is likely that the complex binds to
hydrophobic organelles such as the Golgi body and endoplasmic reticulum.[5c, 17l,m, 30] When a higher dosage (100 mm)
was used, the complex was localized in the perinuclear
region with negligible nuclear uptake. Most importantly,
HeLa cells incubated with the complex at a relatively high
concentration (200 mm) for 2 h still remained viable, and
staining of the perinuclear region with very high emission
intensity was observed. Interestingly, the stained HeLa cells
still survived after further incubation in a complex-free
growth medium. To gain more insight, we have treated four
HeLa cell cultures with complex 2 a (200 mm) for 2 h at various time points in three 24 h incubation periods. The cells
were washed with phosphate-buffered saline (PBS) after
each incubation period before returning to the culture
medium. We found that the emission intensity of the cells
reduced gradually with increasing incubation time in the
complex-free medium (Table 5) and the cell counts were not
much different from that of the control, which was not treat-
Table 4. Number of moles and concentrations of iridium associated with
an average HeLa cell upon incubation with the cyclometalated iridiumACHTUNGRE(III) complexes (5 mm) at 37 8C for 2 h, determined by ICPMS.
Complex
Number of moles of Ir [fmol]
Concentration of Ir [mm]
1a
1b
1c
2a
2b
2c
3
2.56 0.44
3.03 1.12
1.39 0.04
1.38 0.19
1.56 0.06
4.45 0.80
2.88 0.92
754 128
812 330
408 12
404 57
460 18
1308 236
848 267
Chem. Eur. J. 2010, 16, 8329 – 8339
Figure 2. Confocal microscopy images of HeLa cells incubated with complex 2 a at 10 mm for 1 h (left), 100 mm for 1 h (middle), and 200 mm for 2 h
(right) at 37 8C.
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K. K.-W. Lo et al.
Table 5. Flow cytometric analysis and cell counts of HeLa cell cultures
after treatment with complex 2 a (200 mm) for 2 h at various time points
in three incubation 24 h periods.[a]
Entry
Incubation
time before
treatment with
complex [h][b]
Incubation
time after
treatment with
complex [h][b]
Emission
Cell
intensity [a.u.][c] count [mL]
1
2
3
4
control[d]
72
48
24
0
NA[d]
0
24
48
72
NA[d]
74.85 26.40
17.17 6.06
10.04 4.22
9.93 4.16
3.18 1.44
1593 297
1599 23
1347 266
1296 90
1482 102
[a] Cells were washed with PBS after each 24 h incubation period. [b] Incubation in complex-free medium. [c] Data from flow cytometry.
[d] Cells were not treated with the complex; total incubation time = 74 h.
PEI ratio was determined to be 4.2 from the spectroscopic
data. Upon photoexcitation, the PEGylated PEI polymer
exhibited intense and long-lived green emission. On the
basis of the photophysical data (Table 2), we have assigned
the emission to a 3IL (p!p*) (N^C or N^N) state,[17g,l]
which is similar to that of the PEGylated BSA conjugate
3-BSA.
The ability of luminescent PEGylated polymer 3-PEI to
form polyplexes with pDNA has been investigated. The
polyACHTUNGREplex 3-PEI/pCMV-luc was prepared in different N/P
ratios (the number of nitrogen residues of PEI per DNA
phosphate; from 0.5 to 16.0) prior to analysis by agarose gel
electrophoresis (Figure 3). The results showed that the
ed with the complex (Table 5). These results indicate that
the internalized complex did not adversely interfere with
the cell-division process and was transported out of the
cells.[31] These findings also highlight the low cytotoxicity
and high biocompatibility of the PEG complexes, which are
remarkable advantages for live-cell imaging applications.
PEGylation and transfection studies: Because the aldehyde
moiety is reactive toward primary amines, complex 3 can
function as a novel luminescent PEGylation reagent. We
have PEGylated BSA with the complex to give the conjugate 3-BSA. On the basis of spectroscopic data and Bradford assays,[32] the iridium-to-protein ratio was determined
to be 2.3. The PEGylated conjugate 3-BSA was strongly
emissive in aqueous buffer under ambient conditions, and
the emission spectrum was characterized by a vibronically
structured band at 490 (max), 520 nm (Table 2). This, together with a very long lifetime ( 2.61 ms) suggests a 3IL
(p!p*) (N^C or N^N) emissive state.[17g,l]
Gene delivery applications that use PEI as a nonviral
vector have been well documented.[33–35] The polycationic
nature of PEI in aqueous media enables the formation of
polyplexes with plasmid DNA (pDNA), which can be efficiently delivered into eukaryotic cells. However, there are
few details about the intracellular departure of nucleic acid
from the polymer owing to a lack of reporting properties.
Also, because PEI is rather cytotoxic to many cell lines, it is
commonly PEGylated to lower its cytotoxicity and to facilitate the transfection applications.[36–39] In this work, we have
PEGylated PEI (MW = 25 kDa) with complex 3 (Scheme 2)
and isolated the luminescent polymer 3-PEI. The iridium-to-
Figure 3. Gel electrophoresis of polyplexes formed from 3-PEI and
pCMV-luc. The lane numbers correspond to different N/P ratios:
1) DNA only, 2) 3-PEI only, 3) 0.5, 4) 1.0, 5) 2.0, 6) 4.0, 7) 8.0, 8) 16.0.
pDNA band was retarded with increasing the N/P ratio, indicating that the negative charge of the plasmid was neutralized by the positively charged PEI derivative. At an N/P
ratio = 4.0 (lane 6 in Figure 3), the band was completely retarded, indicating the capacity of 3-PEI to condense DNA
effectively. The zeta potentials and mean hydrodynamic diameters of the 3-PEI/pCMV-luc polyplexes have also been
studied by using dynamic light scattering (Table 6). The zeta
potentials of the polyplexes changed from 32 to
+ 42 mV upon increasing the N/P ratio. At N/P 4.0, the
polyplex acquired a positive zeta potential, which is in accordance with the gel electrophoresis results (Figure 3). Interestingly, the hydrodynamic diameters of the polyplexes
formed were 490 to 540 nm with N/P being between 0.5
and 2.0 (Table 6). When N/P was 4.0, the polyplexes
Scheme 2. Synthetic scheme of 3-PEI.
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2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2010, 16, 8329 – 8339
Luminescent IrIII Polypyridine Complexes
Table 6. Zeta potentials and mean hydrodynamic diameters of 3-PEI/
pCMV-luc in Tris-Cl buffer (50 mm, pH 7.4) at various N/P ratios.
N/P ratio
Zeta potential [mV]
Mean hydrodynamic diameter [nm]
0.5
1.0
2.0
4.0
8.0
16.0
31.8 2.87
15.1 0.35
21.5 2.25
38.3 0.36
40.8 0.58
42.2 0.46
488.5 96.42
490.6 35.19
540.0 62.38
245.5 22.88
218.4 13.44
188.0 1.35
FULL PAPER
the polyplex 3-PEI/pCMV-luc (4 mg pCMV-luc; N/P = 4.0)
has been investigated by using conACHTUNGREfocal microscopy. The
images showed
extracellular luminescent granules
(Figure 5), which are probably condensed polyACHTUNGREplexes. However, some of these luminescent spots were located in the
cytoplasm surrounding the nuclei with a low but nonzero intensity inside the nucleus. All these results demonstrated
that complex 3 can retain the transfection properties of PEI,
and the emissive behavior and intracellular uptake of the
shrank to 190 to 250 nm in diameter, which supports the argument that the positively
charged 3-PEI condensed the
negative pDNA and formed a
compact polyplex at such N/P
ratios.[40, 41] We further assessed
the transfection efficacy of
PEGACHTUNGREylated 3-PEI by using
HeLa cells and the same
pDNA that expresses luciferase. Figure 5. Fluorescence (left), brightfield (middle), and overlaid (right) microscopy images of HeLa cells incubated with 3-PEI loaded pCMV-luc (4-mg p-CMV-luc; N/P = 4.0) at 378C for 5 h.
The polyplex 3-PEI/pCMV-luc
was prepared in the same N/P
ratios (from 0.5 to 16.0) as described above, and lipofectamine (Promega)/pCMV-luc and
polymer can be examined by emission spectroscopy and
the naked pDNA were used as a positive and negative conconfocal microscopy, respectively.
trol, respectively. The highest transfection efficacy occurred
at N/P = 4.0 and 8.0 (Figure 4), which is in agreement with
the gel electrophoresis and dynamic light scattering results.
Conclusion
The lower transfection efficacy at the highest N/P ratio we
studied (16.0) is most likely a result of extensive cell death
In summary, we have developed a new class of luminescent
caused by the cytotoxicity of PEI.[42–44] At lower N/P ratios
cyclometalated iridiumACHTUNGRE(III) polypyridine PEG complexes,
which showed very rich photophysical properties, high water
(0.5 to 2.0), the effect of the transfection reagent 3-PEI is
solubility, very low cytotoxicity, excellent biocompatibility,
less significant. Note that the optimal N/P ratio (4.0) is
and high cellular uptake efficiency. One of the complexes
lower than that of untreated PEI (10.0).[45] One possible
has been functionalized with amine-reactive aldehyde moieexplanation is that complex 3 provides additional positive
ACHTUNGREties, which can target proteins and amine-containing polycharges to the PEI molecule, lowering the N/P ratio remers. This renders the complex a luminescent PEGylation
quired for optimum transfection efficacy. However, the
reagent derived from transition-metal complexes, which is
effect originating from the biocompatible PEG pendants of
the first of its kind. Related work on luminescent inorganic
complex 3 should not be neglected. The cellular uptake of
and organometallic transition-metal PEG complexes with a
focus on biological applications is underway.
Experimental Section
Figure 4. Luciferase activity of 3-PEI/pCMV-luc at various N/P ratios in
HeLa cells. Lipofectamine/pCMV-luc and the naked pDNA were used as
a positive and negative control, respectively.
Chem. Eur. J. 2010, 16, 8329 – 8339
Materials, synthesis, and instrumentation: All solvents were of analytical
grade and purified according to standard procedures.[46] All buffer components were of biological grade and used as received. cis-Diamminedichloroplatinum (cisplatin), N,N’-dicyclohexylcarbodiimide, N-hydroxysuccinimide (NHS), KPF6, Na(CN)BH4, and triethylamine were obtained
from Acros. 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Sigma. Branched PEI (MW = 25 kDa),
Hpba, Hppy, Hpq, 4,4’-dimethyl-2,2’-bipyridine, and Ir3·3H2O were supplied by Aldrich. The PEGamines m-dPEG24amine and amino
dPEG4ACHTUNGRE(m-dPEG12)3 were purchased from Quanta Biodesign. BSA was
obtained from Calbiochem. PD-10 size-exclusion columns and YM-30 microcons were received from GE Healthcare and Amicon, respectively.
Autoclaved Milli-Q water was used for the preparation for the aqueous
2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
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K. K.-W. Lo et al.
solutions. BpyNHS,[47] bpyCONHEt,[15b, 48] the dichloro-bridged
dimers [Ir2ACHTUNGRE(ppy)4Cl2],[17a,c] [Ir2(pq)4Cl2],[17c] and [Ir2ACHTUNGRE(pba)4Cl2],[17b] and the
control complexes [IrACHTUNGRE(ppy)2(bpyCONHEt)]ACHTUNGRE(PF6) (1 c) and [IrACHTUNGRE(pq)2ACHTUNGRE(bpyCONHEt)]ACHTUNGRE(PF6) (2 c)[17h] were synthesized according to literature
procedures. HeLa cells were obtained from American Type Culture Collection. UltraPure agarose, Lipofectamine 2000 Reagent, Dulbeccos
modified Eagles medium (DMEM), reduced serum medium (OptiMEM), fetal bovine serum (FBS), phosphate-buffered saline at pH 7.2
(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. Tris(hydroxymethyl)methylACHTUNGREamine (Tris) from USB was used to prepare Tris-Cl (50 mm, pH 7.4).
Plasmid DNA pCMV-luc (6.8 kb) was amplified in E. coli and purified
by HiPure Filter Plasmid Kit, and the concentration of plasmid DNA was
measured spectrophotometrically. Luciferase assay kit was obtained from
Promega and stored at 708C before use. The instruments for characterization and photophysical studies have been described previously.[17j] The
methods by which determination of luminescence quantum yields,[48, 49]
the MTT assay,[50] ICPMS,[17j] and live-cell confocal imaging[17i,j] were undertaken have also been reported previously.
Synthesis of bpyCONHPEG1: A mixture of bpyNHS (57.2 mg,
183.9 mmol), m-dPEG24amine (100.0 mg, 91.9 mmol), and triethylamine
(45.0 mL, 323.0 mmol) in CH2Cl2 (20 mL) was stirred under an inert atmosphere of nitrogen at room temperature for 12 h. The solution was filtered and evaporated by rotary evaporation, leaving a colorless oil. The
crude product was purified by column chromatography on silica gel by
using CH2Cl2/MeOH/NH4OH (10:1:0.1, v/v/v) as the mobile phase. The
portions containing the product were evaporated in vacuo, leaving a colorless oil. Yield: 100.2 mg (85 %); 1H NMR (300 MHz, [D6]acetone,
298 K, TMS): d = 8.75 (d, J = 4.8 Hz, 1 H; H6 pyridyl ring), 8.67 (s, 1 H;
H3 pyridyl ring), 8.51 (d, J = 5.1 Hz, 1 H; H6’ pyridyl ring), 8.22 (s, 1 H;
H3’ pyridyl ring), 7.75 (d, J = 4.8 Hz, 1 H; H5 pyridyl ring), 7.43 (br s, 1 H;
bpy4-CONH), 7.14 (d, J = 5.1 Hz, 1 H; H5’ pyridyl ring), 3.67–3.48 (m,
96 H; OCH2, CONH-CH2), 3.34 (s, 3 H; OCH3), 2.41 ppm (s, 3 H; CH3
pyridyl ring); ESIMS: m/z: 1286 [M+H] + .
Synthesis of bpyCONHPEG3: The procedure was similar to that of
bpyCONHPEG1, except that aminodPEG4ACHTUNGRE(m-dPEG12)3 (200 mg,
90.5 mmol) was used instead of m-dPEG24amine. The desired product
was isolated as a colorless oil. Yield: 197.2 mg (91 %); 1H NMR
(300 MHz, [D6]acetone, 298 K, TMS): d = 8.78 (d, J = 5.1 Hz, 1 H; H6 pyridyl ring), 8.71 (s, 1 H; H3 pyridyl ring), 8.54 (d, J = 4.8 Hz, 1 H; H6’ pyridyl ring), 8.25 (s, 1 H; H3’ pyridyl ring), 7.79 (d, J = 5.1 Hz, 1 H; H5 pyridyl ring), 7.58 (br s, 1 H; bpy4-CONH), 7.18 (d, J = 4.8 Hz, 1 H; H5’ pyridyl ring), 6.88 (t, J = 5.1 Hz, 3 H; m-dPEG12CONH), 6.59 (s, 1 H;
dPEG4CONH), 3.68–3.51 (m, 166 H; OCH2), 3.43–3.42 (m, 8 H; CONHCH2), 3.37 (s, 9 H; OCH3), 2.45 (s, 3 H; CH3 pyridyl ring), 2.40 ppm (t,
J = 5.4 Hz, 8 H; CH2-CONH); ESIMS: m/z: 2406 [M+H] + .
[IrACHTUNGRE(ppy)2(bpyCONHPEG1)]ACHTUNGRE(PF6) (1 a): A mixture of [Ir2ACHTUNGRE(ppy)4Cl2]
(20.9 mg, 19.5 mmol) and bpyCONHPEG1 (50.0 mg, 38.9 mmol) in
MeOH/CH2Cl2 (30 mL, 1:1, v/v) was heated at reflux under an inert atmosphere of nitrogen for 4 h. The reaction mixture was then cooled to
room temperature and KPF6 (20.0 mg, 0.11 mmol) was added. The mixture was stirred for 30 min and was then evaporated under vacuum. The
residual orange oil was dissolved in CH2Cl2 and purified by column chromatography on silica gel. Subsequent recrystallization of the product
from CH2Cl2/diethyl ether afforded complex 1 a as an orange semi-solid.
Yield: 60.0 mg (80 %); 1H NMR (300 MHz, [D6]acetone, 298 K, TMS):
d = 9.08 (s, 1 H; H3 pyridyl ring bpyCONHPEG1), 8.80 (s, 1 H; H3’
pyridyl ring bpyCONHPEG1), 8.25–8.18 (m, 4 H; bpy4-CONH, H6
pyridyl ring bpyCONHPEG1, H3 pyridyl ring ppy), 8.01–7.80 (m, 8 H;
H5, H6’ pyridyl ring bpyCONHPEG1, H4, H6 pyridyl ring ppy, H3
phenyl ring ppy), 7.54 (d, J = 5.4 Hz, 1 H; H5’ pyridyl ring bpyCONH
PEG1), 7.18–7.15 (m, 2 H; H5 pyridyl ring ppy), 7.04–7.00 (m, 2 H; H4
phenyl ring ppy), 6.92–6.90 (m, 2 H; H5 phenyl ring ppy), 6.37–6.32 (m,
2 H; H6 phenyl ring ppy), 3.68–3.59 (m, 96 H; OCH2, CONH-CH2), 3.33
(s, 3 H; OCH3), 2.60 ppm (s, 3 H; CH3 pyridyl ring bpyCONHPEG1);
IR (KBr): n̄ = 3448 (NH), 1655 (C=O), 1109 (CO), 842 cm1 (PF6);
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www.chemeurj.org
HR ESI-TOF MS: m/z calcd for C83H125IrN5O25 : 1784.8293; found
1784.8266 [M] + , 911.8956 [M+K]2 + .
[IrACHTUNGRE(ppy)2(bpyCONHPEG3)]ACHTUNGRE(PF6) (1 b): The synthetic procedure was
similar to that of complex 1 a, except that bpyCOHNPEG3 (98 mg,
40.7 mmol) was used instead of bpyCONHPEG1. The complex was isolated as an orange semi-solid. Yield: 100.0 mg (81 %); 1H NMR
(300 MHz, [D6]acetone, 298 K, TMS): d = 9.13 (s, 1 H; H3 pyridyl ring
bpyCONHPEG3), 8.74 (s, 1 H; H3’ pyridyl ring bpyCONHPEG3),
8.25–8.21 (m, 3 H; H3 pyridyl ring ppy, H6 pyridyl ring bpyCONH
PEG3), 8.07 (br s, 1 H; bpy4-CONH), 8.00–7.80 (m, 8 H; H5, H6’ pyridyl
ring bpyCONHPEG3, H4, H6 pyridyl ring ppy, H3 phenyl ring ppy),
7.56 (m, 2 H; H5’ pyridyl ring bpyCONHPEG3, dPEG4CONH), 7.44
(br s, 3 H; m-dPEG12CONH), 7.20–7.13 (m, 2 H; H5 pyridyl ring ppy),
7.06–7.00 (m, 2 H; H4 phenyl ring ppy), 6.94–6.87 (m, 2 H; H5 phenyl
ring ppy), 6.35–6.29 (m, 2 H; H6 phenyl ring ppy), 3.68–3.51 (m, 166 H;
OCH2), 3.42–3.40 (m, 8 H; CONH-CH2), 3.32 (s, 9 H; OCH3), 2.62 (s,
3 H; CH3 pyridyl ring bpyCONHPEG3), 2.45 ppm (br s, 8 H; CH2CONH); IR (KBr): n̄ = 3432 (NH), 1655 (C=O), 1106 (CO), 845 cm1
(PF6); HR ESI-TOF MS: m/z calcd for C133H221IrN9O48 : 2906.4792;
found 1472.7218 [M+K]2 + , 994.7982 [M + 2K]3 + .
[Ir(pq)2(bpyCONHPEG1)]ACHTUNGRE(PF6) (2 a): The synthetic procedure was
similar to that of complex 1 a, except that [Ir2(pq)4Cl2] (24.8 mg,
19.5 mmol) was used instead of [Ir2ACHTUNGRE(ppy)4Cl2]. The complex was isolated
as an orange semi-solid. Yield: 65.0 mg (82 %); 1H NMR (300 MHz,
[D6]acetone, 298 K, TMS): d = 8.75 (s, 1 H; H3 pyridyl ring bpyCONH
PEG1), 8.54–8.53 (m, 4 H; H3 phenyl ring pq, H3 quinoline ring pq), 8.49
(s, 1 H; H3’ pyridyl ring bpyCONHPEG1), 8.47–8.45 (d, J = 5.7 Hz,
1 H; H6 pyridyl ring bpyCONHPEG1), 8.26 (d, J = 7.5 Hz, 2 H; H4
quinoline ring pq), 8.20–8.18 (m, 2 H; bpy4-CONH, H5 pyridyl ring
bpyCONHPEG1), 8.04–8.02 (m, 1 H; H6’ pyridyl ring bpyCONH
PEG1), 7.96–7.92 (m, 2 H; H8 quinoline ring pq), 7.57 (d, J = 4.5 Hz, 1 H;
H5’ pyridyl ring bpyCONHPEG1), 7.48–7.41 (m, 4 H; H5, H7 quinoline ring pq), 7.20–7.12 (m, 4 H; H4 phenyl ring pq, H6 quinoline ring
pq), 6.86–6.80 (m, 2 H; H5 phenyl ring pq), 6.58–6.52 (m, 2 H; H6 phenyl
ring pq), 3.69–3.58 (m, 96 H; OCH2, CONH-CH2), 3.32 (s, 3 H; OCH3),
2.50 ppm (s, 3 H; CH3 pyridyl ring bpyCONHPEG1); IR (KBr): n̄ =
3448 (NH), 1655 (C=O), 1109 (CO), 843 cm1 (s, PF6); HR ESI-TOF
MS: m/z calcd for C91H129IrN5O25 : 1884.8606; found 1884.8546 [M] + ,
961.9102 [M+K]2 + .
[Ir(pq)2(bpyCONHPEG3)]ACHTUNGRE(PF6) (2 b): The synthetic procedure was
similar to that of complex 2 a, except that bpyCOHNPEG3 (98 mg,
40.7 mmol) was used instead of bpyCONHPEG1. The complex was isolated as an orange semi-solid. Yield: 106.0 mg (80 %); 1H NMR
(300 MHz, [D6]acetone, 298 K, TMS): d = 8.75 (s, 1 H; H3 pyridyl ring
bpyCONHPEG3), 8.54 (m, 4 H; H3 phenyl ring pq, H3 quinoline ring
pq), 8.47–8.44 (m, 2 H; H3’, H6 pyridyl ring bpyCONHPEG3), 8.37
(br s, 1 H; bpy4-CONH), 8.26 (d, J = 6.6 Hz, 2 H; H4 quinoline ring pq),
8.18 (d, J = 5.7 Hz, 1 H; H5 pyridyl ring bpyCONHPEG3), 8.06–8.04
(m, 1 H; H6’ pyridyl ring bpyCONHPEG3), 7.97–7.92 (m, 2 H; H8
quinoline ring pq), 7.57 (d, J = 4.8 Hz, 1 H; H5’ pyridyl ring bpyCONH
PEG3), 7.49–7.38 (m, 7 H; m-dPEG12CONH, H5, H7 quinoline ring
pq), 7.20–7.15 (m, 4 H; H4 phenyl ring pq, H6 quinoline ring pq), 7.00 (s,
1 H; dPEG4CONH), 6.84–6.80 (m, 2 H; H5 phenyl ring pq), 6.57–6.52
(m, 2 H; H6 phenyl ring pq), 3.69–3.59 (m, 166 H; OCH2), 3.56–3.50 (m,
8 H; CONH-CH2), 3.30 (s, 9 H; OCH3), 2.50 (s, 3 H; CH3 pyridyl ring
bpyCONHPEG3), 2.50–2.39 ppm (m, 8 H; CH2-CONH); IR (KBr):
n̄ = 3432 (NH), 1655 (C=O), 1107 (CO), 845 cm1 (PF6); HR ESITOF MS: m/z calcd for C141H225IrN9O48 : 3006.5105; found 1522.7286
[M+K]2 + , 1028.1409 [M+2K]3 + .
[IrACHTUNGRE(pba)2(bpyCONHPEG1)]ACHTUNGRE(PF6) (3): The synthetic procedure was
similar to that of complex 1 a, except that [Ir2ACHTUNGRE(pba)4Cl2] (23.1 mg,
19.5 mmol) was used instead of [Ir2ACHTUNGRE(ppy)4Cl2]. The complex was isolated
as an orange semi-solid. Yield: 42.0 mg (55 %); 1H NMR (300 MHz,
CDCl3, 298 K, TMS): d = 9.70 (s, 2 H; CHO), 8.79 (s, 1 H; H3 pyridyl ring
bpyCONHPEG1), 8.55 (s, 1 H; H3’ pyridyl ring bpyCONHPEG1),
8.06–7.98 (m, 4 H; bpy4-CONH, H3 phenyl ring pba, H6 pyridyl ring
bpyCONHPEG1), 7.92–7.85 (m, 5 H; H3, H3’, H5, H5’, H6’ pyridyl
ring bpyCONHPEG1), 7.72–7.65 (m, 4 H; H3, H6 pyridyl ring pba),
2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2010, 16, 8329 – 8339
Luminescent IrIII Polypyridine Complexes
7.63–7.53 (m, 4 H; H4 phenyl ring pba, H5 pyridyl ring pba), 7.26–7.24
(m, 2 H; H4 pyridyl ring pba), 6.70 (d, J = 6.3 Hz, 2 H; H6 phenyl ring
pba), 3.69–3.55 (m, 96 H; OCH2, CONH-CH2), 3.35 (s, 3 H; OCH3),
2.61 ppm (s, 3 H; CH3 pyridyl ring bpyCONHPEG1); IR (KBr): n̄ =
3434 (NH), 1686 (C=O), 1107 (CO), 844 cm1 (s, PF6); HR ESI-TOF
MS: m/z calcd for C85H125IrN5O27: 1840.8191; found 1840.8159 [M] + ,
939.8861 [M+K]2 + .
Flow cytometry: HeLa cells were seeded at a density of 1 000 000 cells
per dish in five 60 mm cell culture dishes and incubated at 37 8C under a
5 % CO2 atmosphere for three 24 h periods. At a certain time point, the
cells of one of the dishes were incubated with a culture medium that contained complex 2 a (200 mm) for 2 h, followed by gentle washing with PBS
(1 mL 3) prior to further incubation in a complex-free medium. In addition, after each 24 h incubation period, the cells were washed with PBS
and then incubated in a complex-free medium. Finally, the cell layer was
trypsinized and was made 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) with excitation at 488 nm. The
number of cells analyzed for each sample was 9000 to 10 000.
Labeling of BSA with complex 3: The iridiumACHTUNGRE(III) aldehyde complex 3
(3.0 mg, 1.5 mmol) in deionized water (60 mL) was added to BSA
(15.0 mg, 0.227 mmol) that was dissolved in carbonate buffer (600 mL,
50 mm, pH 9.0). After the mixture was stirred slowly in the dark at room
temperature for 12 h, NaCNBH3 (43.4 mg, 0.69 mmol) in 1 m NaOH
(100 mL) was added to the solution. The yellow solution was stirred for
another 24 h in the dark at room temperature. The reaction mixture was
diluted to 2 mL with Tris-Cl buffer (50 mm, pH 7.4), which was then purified with a YM-30 centricon (Amicon) through successive washing with
Tris-Cl buffer (50 mm, pH 7.4) and exchanged into phosphate buffer
(50 mm, pH 7.4). The volume of the solution was finally reduced to
400 mL. The resulting conjugate 3-BSA was stored at 4 8C before use. The
dye-to-BSA ratio was determined to be 2.3 by the Bradford assay.
Labeling of PEI with complex 3: The iridiumACHTUNGRE(III) aldehyde complex 3
(2.6 mg, 1.3 mmol) in deionized water (60 mL) was added to branched PEI
(3.4 mg, 0.136 mmol) that was dissolved in carbonate buffer (600 mL,
50 mm, pH 9.0). After the mixture was stirred slowly in the dark at room
temperature for 12 h, NaCNBH3 (17.0 mg, 0.27 mmol) in 1 m NaOH
(100 mL) was added to the solution. The yellow solution was stirred for
another 24 h in the dark at room temperature. The reaction mixture was
diluted to 2 mL with Tris-Cl buffer (50 mm, pH 7.4), which was then purified with a YM-30 centricon (Amicon) via successive washing with TrisCl buffer (50 mm, pH 7.4) and exchanged into phosphate buffer (50 mm,
pH 7.4). The volume of the solution was finally reduced to 600 mL. The
resulting conjugate 3-PEI was stored at 4 8C before use. The dye-to-PEI
ratio was determined to be 4.2 on the basis of spectroscopic data.
Agarose gel electrophoresis retardation assays: The 3-PEI/pCMV-luc
polyACHTUNGREplexes at various N/P ratios (the number of nitrogen residues of PEI
per DNA phosphate, from 0.5 to 16.0) were prepared by mixing 3-PEI
and pCMV-luc at an appropriate ratio in an elution buffer. The pCMVluc and 3-PEI were used as a positive and negative control, respectively.
After incubation for 30 min at room temperature, the polyplexes were
analyzed by electrophoresis on a 0.9 % (w/v) agarose gel containing
ethidium bromide with Tris-acetate buffer at 100 V for 45 min. The gel
was visualized by using a Bio-Rad Gel Doc imager.
Zeta potentials and mean hydrodynamic diameter measurements: A mixture of the pCMV-luc (4 mg) and 3-PEI at various N/P ratios (from 0.5 to
16.0) in Tris-Cl buffer (80 mL, 50 mm, pH 7.4) was incubated for 30 min at
room temperature. The mixture was then diluted tenfold with the same
buffer before measurements. The zeta potential of the resulting polyplex
was measured by using Zetasizer Nano ZS (Malvern Instruments) with
the following specifications: sampling time, 10–20 s; medium viscosity,
1.0031 cP; dielectric constant, 80.4; temperature, 20 8C; beam mode
F(Ka) = 1.50 (Smoluchowsky). Particle size was determined with the following specifications: sampling time, 180 s; medium viscosity, 1.0031 cP;
refractive index (RI) medium, 1.330; RI particle, 1.450; temperature,
20 8C. All the experiments were carried out in triplicate to ascertain reproducibility.
Chem. Eur. J. 2010, 16, 8329 – 8339
FULL PAPER
In vitro transfection (luciferase assays): HeLa cells were seeded at a density of 100 000 cells per dish in a 35 mm cell culture dish and incubated
for 48 h at 37 8C under a 5 % CO2 atmosphere. The culture medium was
replaced with DMEM (2 mL) containing 10 % FBS 2 h prior to transfection. The transfection experiments were performed with 4 mg pCMV-luc.
At the time of transfection, the medium was replaced with Opti-MEM
(2 mL). 3-PEI/pCMV-luc polyplexes at various N/P ratios were then incubated with the cells for 5 h. The medium was replaced with fresh growth
medium (3 mL) and the cells were further incubated for 43 h. LipofectACHTUNGREamine/pCMV-luc polyplex and the naked pDNA were used as a positive
and negative control, respectively. After the incubation, the cells were
permeabilized with cell lysis buffer (200 mL) (Promega) with one freezethaw cycle. The luciferase activity in cell extracts was measured by using
a luciferase assay kit (Promega) on a microplate reader (BMG FLUOstar
OPTIMA) for an interval of 10 s. All the experiments were carried out in
triplicate to ascertain the reproducibility.
Acknowledgements
We thank The Hong Kong Research Grants Council (Project Nos. CityU
101908 and 102109) for financial support. S.P.-Y.L. and K.Y.Z. acknowledge the receipt of a Postgraduate Studentship administered by the City
University of Hong Kong. K.Y.Z. acknowledges the receipt of Research
Tuition Scholarship and an Outstanding Academic Performance Award
administered by the City University of Hong Kong.
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