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Emissive behavior, cytotoxic activity, cellular uptake, and PEGylation properties of new luminescent rhenium(I) polypyridine poly(ethylene glycol) complexes.
Article
pubs.acs.org/IC
Emissive Behavior, Cytotoxic Activity, Cellular Uptake, and
PEGylation Properties of New Luminescent Rhenium(I) Polypyridine
Poly(ethylene glycol) Complexes
Alex Wing-Tat Choi, Man-Wai Louie, Steve Po-Yam Li, Hua-Wei Liu, Bruce Ting-Ngok Chan,
* *
Tonlex Chun-Ying Lam, Alex Chun-Chi Lin, Shuk-Han Cheng, and Kenneth Kam-Wing Lo
Department of Biology and Chemistry, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong, P.R. China
*
S Supporting Information
ABSTRACT: We report here a new class of biological
reagents derived from luminescent rhenium(I) polypyridine
complexes modified with a poly(ethylene glycol) (PEG)
∧
pendant. The PEG-amine complexes [Re(N N)(CO) (py-
3
PEG-NH )](PF ) (py-PEG-NH = 3-amino-5-(N-(2-(ω-
2 6 2
methoxypoly(1-oxapropyl))ethyl)aminocarbonyl)pyridine,
∧
MW = 5000 Da, PDI < 1.08; N N = 1,10-phenanthro-
PEG PEG
line (phen) (1-PEG-NH ), 3,4,7,8-tetramethyl-1,10-phenan-
2
throline (Me -phen) (2-PEG-NH ), 4,7-diphenyl-1,10-phe-
4 2
nanthroline (Ph -phen) (3-PEG-NH )) and [Re(bpy-PEG)-
2 2
(CO) (py-NH )](PF ) (bpy-PEG = 4-(N-(2-(ω-
3 2 6
methoxypoly(1-oxapropyl))ethyl)aminocarbonyl)-4′-methyl-
2,2′-bipyridine; py-NH = 3-aminopyridine) (4-PEG-NH ) have been synthesized and characterized. The photophysical
2 2
properties,lipophilicity,watersolubility,cytotoxicactivity,andcellularuptakepropertiesofthesecomplexeshavebeencompared
∧
to those of their PEG-free counterparts [Re(N N)(CO) (py-Et-NH )](PF ) (py-Et-NH = 3-amino-5-(N-(ethyl)-
3 2 6 2
∧
aminocarbonyl)pyridine; N N = phen (1-Et-NH ), Me -phen (2-Et-NH ), Ph -phen (3-Et-NH )) and [Re(bpy-Et)-
2 4 2 2 2
(CO) (py-NH )](PF ) (bpy-Et = 4-(N-(ethyl)aminocarbonyl)-4′-methyl-2,2′-bipyridine) (4-Et-NH ). The PEG complexes
3 2 6 2
exhibited significantly higher water solubility and lower cytotoxicity (IC = 6.6 to 1152 μM) than their PEG-free counterparts
50
(IC =3.6to159μM),indicatingthatthecovalentattachmentofaPEGpendanttorhenium(I)polypyridinecomplexesisan
50
effective way to increase their biocompatibility. The amine complexes 1-PEG-NH −4-PEG-NH have been activated with
2 2
∧
thiophosgene to yield the isothiocyanate complexes [Re(N N)(CO) (py-PEG-NCS)](PF ) (py-PEG-NCS = 3-isothiocyanato-
3 6
5-(N-(2-(ω-methoxypoly(1-oxapropyl))ethyl)aminocarbonyl)pyridine; N ∧ N = phen (1-PEG-NCS), Me -phen (2-PEG-NCS),
4
Ph -phen(3-PEG-NCS)),and[Re(bpy-PEG)(CO) (py-NCS)](PF )(py-NCS=3-isothiocyanatopyridine)(4-PEG-NCS)asa
2 3 6
new class of luminescent PEGylation reagents. To examine their PEGylation properties, these isothiocyanate complexes have
∧
been reacted with a model substrate n-butylamine, resulting in the formation of the thiourea complexes [Re(N N)(CO) (py-
3
PEG-Bu)](PF ) (py-PEG-Bu = 3-n-butylthioureidyl-5-(N-(2-(ω-methoxypoly(1-oxapropyl))ethyl)aminocarbonyl)pyridine;
6
∧
N N = phen (1-PEG-Bu), Me -phen (2-PEG-Bu), Ph -phen (3-PEG-Bu)), and [Re(bpy-PEG)(CO) (py-Bu)](PF ) (py-Bu
4 2 3 6
= 3-n-butylthioureidylpyridine) (4-PEG-Bu). Additionally, bovine serum albumin (BSA) and poly(ethyleneimine) (PEI) have
been PEGylated with the isothiocyanate complexes to yield bioconjugates 1-PEG-BSA−4-PEG-BSA and 1-PEG-PEI−4-PEG-
PEI, respectively. Upon irradiation, all the PEGylated BSA and PEI conjugates exhibited intense and long-lived emission in
aqueous buffer under ambient conditions. The DNA-binding and polyplex-formation properties of conjugate 3-PEG-PEI have
been studied and compared with those of unmodified PEI. Furthermore, the in vivo toxicity of complex 3-PEG-NH and its
2
PEG-free counterpart 3-Et-NH has been investigated using zebrafish embryos as an animal model. Embryos treated with the
2
PEGcomplexathighconcentrationsrevealeddelayedhatching,whichhasbeenascribedtohypoxiaasaresultofadheringofthe
complex to the external surface of the chorion.
■
INTRODUCTION interactions with surfaces and other biological entities and
PEGylation is a covalent modification of proteins, peptides, display enhanced biodistribution, pharmacokinetics, and
antibody fragments, and drug molecules with poly(ethylene resistance to undesirable proteolysis.2 Additionally, PEG has
glycol) (PEG). This derivatization procedure significantly been used as a highly flexible spacer-arm for protein-
reduces the toxicity of the molecules without sacrificing their
specific biological or therapeutic properties.1 PEGylated Received: September6, 2012
molecules show reduced aggregation tendency and nonspecific
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Chart 1. Structures of Complexes 1-PEG-NH −4-PEG-NH , 1-Et-NH −4-Et-NH , 1-PEG-NCS−4-PEG-NCS, and 1-PEG-Bu−
2 2 2 2
4-PEG-Bu
conjugation3 and protein-cross-linking.4 Since PEGylation is PEG and the incorporation of related anticancer drugs into
suchausefulbioconjugationprocess,PEGylationreagentswith PEG-containing micelles,11there is anemerging interest inthe
a wide range of molecular weights and shapes, reactive use of transition metal PEG complexes in biological
functional groups for modification, and specific properties applications; for example, the luminescent platinum(II)
have been developed.5 Those reagents containing fluorescent complex [Pt(C ∧ N ∧ N-4-Ph-PEGm)]Cl has been designed as a
unitssuchasthedansylgroup,6BODIPY,7andfluorescein8are sensitive light-switch for proteins.12 G3- and G4-poly-
particularly useful in the modification of biological targets as (amidoamine)(PAMAM)dendrimershavealsobeenmodified
they not only confer the aforementioned properties but also with organorhenium CpRe(CO) units and PEG chains,
3
enable detection and quantitation of the targets by optical yielding immunological reagents for carbonyl metalloimmuno-
methods. assays.13 Additionally, folic acid has been conjugated to a
In fact, transition metal complexes have been modified with tricarbonylrhenium(I) unit with a PEG linker to examine its
PEGtoincreasetheiraqueoussolubility;forexample,thereisa receptor-mediated endocytotic uptake by A2780/AD cells.14
growing interest in using transition metal PEG complexes as Despite these studies, the biological applications of
soluble polymer-supported catalysts.9 Also, reports on the luminescent transition metal PEG complexes are still relatively
electro-optical applications of transition metal PEG complexes unexplored. Recently, we have reported a new class of
with intriguing emission properties have appeared.10 In luminescent iridium(III) polypyridine PEG complexes that
addition to the work on the modification of cisplatin with possess high water solubility and low cytotoxicity as new
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Table 1. Photophysical Data of Complexes 1-PEG-NH −4-PEG-NH , 1-Et-NH −4-Et-NH , 1-PEG-NCS−4-PEG-NCS, and 1-
2 2 2 2
PEG-Bu−4-PEG-Bu
complex medium(T/K) λ /nm τ/μs Φ k/s−1 k /s−1
em o em r nr
1-PEG-NH CHCl (298) 532 2.18 0.11 5.1×104 4.1×105
2 2 2
CHCN(298) 546 0.73 0.048 4.1×104 1.3×106
3
buffer(298)a 543 0.14 0.018 1.3×105 7.0×106
glass(77)b 508 10.13
2-PEG-NH CHCl (298) 491sh,511 16.05 0.29 1.8×104 4.4×104
2 2 2
CHCN(298) 487sh,516 12.9 0.22 1.7×104 6.1×104
3
buffer(298)a 487sh,512 0.85 0.013 1.5×104 1.2×105
glass(77)b 468(max),502,539sh 42.71(22%),150.57(78%)
3-PEG-NH CHCl (298) 545 6.24 0.26 4.2×104 1.2×105
2 2 2
CHCN(298) 559 2.32 0.061 2.6×104 4.1×105
3
buffer(298)a 563 0.61 0.026 4.3×104 1.6×106
glass(77)b 511,540sh 19.50
4-PEG-NH CHCl (298) 552 0.45 0.010 2.2×104 2.2×106
2 2 2
CHCN(298) 578 0.21 0.0041 2.0×104 4.7×106
3
buffer(298)a 580 0.07 0.0019 2.7×104 1.4×107
glass(77)b 517 5.24
1-Et-NH CHCl (298) 530 2.65 0.30 1.1×105 2.6×105
2 2 2
CHCN(298) 547 1.26 0.18 1.4×105 6.5×105
3
buffer(298)c 544 0.07 0.010 1.4×105 1.4×107
glass(77)b 502 11.19
2-Et-NH CHCl (298) 488sh,512 11.33 0.44 3.9×104 4.9×104
2 2 2
CHCN(298) 486sh,514 9.16 0.28 3.1×104 7.9×104
3
buffer(298)c 486sh,513 0.62 0.0087 1.4×104 1.6×106
glass(77)b 467(max),501,539sh 41.92(22%),152.17(78%)
3-Et-NH CHCl (298) 545 8.00 0.33 4.1×104 8.4×104
2 2 2
CHCN(298) 559 3.97 0.20 5.0×104 2.0×105
3
buffer(298)c 558 0.42 0.021 5.0×104 2.3×106
glass(77)b 510,533sh 20.49
4-Et-NH CHCl (298) 547 0.46 0.037 8.0×104 2.1×106
2 2 2
CHCN(298) 578 0.28 0.013 4.6×104 3.5×106
3
buffer(298)c 584 0.02 0.0015 7.5×104 5.0×107
glass(77)b 521 5.46
1-PEG-NCS CHCl (298) 525 2.90 0.26 9.0×104 2.6×105
2 2
CHCN(298) 540 1.88 0.072 3.8×104 4.9×105
3
glass(77)b 508 9.42
2-PEG-NCS CHCl (298) 486sh,507 13.68 0.28 2.1×104 5.3×104
2 2
CHCN(298) 490sh,510 12.08 0.14 1.2×104 7.1×104
3
glass(77)b 470(max),501,535sh 30.07(21%),111.52(79%)
3-PEG-NCS CHCl (298) 539 9.23 0.29 3.1×104 7.7×104
2 2
CHCN(298) 552 3.79 0.050 1.3×104 2.5×105
3
glass(77)b 513,538sh 20.36
4-PEG-NCS CHCl (298) 552 0.63 0.089 1.4×105 1.5×106
2 2
CHCN(298) 572 0.26 0.0050 1.9×104 3.8×106
3
glass(77)b 513 5.99
1-PEG-Bu CHCl (298) 527 3.20 0.28 8.8×104 2.3×105
2 2
CHCN(298) 541 1.68 0.056 3.3×104 5.6×105
3
buffer(298)a 542 1.19 0.043 3.6×104 8.0×105
glass(77)b 510 9.76
2-PEG-Bu CHCl (298) 486sh,511 13.40 0.39 2.9×104 4.6×104
2 2
CHCN(298) 482sh,513 9.73 0.18 1.9×104 8.4×104
3
buffer(298)a 487sh,513 4.21 0.079 1.9×104 2.2×105
glass(77)b 470(max),501,537sh 32.48(18%),146.15(82%)
3-PEG-Bu CHCl (298) 541 9.20 0.30 3.3×104 7.6×104
2 2
CHCN(298) 554 3.83 0.049 1.3×104 2.5×105
3
buffer(298)a 560 2.88 0.045 1.6×104 3.3×105
glass(77)b 516,538sh 19.33
4-PEG-Bu CHCl (298) 554 0.53 0.017 3.2×104 1.9×106
2 2
CHCN(298) 575 0.19 0.014 7.4×104 5.2×106
3
buffer(298)a 588 0.08 0.0060 7.5×104 1.2×107
glass(77)b 514 5.92
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Table 1. continued
a50mMpotassiumphosphatebufferatpH7.4.bInbutyronitrileglass.c50m■MpotassiumphosphatebufferatpH7.4containing10%methanol.
bioprobes and imaging reagents.15 Thus, we envisage that the RESULTS AND DISCUSSION
incorporation of a PEG pendent to rhenium(I) polypyridine
Design and Synthesis of Complexes. Regarding the
complexes would significantly increase their biocompatibility. design of the PEG complexes, different diimine ligands (phen,
Theadvantagesofrhenium(I)polypyridinecomplexesaretheir
Me -phen, Ph -phen, and bpy-PEG) have been used to
ease of emission color-tuning using different diimine ligands inve 4 stigate the 2 effects of the methyl, phenyl, and amide
and their long-lived excited states, which are useful in the
substituentsonvariousphysicalandbiologicalpropertiesofthe
development of multicolor probes for time-resolved applica-
tionssuchasfluorescencelifetimeimagingmicroscopy(FLIM). complexes. The PEG pendant was linked to the complexes via
the use of the monodentate py-PEG-NH and bidentate bpy-
Additionally, since the coordination chemistry of the group 7 2
PEG ligands, which were synthesized from the reactions of 3-
congeners rhenium and technetium is similar, the same set of
amino-5-carboxysuccinimidylpyridine (py-NHS-NH ) and 4-
polypyridine ligands can be coordinated to [Re(CO) ]+ and 2
3 carboxysuccinimidyl-4′-methyl-2,2′-bipyridine (bpy-NHS), re-
[99mTc(CO) ]+ cores to yield luminescent probes and radio-
3 spectively, with α-amino-ω-methoxypoly(ethylene glycol)
imagingreagentsand-pharmaceuticals,respectively.Herein,we
(mPEG -NH ). Complexes 1-PEG-NH −4-PEG-NH and
report the synthesis, characterization, and photophysical 5000 2 2 2
their PEG-free counterparts 1-Et-NH −4-Et-NH were
properties of a new class of luminescent rhenium(I) PEG- 2 2
∧
∧ obtained from the reaction of [Re(N N)(CO) (CH CN)]-
amine complexes [Re(N N)(CO) (py-PEG-NH )](PF ) (py- 3 3
PEG-NH = 3-amino-5-(N-(2-(ω-m 3 ethoxypoly(1 2 -oxapro 6 pyl))- (CF 3 SO 3 ) with the respective pyridine ligands, followed by
2 anionexchangewithKPF andpurification.Theisothiocyanate
ethyl)aminocarbonyl)pyridine, MW = 5000 Da, PDI < 6
∧ PEG PEG complexes 1-PEG-NCS−4-PEG-NCS were obtained from the
1.08; N N = 1,10-phenanthroline (phen) (1-PEG-NH ),
2 reaction of the amine complexes 1-PEG-NH −4-PEG-NH
3,4,7,8-tetramethyl-1,10-phenanthroline (Me -phen) (2-PEG- 2 2
4 withthiophosgeneinacetoneatroomtemperature.Toexamine
NH ), 4,7-diphenyl-1,10-phenanthroline (Ph -phen) (3-PEG-
2 2 the reactivity of the isothiocyanate complexes toward primary
NH )), and [Re(bpy-PEG)(CO) (py-NH )](PF ) (bpy-PEG
= 4 2 -(N-(2-(ω-methoxypoly 3 (1-oxa 2 propy 6 l))ethyl)- amines, they have been reacted with a model substrate, n-
aminocarbonyl)-4′-methyl-2,2′-bipyridine; py-NH = 3-amino- butylamine, yielding the thiourea complexes 1-PEG-Bu−4-
2 PEG-Bu. These complexes were purified by size exclusion
pyridine) (4-PEG-NH ) (Chart 1). The lipophilicity, water
2 chromatography and microfiltration. All the complexes have
solubility, cytotoxic activity, and cellular uptake properties of
been characterized by 1H NMR, IR spectroscopy, ESI-MS (or
these complexes have been compared to those of their PEG-
∧ MALDI-TOF-MS for the PEG complexes), and elemental
free counterparts [Re(N N)(CO) (py-Et-NH )](PF ) (py-Et-
3 2 6
∧ analysis (for the non-PEG complexes). The molecular weight
NH = 3-amino-5-(N-(ethyl)aminocarbonyl)pyridine; N N =
phen 2 (1-Et-NH ), Me -phen (2-Et-NH ), Ph -phen (3-Et- difference of complexes 1-PEG-NH 2 −3-PEG-NH 2 and the
2 4 2 2
NH 2 )) and [Re(bpy-Et)(CO) 3 (py-NH 2 )](PF 6 ) (bpy-Et = 4- ligand py-PEG-NH 2 , respectively, was found to be about 500
(N-(ethyl)aminocarbonyl)-4′-methyl-2,2′-bipyridine) (4-Et- DaintheMALDI-TOFmassspectra(SupportingInformation,
NH ) (Chart 1). The amine complexes 1-PEG-NH −4-PEG- Figure S1), which is in agreement with the mass of the
2 2 ∧
NH 2 have been activated with thiophosgene to yield the corresponding [Re(N N)(CO) 3 ] unit.
isothiocyanate complexes [Re(N ∧ N)(CO) (py-PEG-NCS)]- Electronic Absorption and Emission Properties. The
3
(PF ) (py-PEG-NCS = 3-isothiocyanato-5-(N-(2-(ω- electronic absorption spectral data of all the complexes are
6
methoxypoly(1-oxapropyl))ethyl)aminocarbonyl)pyridine; summarized in Supporting Information, Table S1. All the
N ∧ N = phen (1-PEG-NCS), Me -phen (2-PEG-NCS), Ph - complexes displayed intense absorption bands at about 250−
phen (3-PEG-NCS)) and [Re(b 4 py-PEG)(CO) (py-NCS)] 2 - 345 nm with extinction coefficients on the order of 104 dm3
(PF ) (py-NCS = 3-isothiocyanatopyridine) (4
3
-PEG-NCS) mol
−1
cm
−1,
which have been assigned to spin-allowed
(Ch 6 art 1) as a new class of luminescent PEGylation reagents. intraligand (1IL) (π → π*) (N ∧ N and pyridine ligands)
To examine their PEGylation properties, these isothiocyanate
transitions.16−26
The lower-energy absorption shoulders at
complexes have been reacted with a model substrate n-
about366−396nm,withextinctioncoefficientsontheorderof
butylamine, resulting in the formation of the thiourea
103dm3mol −1cm −1,havebeenassignedtospin-allowedmetal-
complexes [Re(N ∧ N)(CO) (py-PEG-Bu)](PF ) (py-PEG-Bu to-ligand charge-transfer (1MLCT) (dπ(Re) → π*(N ∧ N))
3 6
= 3-n-butylthioureidyl-5-(N-(2-(ω-methoxypoly(1- transitions.
oxapropyl))ethyl)aminocarbonyl)pyridine; N ∧ N = phen (1- Upon photoexcitation, all the complexes displayed intense
PEG-Bu),Me -phen(2-PEG-Bu),Ph -phen(3-PEG-Bu))and and long-lived green to orange emission. The photophysical
4 2
[Re(bpy-PEG)(CO) (py-Bu)](PF ) (py-Bu = 3-n-butylthiour- dataaresummarizedinTable1,andtheemissionspectraofthe
eidylpyridine) (4-PE 3 G-Bu) (Cha 6 rt 1). Additionally, bovine PEG-amine complexes 1-PEG-NH −3-PEG-NH in CH CN
2 2 3
serum albumin (BSA) and poly(ethyleneimine) (PEI) have at 298 K are shown in Figure 1. In fluid solutions at 298 K,
beenPEGylatedwiththeisothiocyanatecomplexestoyieldthe most of the complexes displayed reduced emission energy,
luminescent bioconjugates 1-PEG-BSA−4-PEG-BSA and 1- quantum yields, and lifetimes upon increasing the polarity of
PEG-PEI−4-PEG-PEI, respectively. The DNA-binding and the solvents. These findings, together with the dependence of
polyplex-formation properties of conjugate 3-PEG-PEI have the emission energy onthe π*orbital energy level ofthe N ∧ N
been studied and compared with those of unmodified PEI. ligands, point to an emissive state of 3MLCT (dπ(Re) →
Furthermore, the in vivo toxicity of complex 3-PEG-NH and π*(N ∧ N))character.Thestructuralfeaturesandlongemission
2
itsPEG-freecounterpart3-Et-NH hasbeeninvestigatedusing lifetimes of the Me -phen complexes in fluid solutions under
2 4
zebrafish embryos as an animal model. ambient conditions suggest the involvement of 3IL (π → π*)
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determined by the flask-shaking method, and the results are
listed in Table 2. We found that the lipophilicity of the
Table 2. Lipophilicity (log P ) and Water Solubility of
o/w
Complexes 1-PEG-NH −4-PEG-NH and 1-Et-NH −4-Et-
2 2 2
NH
2
complex logP watersolubility/mM
o/w
1-PEG-NH −1.70±0.04 47.5
2
2-PEG-NH −0.94±0.02 13.1
2
3-PEG-NH −0.87±0.05 39.7
2
4-PEG-NH −1.04±0.01 28.7
2
1-Et-NH 0.81±0.03 0.36
2
2-Et-NH 1.09±0.03 0.19
2
3-Et-NH 2.04±0.05 0.016
2
4-Et-NH 1.37±0.06 2.66
Figure1.Emissionspectraofcomplexes1-PEG-NH (green),2-PEG- 2
2
NH (blue),and 3-PEG-NH (red) in CHCNat 298 K.
2 2 3 complexes depended on the diimine ligands and followed the
order: Ph -phen > Me -phen ≈ bpy-amide > phen, which is in
2 4
accordance with the hydrophobic character ofthe ligands. The
(Me -phen) character in their emissive states. In low-temper-
4 PEG complexes 1-PEG-NH −4-PEG-NH (log P = −0.87
ature glass, the Me -phen complexes displayed even richer 2 2 o/w
structural features in 4 their emission spectra, as exemplified by to −1.70) exhibited substantially lower lipophilicity than their
PEG-free counterparts 1-Et-NH −4-Et-NH (log P = 0.81
that of complex 2-PEG-NH (Supporting Information, Figure 2 2 o/w
2 to 2.04), reflecting the highly hydrophilic nature of the PEG
S2).Also,double-exponentialdecaywasobserved,withshorter-
pendants. Importantly, the water solubility of the PEG-NH
andlonger-livedcomponentsofabout30to40and110to150 2
μs, respectively (Table 1), which have been attributed to complexes (from 13.1 to 47.5 mM) is significantly higher than
3MLCT (dπ(Re) → π*(Me -phen)) and 3IL (π → π*) (Me - that of the PEG-free analogues (from 0.02 to 2.7 mM) (Table
4 4 2).ThehighwatersolubilityofthePEGcomplexesisobviously
phen) emissive states. Interestingly, while the PEG pendants
did not significantly perturb the emission energy of the an advantage that would render the complexes useful reagents
rhenium−aminecomplexes,theemissionquantumyieldsofthe for various biological applications.
Cellular Uptake Properties and Live-Cell Confocal
PEG-amine complexes are lower than those of their PEG-free
Imaging. The cellular uptake properties of the PEG-amine
counterparts in CH Cl andCH CN. Itis conceivable that the
long and flexible P 2 EG 2 pendant 3 facilitates the nonradioactive complexes 1-PEG-NH 2 −4-PEG-NH 2 and their PEG-free
decayofthePEGcomplexes,asreflectedbythegenerallylarge counterparts 1-Et-NH 2 −4-Et-NH 2 have been studied by ICP-
MS. Upon incubation with the complexes at 37 °C for 3 h, an
k values of these complexes compared to the PEG-free
co nr mplexes (Table 1),26e which subsequently lead to lower average HeLa cell (volume = 3.4 pL) contained 0.09 to 2.99
fmol of rhenium (Table 3), which is comparable to related
emission quantum yields. Although complex 2-PEG-NH
2
displayed a lower emission quantum yield, unexpectedly, it
Table 3. Numbers of Moles of Rhenium(I) Associated with
exhibited a longer emission lifetime than its PEG-free
counterpart 2-Et-NH , which may be related to the 3IL an Average HeLa Cell upon Incubation with Complexes 1-
characteroftheemissi 2 vestates.Inaqueousbuffer,allthePEG- PEG-N a H 2 −4-PEG-NH 2 and 1-Et-NH 2 −4-Et-NH 2 at 37 °C
for 3 h
amine complexes displayed smaller k values and higher
nr
emission quantum yields than their PEG-free counterparts
complex no.ofmol/fmol concentration/mM
(Table 1). We have tentatively attributed this observation to
1-PEG-NH 0.21±0.03 0.06±0.008
the self-wrapping of the rhenium(I)-diimine core by the PEG 2
2-PEG-NH 0.37±0.02 0.11±0.006
chain, which is expected to shield the luminophore from 2
interacting with the water molecules and buffer ions, resulting 3-PEG-NH 2 0.40±0.01 0.12±0.003
in less efficient nonradiative decay and an increased emission
4-PEG-NH
2
0.09±0.01 0.03±0.001
quantum yield. Similar findings have also been observed in
1-Et-NH
2
0.27±0.03 0.08±0.009
2-Et-NH 0.82±0.02 0.24±0.006
related rhenium(I) polypyridine complexes modified with long 2
3-Et-NH 2.99±0.07 0.88±0.020
aliphatic chains.27 It is noteworthy that the isothiocyanate 4-Et-NH 2 0.12±0.03 0.04±0.009
complexes1-PEG-NCS−4-PEG-NCSemittedathigherenergy 2
than their amine 1-PEG-NH −4-PEG-NH and thiourea 1- a[Re] = 10 μM in the incubation medium.
2 2
PEG-Bu−4-PEG-Bucounterparts.Wehaveascribedthistothe
strongly electron-withdrawing isothiocyanate group, which rhenium(I) complexes in other studies such as [Re(phen)-
stabilizes the dπ(Re) orbitals and hence causes higher (CO) (py-TU-DPAT)](CF SO ) (py-TU-DPAT = 3-(2-(4-
3 3 3
3MLCT emission energy. Nonetheless, the energy difference hydroxy-3-(2,2′-dipicolylaminomethyl)phenyl)-
issmallbecausetheelectrondensityoftherhenium(I)centeris ethylthioureidyl)pyridine) (2.8 fmol of rhenium)24 and [Re-
only remotely influenced by the substituents on the pyridine (phen)(CO) (py-3-glu)](PF ) (py-3-glu = 3-(N-(6-(N′-(4-(α-
3 6
ligand. D-glucopyranosyl)phenyl)thioureidyl)hexyl)thioureidyl)-
LipophilicityandWaterSolubility.Thelipophilicity(log pyridine) (1.10 fmol of rhenium).26i The PEG-amine
P ) of the PEG-amine complexes 1-PEG-NH −4-PEG-NH complexes displayed less effective cellular uptake than their
o/w 2 2
andtheirPEG-freecounterparts1-Et-NH −4-Et-NH hasbeen PEG-free counterparts, which is probably due to their lower
2 2
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Inorganic Chemistry Article
Figure2.Laser-scanningconfocalmicroscopyimagesofHeLacellsincubatedwithcomplexes3-PEG-NH (toprow)and3-Et-NH (bottomrow)
2 2
(10μM) at 37°C for 1 h.
Figure3.Laser-scanningconfocalmicroscopyimagesofHeLacellstreatedwithcomplex3-PEG-NH and(a)MitoTrackerDeepRedFMand(b)
2
Alexa Fluor633-conjugated transferrin at 37 °C, respectively.
lipophilicity and larger molecular size. It is important to point were effectively internalized and localized in the cytoplasmic
out that the intracellular rhenium concentrations of all the region(Figure 2).Thus, the PEGpendantofcomplex3-PEG-
complexes (0.03 to 0.88 mM) were much higher than that in NH did not significantly affect its intracellular localization
2
the medium before the uptake (10 μM), indicative of cellular properties. Also, the nuclei showed much weaker or no
accumulation of the complexes. emission, indicative of negligible nuclear uptake. HeLa cells
ThebiologicalpropertiesofthePh -phencomplexes3-PEG- incubated with complex 3-PEG-NH emitted weaker than
2 2
NH and 3-Et-NH have been studied in more detail. The those loaded with complex 3-Et-NH , which is a result of the
2 2 2
laser-scanning confocal microscopy images of HeLa cells loweruptakeefficiencyoftheformercomplex.Toexaminethe
treated with these complexes (10 μM, 1 h) showed that they intracellular localization properties of the PEG complexes,
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Inorganic Chemistry Article
HeLacellshavebeenco-stainedwithcomplex3-PEG-NH and
2
MitoTracker Deep Red FM (a mitochondrial marker) and
AlexaFluor633-conjugatedtransferrin(anendosomalmarker),
respectively. The images showed that the complex was co-
localized with the mitochondrial and endosomal markers with
co-localization coefficients of 71.6% and 18.2%, respectively,
suggesting that the PEG complex 3-PEG-NH is enriched in
2
the mitochondria (Figure 3). Similar mitochondria-targeting
behavior has been observed in other rhenium(I) polypyridine
complexes.26j
Cytotoxicity.ThecytotoxicityofthePEG-aminecomplexes
1-PEG-NH −4-PEG-NH and their PEG-free counterparts 1-
2 2
Et-NH −4-Et-NH toward HeLa cells over an incubation
2 2
periodof48hhavebeeninvestigatedbytheMTTassay(Table
4). In both series of complexes, the Ph -phen and Me -phen
2 4
Table 4. Cytotoxicity (IC , 48 h) of Complexes 1-PEG-
NH −4-PEG-NH and 1-E 50 t-NH −4-Et-NH and Cisplatin Figure 4. Electronic absorption (red) and emission (blue) spectra of
2 2 2 2 bioconjugate 1-PEG-BSA in degassed 50 mM potassium phosphate
toward HeLa Cells bufferat pH 7.4 at 298 K.
complex IC /μM
50 3.8, respectively. Upon photoexcitation, all the bioconjugates
1-PEG-NH 26.3±1.6
2 showed intense and long-lived green to orange-yellow
2-PEG-NH 2 11.9±1.6 3MLCT/3IL emission in 50 mM potassium phosphate buffer
3-PEG-NH 6.6±0.4
2 pH 7.4 at 298 K (Table 5). The emission spectrum of
4-PEG-NH >1151.7
2
1-Et-NH 15.0±4.8
2-Et-NH 2 5.0±0.4 Table 5. Photophysical Data of Bioconjugates 1-PEG-BSA−
3-Et-NH 2 3.6±0.4 4-PEG-BSA and 1-PEG-PEI−4-PEG-PEI in Degassed 50
4-Et-NH 2 159.1±8.0 mM Potassium Phosphate Buffer at pH 7.4 at 298 K
2
cisplatin 11.9±0.3 bioconjugate λ /nm τ/μs Φ
em o em
1-PEG-BSA 541 0.58(39%),1.29(61%) 0.018
complexesshowedhighercytotoxicity,whichhasbeenascribed 2-PEG-BSA 485sh,516 3.26(23%),10.78(77%) 0.041
to their higher lipophilicity and uptake efficiency. Similar 3-PEG-BSA 562 1.66(26%),4.70(74%) 0.013
observations have been reported in related studies.26i,j We also 4-PEG-BSA 566 0.10(18%),0.41(82%) 0.0071
noted that the PEG complexes were less cytotoxic than their 1-PEG-PEI 552 0.96 0.014
PEG-free counterparts, which has been attributed to the long 2-PEG-PEI 484sh,520 3.89 0.012
and flexible PEG pendants which prevent the complexes from 3-PEG-PEI 565 1.86 0.0045
interacting nonspecifically with extracellular proteins and 4-PEG-PEI 584 0.08 0.0022
triggering immunogenicity and antigenicity inside the cells.28
Remarkably, the bpy-PEG complex 4-PEG-NH displayed bioconjugate 1-PEG-BSA is shown in Figure 4. While the
2
extraordinarily low cytotoxicity, with an IC value of >1152 emission wavelengths of bioconjugates 1-PEG-BSA−3-PEG-
50
μM, which is at least 7 times lower than that of its PEG-free BSA were similar to those of their thiourea counterparts 1-
counterpart4-Et-NH (IC =159.1 μM).Apparently, thelow PEG-Bu−3-PEG-Bu, respectively, bioconjugate 4-PEG-BSA
2 50
lipophilicityanduptakeefficiencyofthiscomplexalonecannot emitted at higher energy than 4-PEG-Bu (Table 1). This
account for this result. One possible reason is that the PEG observation may originate from the hydrophobic nature of the
∧
pendant of this complex is linked to the diimine ligand, which protein surface since the emission of common [Re(N N)-
may provide more effective wrapping of the complex, resulting (CO) (py)]+ complexes occurs at higher energy in more
3
in much enhanced biocompatibility. nonpolar solvents, with higher environment-sensitivity of the
PEGylationofBSAandPEI.ToevaluatetheirPEGylation bpy-amideligandcomparedtotheotherdiimineligands(phen,
properties,theisothiocyanatecomplexes1-PEG-NCS−4-PEG- Me -phen, and Ph -phen).26e,g Another interesting observation
4 2
NCS have been used to label a model protein, BSA, via the is that all the bioconjugates displayed biexponential decay
reactionoftheisothiocyanategroupwiththeprimaryaminesof (Table 5), which is common for biomolecules labeled with
the lysine residues. The resultant bioconjugates 1-PEG-BSA− luminescent transition metal complexes.25a,b,29 It is noteworthy
4-PEG-BSA were purified by size exclusion chromatography that the average emission lifetimes of bioconjugates 1-PEG-
and ultrafiltration. The electronic absorption spectra of the BSA−4-PEG-BSA (from 0.39 to 10.16 μs) are longer than
bioconjugates have been measured and that of bioconjugate 1- those of the thiourea complexes 1-PEG-Bu−4-PEG-Bu (from
PEG-BSA is shown in Figure 4 as an example. The spectra 0.08 to 4.21 μs) as a result of the more hydrophobic and rigid
displayed an intense absorption band at 280 nm, which is local environment associated with the protein molecules.
attributed to both the protein and the complexes, and a The use of polyamines in gene delivery and cancer therapy
shoulder at about 328−371 nm, which is solely due to the has attracted much attention. In this context, PEI has been
rhenium(I) complexes. On the basis of the spectroscopic data, commonly used as a DNA condensing and gene delivery
therhenium-to-proteinratiosofbioconjugates1-PEG-BSA−4- reagent because of its high positive charge density and high
PEG-BSA have been determined to be about 2.2, 2.7, 2.9, and proton buffer capacity over a wide pH range.30 Since PEI does
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Inorganic Chemistry Article
not absorbor emitin the UV−vis region, it has beenmodified
with various reporters so that its cellular uptake and gene
delivery properties can be readily investigated by fluorescence
methods; for example, PEI has been conjugated with
fluorescent organic dyes and phosphorescent transition metal
complexes to study the cellular uptake and intracellular
transport pathways of PEI/DNA polyplexes.15,31 Also, since
PEI is cytotoxic to many cell types, it is commonly PEGylated
to lower its cytotoxicity, to increase the water solubility of the
PEI/DNA polyplexes, and to facilitate transfection applica-
tions.32Inthiswork,wehavePEGylatedPEI(25kDa)withthe
isothiocyanate complexes 1-PEG-NCS−4-PEG-NCS to gen-
erateanewclassofluminescentvectorsfortransfectionstudies. Figure5.Gelelectrophoresisofpolyplexes3-PEG-PEI/pRL-TKand
The resultant conjugates 1-PEG-PEI−4-PEG-PEI were PEI/pRL-TK with different N/P ratios. Lane 1: DNA only; Lane 2:
purified by size exclusion chromatography and ultrafiltration. conjugate3-PEG-PEIonly;Lane3to8:polyplexes3-PEG-PEI/pRL-
On the basis of the spectroscopic data, the rhenium-to-PEI TKwithN/Pratios=1,2,4,8,16,and32,respectively;Lane9:DNA
ratios of conjugates 1-PEG-PEI−4-PEG-PEI have been only;Lane10:PEIonly;Lane11to16:polyplexesPEI/pRL-TKwith
N/P ratios = 1, 2,4, 8, 16, and 32, respectively.
determined to be about 21.6, 13.6, 8.4, and 13.7, respectively.
These are larger than those of the BSA conjugates 1-PEG-
BSA−4-PEG-BSA (vide supra), most likely because of the
highernumberofprimaryaminesavailableonthePEIpolymer. ratio arrives at 8. This is in agreement with the gel
Upon photoexcitation, all the PEI conjugates displayed long- electrophoresis results since the polyplexes at N/P ratios ≥8
lived green to orange-yellow 3MLCT/3IL emission in 50 mM or 4 were retarded in the agarose gel for 3-PEG-PEI/pRL-TK
potassium phosphate buffer pH 7.4 at 298 K. The emission and unmodified PEI/pRL-TK, respectively (Figure 5). The
maxima of these PEI conjugates (520−584 nm) occurred at hydrodynamic diameters of the polyplexes 3-PEG-PEI/pRL-
slightly lower energy than their BSA counterparts (Table 5). TK increased from about 260 to 390 nm upon increasing the
These results illustrate the highly polar nature of the N/P ratiofrom1to8,anddecreasedat higherN/Pratios(16
protonated amine groups of the PEI moiety in an aqueous and 32). Similarly, the size of unmodified PEI/pRL-TK
environment. Interestingly, these findings are also in increased from 226 to 816 nm (with N/P ratios from 1 to 4)
accordance with the shorter emission lifetimes and lower and subsequently decreased to 249 nm at N/P ratio = 32.
quantum yields of the PEI conjugates compared to both the These observations support the argument that the positively
corresponding BSA conjugates (Table 5) and the thiourea charged PEI conjugate condensed the negative pDNA
complexes1-PEG-Bu−4-PEG-Bu(Table1).Thus,inaqueous efficiently and formed more compact polyplexes at N/P ratios
buffer,therhenium(I)polypyridinePEGcomplexesexperience ≥8 and 4, respectively for the two series of polyplexes. Similar
themosthydrophobicenvironmentonBSAmolecule,whereas expansion and shrinking of hydrodynamic diameters are
the polycationic nature of the PEI polymer would offer the commonly observed in other amphiphile/DNA polyplexes.33
most hydrophilic surroundings. This also highlights the Interestingly, the increase is less prominent for the polyplexes
environment-sensitive emission properties of this class of 3-PEG-PEI/pRL-TKthantheunmodifiedPEI/pRL-TK,which
complexes, which render them useful reporters of their local isattributabletothefactthataggregationofthepolyplexescan
environments. be prevented by PEG pendants.
DNA-Binding and Transfection Properties. We have The in vitro transfection efficiency of conjugate 3-PEG-PEI
investigated the DNA-binding properties of conjugate 3-PEG- has been examined using HeLa cells and the plasmid pRL-TK
PEIbyagarosegelelectrophoresis.Polyplexesformedfromthis that expresses luciferase via formation of polyplexes with N/P
conjugateandthepDNApRL-TKwithN/Pratiosfrom1to32 ratios from 1 to 32. Lipofactamine/pRL-TK and naked pDNA
have been prepared prior to the analysis. Polyplexes prepared were used as a positive and negative control, respectively, and
with unmodified PEI and pRL-TK were used as a control. As theresultsareshowninFigure6.Thetransfectionefficiencyof
shown in Figure 5, conjugate 3-PEG-PEI retarded the pDNA polyplexes 3-PEG-PEI/pRL-TK and unmodified PEI/pRL-TK
withincreasingN/Pratios,revealingthatthepositivelycharged wasthehighestatN/Pratios=16and8,respectively,whichis
PEI conjugate neutralizes the negative charge of the pDNA. generallyinlinewiththegelelectrophoresisresults(Figure5).
The migration of DNA bands of polyplexes 3-PEG-PEI/pRL- Although the transfection efficiency of conjugate 3-PEG-PEI
TKandPEI/pRL-TKwascompletelyretardedatN/Pratios= was weaker than PEI at N/P ratio = 8, its transfection ability
8 and 4, respectively (Figure 5). This result illustrates that the washigherathigherN/Pratios, thatis,16and32.Actually,at
DNA condensation ability of PEI was reduced upon N/P=32,thetransfectionabilityofunmodifiedPEIwasmuch
conjugation with the PEG complex. lower. All these observations can be ascribed to the lower
Thezetapotentialsandmeanhydrodynamicdiametersofthe cytotoxicity of conjugate 3-PEG-PEI due to the PEG units.
polyplexes 3-PEG-PEI/pRL-TK and unmodified PEI/pRL-TK Additionally, the intracellular localization of the polyplex 3-
have been studied by dynamic light scattering. The polyplexes PEG-PEI/pRL-TK (4 μg pDNA; N/P = 16) has been
3-PEG-PEI/pRL-TK displayed negative zeta potentials from investigated by laser-scanning confocal microscopy. HeLa
−31.8to−1.0mVwithN/Pratiosfrom1to8,andacquireda cells treated with the polyplex showed punctate cytoplasmic
significant increase of zeta potentials to +1.1 and +5.8 mV at staining with negligible nuclear uptake (Figure 7). All these
N/P ratios = 16 and 32, respectively (Table 6). The trend resultsdemonstratedthatthetransfectionpropertiesofPEIare
observed for the unmodified PEI/pRL-TK is similar, except retained after PEGylation by complex 3-PEG-NCS and the
thatapositivezetapotential(+3.4mV)appearswhentheN/P intracellular localization properties of the PEGylated PEI
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Inorganic Chemistry Article
Table 6. Zeta Potentials and Mean Hydrodynamic Diameters of Polyplexes 3-PEG-PEI/pRL-TK and PEI/pRL-TK with
Different N/P Ratios in Tris-Cl Buffer (50 mM, pH = 7.4)
zetapotential/mV meanhydrodynamicdiameter/nm
N/Pratio 3-PEG-PEI/pRL-TK PEI/pRL-TK 3-PEG-PEI/pRL-TK PEI/pRL-TK
1 −31.8±1.8 −33.3±8.5 259.9±5.3 225.5±12.8
2 −30.1±0.2 −25.5±1.7 265.2±42.9 234.2±3.6
4 −24.5±0.4 −7.8±2.0 352.9±10.7 815.5±148.6
8 −1.0±0.2 +3.4±2.1 389.8±20.3 713.0±64.3
16 +1.1±0.2 +28.3±1.7 291.0±43.8 234.9±16.4
32 +5.8±0.4 +37.9±2.3 207.1±25.9 248.5±20.9
complexes 3-PEG-NH and 3-Et-NH , and the results are
2 2
shown in Figure 8. The hatching percent was calculated from
thetotalnumberofhatchedlarvaerelativetothetotalnumber
of surviving embryos and larvae at each concentration. In the
control experiments without anyrhenium(I)complexes added,
about 60% of the embryos hatched in 72 hpf and over 95% of
them in 96 hpf. It is noteworthy that the embryos incubated
with the complexes at lower concentrations (from 0.31 to 5
μM) showed a similar hatching rate to the controls. However,
at highercomplex concentrations (10 and20μM),whilenone
of the embryos incubated with complex 3-Et-NH hatched
2
successfully,mostofthoseincubatedwiththePEGcomplex3-
PEG-NH hatched but with a delayed hatching time. The
2
hatchingpercentageoftheembryosat[3-PEG-NH ]=10μM
2
Figure 6. Luciferase activity (RLU/mg of protein) of HeLa cells wasonlyabout20%in72hpfand80%in120hpf.At[3-PEG-
incubatedwithpolyplexes3-PEG-PEI/pRL-TK(red)andunmodified
NH ] = 20 μM, the hatching of the embryos was suppressed
PEI/pRL-TK (blue) with different N/P ratios. Lipofectamine/pRL- 2
significantly, and less than 60% of the embryos hatched in 120
TK (orange) and naked DNA (green) were used as a positive and
negativecontrol, respectively. hpf. It is likely that the delayed hatching is due to hypoxia
because the PEG complex was found to adhere to the external
surface of the chorion upon incubation, as revealed by the
conjugate can be readily studied by optical spectroscopy and fluorescenceandbrightfieldmicroscopyimagesinFigure9(top
microscopy.
InVivoToxicity.Wehaveselectedzebrafishembryosasan row), which is likely to interfere with oxygen and nutrients
animal model to examine the effects of the PEG pendants on exchange. Related studies have also shown that hypoxia
the in vivo toxicity of the complexes. Selected 4 hours post significantly affects embryonic development; for example,
fertilization (hpf) embryos were exposed to the PEG complex sand snail (Polinices sordidus) displays delayed hatching under
3-PEG-NH and its PEG-free counterpart 3-Et-NH , respec- hypoxic conditions.34 Similar hatching delay has also been
2 2
tively, and the LD 50 values have been determined at five observed for the zebrafish embryos incubated with single-wall
incubationtimepoints(24,48,72,96,and120hpf).TheLD50 carbon nanotubes.35 It is important to point out that although
values for both complexes did not vary much in first 4 time
the PEG complex caused delayed hatching, all the successfully
points(24to96hpf)(Table7).Asexpected,thePEGcomplex
hatched larvae did not show any defects. These results further
exhibited higher LD values than its PEG-free counterpart at
all time points stud 5 i 0 ed, indicating that modification of the support that modification of the rhenium complex with a PEG
complex with a PEG unit reduces its in vivo toxicity. We have unit is an efficient method to lower its in vivo toxicity and
studied the hatching rates of the embryos incubated with enhance its biocompatibility.
Figure7.Fluorescence(left),overlaid(middle),andbrightfield(right)microscopyimagesofHeLacellsincubatedwiththepolyplex3-PEG-PEI/
pRL-TK (4 μg pDNA, N/P = 16)at 37 °C for 5h.
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Inorganic Chemistry Article
Table 7. In Vivo Toxicity of Complexes 3-PEG-NH and 3-Et-NH toward Zebrafish Embryos at Different Incubation Time
2 2
Points
LD atdifferentincubationtimepoints/μM
50
complex 24hpf 48hpf 72hpf 96hpf 120hpf
3-PEG-NH >40 >40 >40 >40 20.7±3.0
2
3-Et-NH 5.4±0.3 5.4±0.3 5.4±0.3 5.4±0.3 5.3±0.2
2
Figure9.Brightfield(left)andfluorescence(right)microscopyimages
of24-hpfzebrafishembryosincubatedwithcomplex3-PEG-NH (20
2
μM)at28.5°Cfor20h(toprow).Bottomrow:acontrolexperiment
in whichthe embryowas nottreated with the complex.
the transfection properties of conjugate 3-PEG-PEI have been
investigatedbyluciferaseassays,andtheresultsshowedthatthe
transfection properties of PEI are retained after PEGylation by
complex 3-PEG-NCS. Thus, conjugate 3-PEG-PEI serves as a
Figure 8. Hatching efficiency of zebrafish embryos after exposure to novel phosphorescent transfection reagent for eukaryotic cells,
complexes3-PEG-NH 2 (top)and3-Et-NH 2 (bottom),respectively,at which are expected to offer new insights in understanding the
72(red), 96(blue), and 120 (green) hpf at 28.5°C. cellular uptake, intracellular trafficking, and DNA-delivery
■ properties of PEI molecules.
Compared with their iridium(III) counterparts,15 the effect
CONCLUSION
of the appended PEG unit on the biocompatibility of the
In this work, a series of luminescent rhenium(I) polypyridine luminescent rhenium(I) PEG complexes in this work is less
PEG-amine complexes 1-PEG-NH −4-PEG-NH has been significant; for example, modification of the monodentate
2 2
synthesized and characterized. Upon photoexcitation, these pyridineligandwithaPEGchaindidnotsubstantiallylowerthe
complexes displayed intense and long-lived 3MLCT/3IL cytotoxicity of the rhenium(I) complexes, which we believe is
emission. The PEG complexes exhibited increased water due to the less effective coverage of the complex by the PEG.
solubility and biocompatibility compared to their PEG-free Unlike the iridium(III) system, where the attached PEG
counterparts 1-Et-NH −4-Et-NH . The amine group of pendant alters the emissive state nature of the complexes, the
2 2
complexes 1-PEG-NH −4-PEG-NH was activated by thio- rhenium(I) PEG complexes in this work emit at very similar
2 2
phosgene to yield the amine-specific PEGylation reagents 1- energytotheirPEG-freecounterparts.However,bothclassesof
PEG-NCS−4-PEG-NCS, which have been used to label n- complexesrevealincreasedemissionquantumyieldsinaqueous
butylamine, BSA, and PEI, respectively. All the resultant buffer solutions, which is a consequence of the protection of
conjugateshavebeenisolated,purified,andtheirphotophysical excitedcomplexesthroughwrappingbythePEGunits.Itisalso
propertieshavebeeninvestigated.TheDNA-bindingproperties obvious that the attachment of PEG to these transition metal
of the PEI conjugate 3-PEG-PEI have been studied, and complexes has substantially increased their water solubility,
polyplexes formed from this conjugate and pDNA with which is an important requirement for the design of biological
differentN/Pratioshavealsobeencharacterized.Furthermore, probes. Related work on phosphorescent inorganic and
J dx.doi.org/10.1021/ic301948d|Inorg.Chem.XXXX,XXX,XXX−XXX
Inorganic Chemistry Article
organometallic transition metal PEG and PEI complexes is in 7.5Hz,NHCHCH).Positive-ionESI-MSionclusteratm/z166{M
2 3
progress. + H+}+.
■
Bpy-PEG.Bpy-NHS(94mg,0.40mmol)wasdissolvedinhotDMF
(20mL)underaninertatmosphereofnitrogen.Afterthesolutionwas
EXPERIMENTAL SECTION
cooled to room temperature, both mPEG -NH (500 mg, 0.10
5000 2
Materials and Synthesis. All solvents were of analytical reagent mmol)(MALDI-TOF-MS:M n =5254.95Da,M w =5315.32Da,PDI
gradeandpurifiedaccordingtostandardprocedures.36Diimineligands = 1.012) and triethylamine (0.1 mL) were added. The mixture was
including phen, Ph-phen, and Me-phen, AgCFSO, 3-amino- stirredunderaninertatmosphereofnitrogenatroomtemperaturefor
2 4 3 3
pyridine, KPF, CaCO, ethylamine, triethylamine, thiophosgene, 48 h. The mixture was evaporated to dryness yielding a pale yellow
6 3
and cisplatin were purchased from Acros. Re(CO)Cl and branched solid, which was redissolved in deionized water (10 mL). Any
5
PEI(MW=25kDa)wereobtainedfromAldrich.MTT,tricaine,and undissolved solid (excess bpy-NHS) was removed by centrifugation.
n-butylamine were purchased from Sigma. α-Amino-ω-methoxypoly- ThesupernatantwasloadedontoaPD-10size-exclusioncolumnthat
(ethylene glycol) (mPEG -NH) (MW = 5000 Da, PDI < had been equilibrated with water, and the solution containing the
5000 2 PEG PEG
1.08) was purchased from Nanocs. All these chemicals were used ligand was collected and lyophilized to give the product as a white
withoutfurther purification. Py-NHS-NH,37bpy-NHS,38[Re(N∧N)- solid. Yield: 416 mg (81%). 1H NMR (300 MHz, CDCl, 298 K) δ
2 3
(CO) (CH CN)](CF SO ),39 and bpy-Et40 were prepared as 8.77(d,1H,J=4.8Hz,H6ofbpy),8.67(s,1H,H3ofbpy),8.52(d,
describ
3
ed p
3
reviously. B
3
SA
3
was obtained from Calbiochem. PD-10
1H,J=5.4Hz,H6′ofbpy),8.23(s,1H,H3′ofbpy),7.77(d,1H,J
size-exclusion columns and YM-30 centricons were purchased from =5.7Hz,H5ofbpy),7.44(s,1H,bpy-CONH),7.16(d,1H,J=5.1
GEHealthcareandMillipore,respectively.Allbuffercomponentswere Hz,H5′ofbpy),3.87−3.38(m,ca.452H,CONHCH andOCH of
2 2
ofbiologicalgradeandusedasreceived.AutoclavedMilli-Qwaterwas PEG),3.35(s,3H,OCH),2.65(s,3H,CH ofbpy).MALDI-TOF-
3 3
used for preparation of aqueous solutions. HeLa cells were obtained MS: M = 5407.26 Da, M = 5420.97 Da, PDI = 1.003.
n w
from American Type Culture Collection. Mature zebrafish were Rhenium(I) Polypyridine Amine Complexes 1-PEG-NH 2 −4-PEG-
purchased from Chong Hing Aquarium, Hong Kong, China, and NH 2 and 1-Et-NH 2 −4-Et-NH 2. A mixture of [Re(N∧N)-
maintainedasdescribedbyWesterfield.41Thezebrafishembryoswere (CO)
3
(CH
3
CN)](CF
3
SO
3
) (0.039 mmol) and the pyridine ligand
obtained by photoinduced spawning over green plants and then py-PEG-NH 2 or py-Et-NH 2 (0.039 mmol) in THF (30 mL) was
cultured at 28.5 °C in filtered tap water with Instant Ocean (60 ng/ refluxedunderaninertatmosphereofnitrogenfor12h.Themixture
mL), which was purchased from Aquatic System. MitoTracker Deep wasthenevaporatedtodryness.ThecomplexwasdissolvedinMeOH,
Red FM, Alexa Fluor 633-conjugated transferrin, UltraPure agarose, converted to the hexafluorophosphate salt by anion exchange with
Dulbecco’s modified Eagle’s medium (DMEM), reduced serum KPF,andthenpurifiedbycolumnchromatographyonalumina.The
6
medium(Opti-MEM), fetalbovineserum(FBS),phosphatebuffered desired product was eluted with a mixture of CHCl and methanol,
2 2
saline (PBS), Lipofectamine 2000, trypsin-EDTA, and penicillin/ and it was subsequently recrystallized from a mixture of CHCl and
2 2
streptomycin were purchased from Invitrogen. Tris(hydroxymethyl)- diethyl ether.
aminomethane(Tris)fromUSBwasusedtoprepareTris-Cl(50mM,
Rhenium(I)PolypyridineIsothiocyanateComplexes1-PEG-NCS−
pH7.4).TheplasmidDNA(pDNA)pRL-TK(4.0kb)wasamplified 4-PEG-NCS. Thiophosgene (15.6 μL, 0.20 mmol) was added to a
in Escherichia coli and purified by HiPure Filter Plasmid Kit, and the mixtureofthePEG-aminecomplex(0.020mmol)andfinelycrushed
concentration of the pDNA was measured spectrophotometrically. CaCO 3 (26 mg, 0.20 mmol) in acetone (10 mL) under an inert
TheRenillaLuciferaseAssaySystemwasobtainedfromPromegaand atmosphere of nitrogen. The suspension was stirred in the dark at
stored at −70 °C before use. The growth medium for cell culture room temperature for 4 h. The mixture was filtered, and the filtrate
contained DMEM with 10% FBSand 1% penicillin/streptomycin. was evaporated to dryness togive the product asa yellow solid.
Py-PEG-NH.Py-NHS-NH (114mg,0.48mmol)wasdissolvedin
Rhenium(I)PolypyridineThioureaComplexes1-PEG-Bu−4-PEG-
hot DMF (20
2
mL) under an
2
inert atmosphere of nitrogen. After the
Bu.AmixtureofthePEGisothiocyanatecomplex(0.01mmol)andn-
solution was cooled to room temperature, both mPEG -NH (800 butylamine (0.04 mmol) in acetone (30 mL) was stirred under an
5000 2
mg,0.16mmol)(MALDI-TOF-MS:numberaveragemolecularweight inertatmosphereofnitrogenfor12h.Themixturewasevaporatedto
(M )=5253.95Da,weightaveragemolecularweight(M )=5315.32 dryness to give a yellow solid, which was redissolved in deionized
n w
Da, PDI = 1.012) and triethylamine (0.1 mL) were added. The water (5 mL). Any undissolved solid was removed by centrifugation.
mixture was stirred under an inert atmosphere of nitrogen at room ThesupernatantwasloadedontoaPD-10size-exclusioncolumnthat
temperaturefor48h.Themixturewasevaporatedtodrynessyielding had been equilibrated with water, and the solution containing the
apaleyellowsolidwhichwasredissolvedindeionizedwater(10mL). complex was collected and lyophilized. Recrystallization of the crude
ThesolutionwasloadedontoaPD-10size-exclusioncolumnthathad product from CH 2 Cl 2 /diethyl ether afforded the complex as pale
been equilibrated with water, and the solution containing the ligand yellow crystals.
was collected and lyophilized to give the product as a white solid. The characterization data of all the complexes are included in the
Yield:744mg(91%).1HNMR(300MHz,CDCl,298K)δ8.26(d, Supporting Information.
3
1 H, J = 1.5 Hz, H6 of pyridine), 8.05 (d, 1 H, J = 2.7 Hz, H2 of InstrumentationandMethods.1HNMRspectrawererecorded
pyridine),7.40(t,1H,J=2.1Hz,H4ofpyridine),7.21(s,1H,py-3- onaVarianMercury300MHzNMRspectrometerat298K.Positive-
CONH),3.78−3.30(m,ca.452H,CONHCH andOCH ofPEG), ionESImassspectrawererecordedonaPerkin-ElmerSciexAPI365
2 2
3.27 (s, 3 H, OCH). MALDI-TOF-MS: M = 5358.37 Da, M = mass spectrometer. MALDI-TOF mass spectra were recorded on an
3 n w
5372.21 Da, PDI = 1.003. AppliedBiosystems4800plusMALDI-TOF/TOFanalyzer.IRspectra
Py-Et-NH . Py-NHS-NH (300 mg, 1.28 mmol) was dissolved in were recorded on a Perkin-Elmer 1600 series FT-IR spectropho-
2 2
hot DMF (20 mL) under an inert atmosphere of nitrogen. After the tometer.ElementalanalyseswerecarriedoutonaVarioELIIICHN
solutionwascooledtoroomtemperature,bothethylamineinTHF(2 elemental analyzer. Electronic absorption and steady-state emission
M,1.92mL,3.84mmol)andtriethylamine(0.1mL)wereadded.The spectra were recorded on a Hewlett-Packard 8453 diode array
mixture was stirred under an inert atmosphere of nitrogen at room spectrophotometer and a SPEX FluoroLog 3-TCSPC spectropho-
temperaturefor24h.Thesolutionwasevaporatedtodrynessyielding tometer equipped with a Hamamatsu R928 PMT detector,
apaleyellowsolid,whichwaspurifiedbycolumnchromatographyon respectively. Emission lifetimes were measured in the Fast MCS or
silicagel.ThedesiredproductwaselutedwithCHCl/MeOH(30:1, the MCS lifetime mode with a NanoLED N-375 as the excitation
2 2
v/v).Thesolventwasremovedundervacuumtogivetheproductasa source.Allthesolutionsforphotophysicalstudiesweredegassedwith
white solid. Yield: 154 mg (73%). 1H NMR (300 MHz, CDCl, 298 atleastfoursuccessivefreeze−pump−thawcyclesandstoredina10-
3
K)δ8.28(d,1H,J=1.5Hz,H6ofpyridine),8.16(d,1H,J=2.4Hz, cm3round-bottomedflaskequippedwithasidearm1-cmfluorescence
H2ofpyridine),7.43(t,1H,J=2.1Hz,H4ofpyridine),6.17(s,1H, cuvette and sealed from the atmosphere by a Rotaflo HP6/6 quick-
py-3-CONH),3.50(q,2H,J=7.2Hz,NHCHCH),1.26(t,3H,J= releaseTeflonstopper.Luminescencequantumyieldsweremeasured
2 3
K dx.doi.org/10.1021/ic301948d|Inorg.Chem.XXXX,XXX,XXX−XXX
Inorganic Chemistry Article
bytheopticallydilutemethod42usingadegassedacetonitrilesolution 20 s; medium viscosity, 1.0031 cP; dielectric constant, 80.4;
of[Re(phen)(CO)(pyridine)](CFSO)(Φ =0.18,λ =355nm) temperature, 20 °C; beam mode F(Ka) = 1.50 (Smoluchowsky).
3 3 3 em ex
as the standard solution.43 Details on the MTT assays and ICP-MS The particle size was determined with the following specifications:
havebeen reported previously.26h sampling time, 180 s; medium viscosity, 1.0031 cP; refractive index
Live-Cell Confocal Microscopy. HeLa cells in growth medium (RI) medium, 1.330; RI particle, 1.450; temperature, 20 °C. All the
were seeded on a sterilized coverslip in a 60-mm tissue culture dish experiments were carried out in triplicate.
andgrownat37°Cundera5%CO atmospherefor48h.Theculture In Vitro Transfection (Luciferase Assays). HeLa cells were
2
mediumwasthenremovedandreplacedwithmedium/DMSO(99:1 seeded at a density of 100,000 cells per dish in a 35 mmcell culture
v/v)containingcomplex3-PEG-NH (10μM)or3-Et-NH (10μM). dish and incubated for 48 h at 37 °C under a 5% CO atmosphere.
2 2 2
Afterincubationfor1h,themediumwasremoved,andthecelllayer The culture medium was replaced with DMEM (2 mL) containing
waswashedwithPBS(1mL×3).Thecoverslipwasmountedontoa 10% FBS 2 h prior to the transfection. The transfection experiments
sterilizedglassslideandthenimagedusingaLeicaTCSSPEconfocal
wereperformedwithpRL-TK(4μg).Atthetimeoftransfection,the
microscope. In the mitochondria-colocalization experiments, HeLa mediumwasreplacedwithOpti-MEM(2mL).Polyplexescomposed
cellsweretreatedwithcomplex3-PEG-NH (10μM)for1handthen of conjugate 3-PEG-PEI (or unmodified PEI) and pRL-TK with
incubated with MitoTracker Deep Red F
2
M (100 nM) in FBS-free
differentN/Pratioswereincubatedwiththecellsfor5h.Themedium
mediumfor20min,followedbywashingwithPBS(1mL×3).Inthe was replaced with fresh growth medium (3 mL), and the cells were
endosome-colocalizationexperiments,HeLacellswereincubatedwith furtherincubatedfor43h.ThepolyplexLipofectamine/pRL-TK and
complex 3-PEG-NH (10 μM) and Alexa Fluor 633-conjugated the naked pDNA were used as a positive and negative control,
2
transferrin(50μg/mL)inmediumfor1h,followedbywashingwith respectively. After incubation, the cells were permeabilized with cell
PBS(1mL ×3).Thecolocalization coefficientswere determinedby lysis buffer (200 μL) (Promega) with one freeze−thaw cycle. The
the program ImageJ (Version1.4.3.67). luciferase activity in the cell lysate was measured using a Luciferase
Lipophilicity (Log P ). The lipophilicity of the complexes was Assay Kit (Promega) on a microplate reader (BMG FLUOstar
o/w
determined using the flask-shaking method where n-octanol and an OPTIMA). All the experiments were carried out in triplicate.
aqueous sodium chloride solution were used as the organic and In Vivo Toxicity. To determine the 50% lethal concentration
aqueous phase, respectively. n-Octanol was presaturated with an (LD 50 ), selected 4-hpf zebrafish (Danio rerio) embryos were exposed
aqueoussolutionofsodiumchloride(0.9%w/v)byswirlingat45rpm to complex 3-PEG-NH 2 and 3-Et-NH 2 , respectively, at a series of
for24h.Thecomplexwasdissolvedintheisolatedorganicphaseata concentrations (0.3125, 0.625, 1.25, 2.5, 5, 10, 20, and 40 μM)
concentrationof50μM.Anequalvolumeofaqueoussodiumchloride dispersedinfilteredtapwater(pH7,28.5°C)withInstantOcean(60
solution (0.9% w/v) was added, and the mixture was swirled for 30 ng/mL). DMSOwas usedasacarriersolventforthecomplexinthe
minat45rpm.Thesolutionwasthencentrifuged,andtheamountsof embryo medium.The finalconcentration ofDMSO inthe treatment
complexin both layers were determined by emission spectroscopy. medium was 1% (v/v), a concentration which was determined to be
PEGylationofBSAwithComplexes1-PEG-NCS−4-PEG-NCS. nontoxictozebrafishembryos.44Threereplicatesweresetupforeach
The isothiocyanate complex (2.0 μmol) in anhydrous dimethylsulf- concentration,includingthecontrolexperimentsinwhichnocomplex
oxide(DMSO,50μL)wasaddedtoBSA(13.0mg,0.2μmol)in50 waspresent;eachreplicateconsistedof20embryosexposedto6mL
mMcarbonatebuffer(450μL)atpH10.Themixturewasstirredfor oftestingmediuminaPetridish(diameter=55mm).Exposuretothe
12hindarkatroomtemperature.Thesolutionwasthendilutedto1.0 complexes lasted from 4 to 120 hpf. Mortality of the embryos was
mL with 50 mM potassium phosphate buffer at pH 7.4 and loaded monitoredatfivetimepoints:24,48,72,96,and120hpf.TheLD 50
onto a PD-10 column equilibrated with the same buffer. The first valuesweredeterminedfromthedependenceofthemortalityonthe
elutionbandwithyellowtoorangeemissionwascollected.Finally,the concentration of the complexes.
bioconjugates 1-PEG-BSA−4-PEG-BSA were washed successively Fluorescence Imaging of Zebrafish Embryos. Selected 4-hpf
with potassium phosphate buffer using an YM-30 centricon, zebrafishembryoswereexposedtocomplex3-PEG-NH 2 (20μM)in
concentrated to 1.5 mL,and stored at 4 °C. filtered tap water (pH 7, 28.5 °C) with Instant Ocean (60 ng/mL).
PEGylation of PEI with Complexes 1-PEG-NCS−4-PEG-NCS. Afterincubationfor20h,themediumwasremoved,andtheembryos
Theisothiocyanatecomplex(5.0μmol)inanhydrousDMSO(50μL) were washed twice thoroughly with deionized water. The embryos
was added to PEI (12.5 mg, 0.5 μmol) in 50 mM carbonate buffer werethentransferredto6mLoftestingmediumwith0.016Mtricaine
(450μL)atpH10.Themixturewasstirredfor12hindarkatroom for anesthetization and imaged using a fluorescence microscope
temperature. The solution was then diluted to 1.0 mL with 50 mM (Olympus SZX 12) equipped with a mercury lamp (EXFO X-cite
potassium phosphate buffer at pH 7.4 and loaded onto a PD-10 1 ■ 20Q) asthe excitation source.
columnequilibratedwiththesamebuffer.Thefirstelutionbandwith
yellow to orange emission was collected. Finally, conjugates 1-PEG- ASSOCIATED CONTENT
PEI−4-PEG-PEIwerewashedsuccessivelywithpotassiumphosphate
*
bufferusinganYM-30centricon,concentratedto1.5mL,andstored S Supporting Information
at 4°C. Characterization and electronic absorption spectral data of the
Agarose Gel Electrophoresis Retardation Assays. Polyplexes rhenium(I) polypyridine complexes, MALDI-TOF mass
composed of conjugate 3-PEG-PEI (or unmodified PEI) and the spectra of the ligands py-PEG-NH and bpy-PEG and
2
pDNA pRL-TK with N/P ratios (the number of PEI nitrogen per complexes 1-PEG-NH −4-PEG-NH , and emission spectrum
DNA phosphate) from 1 to 32 were prepared by mixing different 2 2
of complex 2-PEG-NH in butyronitrile glass at 77 K. This
amountsofthePEI-PEGconjugateandpRL-TKinTris-Clbuffer(50 2
material is available free of charge via the Internet at http://
mM, pH 7.4). After incubation for 30 min at room temperature, the
pubs.acs.org.
polyplexeswereanalyzedbyelectrophoresisona0.9%(w/v)agarose ■
gelcontainingethidiumbromidewithTris-acetatebufferat100Vfor
45min. Thegel was visualized using a Bio-Rad Gel Doc imager. AUTHOR INFORMATION
Zeta Potentials and Mean Hydrodynamic Diameter Meas-
urements. Polyplexes composed of conjugate 3-PEG-PEI (or Corresponding Author
unmodified PEI) and pRL-TK (4 μg) with N/P ratios from 1 to 32 *E-mail: bhcheng@cityu.edu.hk (S.H.C.), bhkenlo@cityu.edu.
in Tris-Cl buffer (80 μL, 50 mM, pH 7.4) were prepared and hk (K.K.-W.L.). Fax: (+852) 3442 0522. Tel: (+852) 3442
incubated for 30 min at room temperature. The mixture was then 7231.
diluted 10-fold with the same buffer. The zeta potentials of the
Notes
polyplexes were measured using Zetasizer Nano ZS (Malvern
Instruments) with the following specifications: sampling time, 10− The authors declare no competing financial interest.
L dx.doi.org/10.1021/ic301948d|Inorg.Chem.XXXX,XXX,XXX−XXX
■Inorganic Chemistry Article
ACKNOWLEDGMENTS (16) (a)Wrighton, M.S.; Morse,D. L.J.Am. Chem. Soc. 1974,96,
998−1003.(b)Fredericks,S.M.;Luong,J.C.;Wrighton,M.S.J.Am.
We thank the Hong Kong Research Grants Councils (Project Chem. Soc. 1979, 101,7415−7417.
No. CityU 102410) and the City University of Hong Kong (17) (a) Connick, W. B.; Di Bilio, A. J.; Hill, M.G.; Winkler, J. R.;
(Project No. 7008174) for financial support. A.W.-T.C., M.- Gray,H.B.Inorg.Chim.Acta1995,240,169−173.(b)Wenger,O.S.;
W.L., and S.P.-Y.L. acknowledge the receipt of a Postgraduate Henling, L. M.; Day, M.W.; Winkler, J. R.; Gray, H. B. Inorg. Chem.
Studentship, a Research Tuition Scholarship, and an Out- 2004, 43, 2043−2048.
standing Academic Performance Award administered by the (18)(a)Yam,V.W.-W.;Lau,V.C.-Y.;Wu,L.-X.J.Chem.Soc.,Dalton
City University of Hong Kong.
Trans.1998,1461−1468.(b)Lam,S.C.-F.;Yam,V.W.-W.;Wong,K.
■ M.-C.;Cheng,E.C.-C.;Zhu,N.Organometallics2005,24,4298−4305.
(19) (a) Guo, X.-Q.; Castellano, F. N.; Li, L.; Szmacinski, H.;
REFERENCES Lakowicz,J.R.;Sipior,J.Anal.Biochem.1997,254,179−186.(b)Guo,
(1)Veronese,F.M.;Pasut,G.DrugDiscoveryToday2005,10,1451− X.-Q.;Castellano,F.N.;Li,L.;Lakowicz,J.R.Anal.Chem.1998,70,
1458.
632−637.(c)Shen,Y.;Maliwal,B.P.;Lakowicz,J.R.J.Fluoresc.2001,
(2) (a) Harris, J. M.; Martin, N. E.; Modi, M. Clin. Pharmacokinet. 11, 315−318. (d) Kusb́ a, J.; Li, L.; Gryczynski, I.; Piszczek, G.;
2001,40,539−551.(b)Michaelis,M.;Cinatl,J.;Pouckova,P.;Langer, Johnson, M.;Lakowicz, J. R.Biophys. J. 2002,82,1358−1372.
K.; Kreuter, J.; Matousek, J. Anti-Cancer Drugs 2002, 13, 149−154. (20) (a) Busby, M.; Gabrielsson, A.; Matousek, P.; Towrie, M.; Di
(c)Mok,H.;Palmer,D.J.;Ng,P.;Barry,M.A.Mol.Ther.2005,11, Bilio, A. J.; Gray, H. B.; Vlcě k, A., Jr. Inorg. Chem. 2004, 43, 4994−
66−79.(d)Pai,S.S.;Przybycien,T.M.;Tilton,R.D.AAPSJ.2009, 5002.(b)Gabrielsson,A.;Matousek,P.;Towrie,M.;Hartl,F.;Zaĺi,S.;
11,88−98.(e)Knop,K.;Hoogenboom,R.;Fischer,D.;Schubert,U. Vlcě k, A., Jr. J. Phys. Chem. A 2005, 109, 6147−6153. (c) Blanco-
S.Angew. Chem., Int.Ed. 2010,49,6288−6308. Rodrkguez,A.M.;Busby,M.;Grdinaru,C.;Crane,B.R.;DiBilio,A.
(3) (a) Veronese, F. M.; Sacca,̀ B.; Polverino de Laureto, P.; Sergi, J.;Matousek,P.;Towrie,M.;Leigh,B.S.;Richards,J.H.;Vlcě k,A.,Jr.;
M.;Caliceti,P.;Schiavon,O.;Orsolini,P.BioconjugateChem.2001,12, Gray, H. B. J. Am. Chem. Soc. 2006,128,4365−4370.
62−70.(b)Manta,C.;Ferraz, N.;Betancor,L.;Antunes,G.;Batista- (21) (a) Wallace, L.; Rillema, D. P. Inorg. Chem. 1993, 32, 3836−
Viera,F.;Carlsson,J.;Caldwell,K.EnzymeMicrob.Technol.2003,33, 3843. (b) Villegas, J. M.; Stoyanov, S. R.; Huang, W.; Rillema, D. P.
890−898. (c) Ozyilmaz, G. J. Mol. Catal. B: Enzym. 2009, 56, 231− Dalton Trans. 2005, 1042−1051.
236. (22) (a) Zipp, A. P.; Sacksteder, L.; Streich, J.; Cook, A.; Demas, J.
(4)Fujii,N.;Jacobsen,R.B.;Wood,N.L.;Schoeniger,J.S.;Guy,R.
N.;DeGraff,B.A.Inorg.Chem.1993,32,5629−5632.(b)Sacksteder,
K. Bioorg. Med.Chem. Lett. 2004,14,427−429. L.;Lee,M.;Demas,J.N.;DeGraff,B.A.J.Am.Chem.Soc.1993,115,
(5) (a) Duncan, R. Nat. Rev. Drug Discovery 2003, 2, 347−360. 8230−8238. (c) Kneas, K. A.; Xu, W.; Demas, J. N.; Zipp, B. A.;
(b)Thompson,M.S.;Vadala,T.P.;Vadala,M.L.;Lin,Y.;Riffle,J.S. DeGraff, A.P.J. Fluoresc. 1998,8, 295−300.
Polymer 2008, 49,345−373. (23) Smithback, J. L.; Helms, J. B.; Schutte, E.; Woessner, S. M.;
(6)(a)Pendri,A.;Martinez,A.;Xia,J.;Shorr,R.G.L.;Greenwald,R. Sullivan, B. P.Inorg. Chem. 2006,45, 2163−2174.
B.BioconjugateChem.1995,6,596−598.(b)Mueller,C.;Capelle,M. (24)(a)Lees,A.J.Chem.Rev.1987,87,711−743.(b)Kotch,T.G.;
A. H.; Arvinte, T.; Seyrek, E.; Borchard, G. J. Pharm. Sci. 2011, 100,
Lees,A.J.;Fuerniss,S.J.;Papathomas,K.I.Chem.Mater.1991,3,25−
1648−1662. 27. (c) Kotch, T. G.; Lees, A. J.; Fuerniss, S. J.; Papathomas, K. I.
(7) (a) Viht, K.; Padari, K.; Raidaru, G.; Subbi, J.; Tammiste, I.;
Chem.Mater.1992,4,675−683.(d)Kotch,T.G.;Lees,A.J.;Fuerniss,
Pooga, M.; Uri, A. Bioorg. Med. Chem. Lett. 2003, 13, 3035−3039. S.J.; Papathomas,K.I.;Snyder,R.W.Inorg.Chem.1993,32,2570−
(b)Erbas,S.;Gorgulu,A.;Kocakusakogullari,M.;Akkaya,E.U.Chem. 2575. (e) Sun, S.-S.; Lees, A.J. Organometallics 2002, 21,39−49.
Commun.2009, 4956−4958. (25)Stephenson,K.A.;Banerjee,S.R.;Besanger,T.;Sogbein,O.O.;
(8)(a)Rapozzi,V.;Cogoi,S.;Spessotto,P.;Risso,A.;Bonora,G.M.; Levadala, M. K.; McFarlane, N.; Lemon, J. A.; Boreham, D. R.;
Quadrifoglio, F.; Xodo, L. E. Biochemistry 2002, 41, 502−510. Maresca,K.P.;Brennan,J.D.;Babich,J.W.;Zubieta,J.;Valliant,J.F.
(b) Rakestraw, J. A.; Baskaran, A. R.; Wittrup, K. D. Biotechnol. Prog. J. Am. Chem. Soc. 2004, 126,8598−8599.
2006,22,1200−1208. (26) (a) Lo, K. K.-W.; Ng, D. C.-M.; Hui, W.-K.; Cheung, K.-K. J.
(9)(a)Corma,A.;Garcìa,H.;Leyva,A.J.Catal.2006,240,87−99. Chem. Soc., Dalton Trans. 2001, 2634−2640. (b) Lo, K. K.-W.; Hui,
(b) Samanta, D.; Kratz, K.; Zhang, X.; Emrick, T. Macromolecules W.-K.; Ng, D. C.-M.; Cheung, K.-K. Inorg. Chem. 2002, 41, 40−46.
2008,41,530−532. (c) Lo, K. K.-W.; Hui, W.-K.; Ng, D. C.-M. J. Am. Chem. Soc. 2002,
(10) (a) Marin, V.; Holder, E.; Meier, M. A. R.; Hoogenboom, R.;
124,9344−9345.(d)Lo,K.K.-W.;Tsang,K.H.-K.;Hui,W.-K.;Zhu,
Schubert, U. S. Macromol. Rapid Commun. 2004, 25, 793−798. N.Chem.Commun.2003,21,2704−2705.(e)Lo,K.K.-W.;Tsang,K.
(b)Holder,E.;Marin,V.;Meier,M.A.R.;Schubert,U.S.Macromol.
H.-K.;Sze,K.-S.Inorg.Chem.2006,45,1714−1722.(f)Lo,K.K.-W.;
Rapid Commun.2004, 25, 1491−1496. Tsang, K. H.-K.; Zhu, N. Organometallics 2006, 25, 3220−3227.
(11)(a)Yokoyama,M.;Okano,T.;Sakural,Y.;Suwa,S.;Kataoka,K. (g)Lo,K.K.-W.;Sze,K.-S.;Tsang,K.H.-K.;Zhu,N.Organometallics
J. Controlled Release 1996, 39, 351−356. (b) Nishiyama, N.; 2007,26,3440−3447.(h)Louie,M.-W.;Lui,H.-W.;Lam,M.H.-C.;
Yokoyama, M.; Aoyagi, T.; Okano, T.; Sakurai, Y.; Kataoka, K. Lau, T.-C.; Lo, K. K.-W. Organometallics 2009, 28, 4297−4307.
Langmuir1999,15,377−383.(c)Iinuma,H.;Maruyama,K.;Okinaga, (i)Louie,M.-W.;Liu,H.-W.;Lam,M.H.-C.;Lam,Y.-W.;Lo,K.K.-W.
K.; Sasaki, K.; Sekine, T.; Ishida, S.; Ogiwara, N.; Johkura, K.; Chem.Eur. J. 2011, 17,8304−8308. (j) Louie, M.-W.;Fong, T. T.-
Yonemura, Y. Int. J. Cancer 2002, 99, 130−137. (d) Aronov, O.; H.; Lo, K. K.-W. Inorg. Chem. 2011,50, 9465−9471.
Horowitz,A.T.;Gabizon,A.;Fuertes,M.A.;Perez,J.M.;Gibson,D. (27)(a)Reitz,G.A.;Demas,J.N.;DeGraff,B.A.;Stephens,E.M.J.
Bioconjugate Chem. 2004, 15, 814−823. (e) Garmann, D.; Warnecke, Am.Chem.Soc.1988,110,5051−5059.(b)Coogan,M.P.;Fernań dez-
A.;Kalayda,G.V.;Kratz,F.;Jaehde,U.J.ControlledRelease2008,131, Moreira,V.;Hess,J.B.;Pope,S.J.A.;Williams,C.NewJ.Chem.2009,
100−106. 33,1094−1099.
(12) Che, C.-M.; Zhang, J.-L.; Lin, L.-R. Chem. Commun. 2002, (28)(a)Bailon,P.;Berthold,W.Pharm.Sci.Technol.Today1998,1,
2556−2557. 352−356. (b) Harris, J. M.; Chess, R. B. Nat. Rev. Drug. Discovery
(13) Heldt, J.-M.; Fischer-Durand, N.; Salmain, M.; Vessier̀es, A.; 2003, 2,214−221.
Jaouen, G. J. Organomet. Chem. 2004,689,4775−4782. (29) (a) Lo, K. K.-W.; Ng, D. C.-M.; Chung, C.-K. Organometallics
(14)Viola-Villegas,N.;Rabideau,A.E.;Cesnavicious,J.;Zubieta,J.; 2001,20,4999−5001.(b)Lo,K.K.-W.;Chung,C.-K.;Lee,T.K.-M.;
Doyle,R. P.ChemMedChem 2008,3,1387−1394. Lui, L.-H.; Tsang, K. H.-K.; Zhu, N. Inorg. Chem. 2003, 42, 6886−
(15)Li,S.P.-Y.;Liu,H.-W.;Zhang,K.Y.;Lo,K.K.-W.Chem.Eur. 6897.(c)Lo,K.K.-W.;Chan,J.S.-W.;Chung,C.-K.;Tsang,V.W.-H.;
J. 2010,16,8329−8339. Zhu, N. Inorg. Chim. Acta 2004, 357, 3109−3118. (d) Leung, S.-K.;
M dx.doi.org/10.1021/ic301948d|Inorg.Chem.XXXX,XXX,XXX−XXX
Inorganic Chemistry Article
Kwok,K.Y.;Zhang,K.Y.;Lo,K.K.-W.Inorg.Chem.2010,49,4984−
4995.(e)Lee,P.-K.;Liu,H.-W.;Yiu,S.-M.;Louie,M.-W.;Lo,K.K.-
W.Dalton Trans. 2011,40,2180−2189.
(30) (a) Boussif, O.; Lezoualc’h, F.; Zanta, M. A.; Mergny, M. D.;
Scherman, D.; Demeneix, B.; Behr, J. P. Proc. Natl. Acad. Sci. U.S.A.
1995, 92, 7297−7301. (b) Rudolph, C.; Lausier, J.; Naundorf, S.;
Müller, R. H.; Rosenecker, J. J. Gene Med. 2000, 2, 269−278.
(c) Akinc, A.; Thomas, M.; Klibanov, A. M.; Langer, R. J. Gene Med.
2004,7, 657−663.
(31)(a)Bieber,T.;Meissner,W.;Kostin,W.;Niemann,A.;Elsasser,
H.-P.J.ControlledRelease2002,82,441−454.(b)Grosse,S.;Aron,Y.;
Thev́ enot,G.;Monsigny,M.;Fajac,I.J.ControlledRelease2007,122,
111−117.(c)Sunoqrot,S.;Bae,J.W.;Jin,S.-E.;Pearson,R.M.;Liu,
Y.;Hong,S.BioconjugateChem.2011,22,466−474.(d)Louie,M.-W.;
Choi, A. W.-T.; Liu, H.-W.; Chan, B. T.-N.; Lo, K. K.-W.
Organometallics 2012, 31, 5844−5855. (e) Li, S. P.-Y.; Tang, T. S.-
M.; Yiu, K. S.-M.; Lo, K. K.-W. Chem.Eur. J. 2012, 18, 13342−
13354.
(32) (a) Erbacher, P.; Bettinger, T.; Belguise-Valladier, P.; Zou, S.;
Coll, J.-L.; Behr, J.-P.; Remy, J.-S. J. Gene Med. 1999, 1, 210−222.
(b) Nguyen, H.-K.; Lemieux, P.; Vinogradov, S. V.; Gebhart, C. L.;
Guerin,N.;Paradis,G.;Bronich,T.K.;Alakhov,V.Y.;Kabanov,A.V.
Gene Ther. 2000, 7, 126−138. (c) Vinogradov, S. V.; Bronich, T. K.;
Kabanov, A. V. Bioconjugate Chem. 1998, 9, 805−812. (d) Petersen,
H.;Fechner,P.M.;Martin,A.L.;Kunath,K.;Stolnik,S.;Roberts,C.
J.;Fischer,D.;Davies,M.C.;Kissel,T.BioconjugateChem.2002,13,
845−854.
(33)Hyvönen,Z.;Plotniece,A.;Reine,I.;Chekavichus,B.;Duburs,
G.;Urtti, A. Biochim.Biophys. Acta 2000, 1509, 451−466.
(34) Booth,D. T. J. Exp. Biol. 1995,198, 241−247.
(35) Cheng, J.; Flahaut, E.; Cheng, S. H. Environ. Toxicol. Chem.
2007,26,708−716.
(36) Perrin, D. D.; Armarego, W. L. F. Purification of Laboratory
Chemicals,3rd ed.;Pergamon Press: NewYork, 1988.
(37)Lo,K.K.-W.;Louie,M.-W.;Sze,K.-S.;Lau,J.S.-Y.Inorg.Chem.
2008,47,602−611.
(38)Peek,B.M.;Ross,G.T.;Edwards,S.W.;Meyer,G.J.;Meyer,
T.J.; Erickson, B. W.Int.J. Pept. Protein Res. 1991,38,114−123.
(39) Lo, K.K.-W.;Hui, W.-K. Inorg. Chem. 2005,44,1992−2002.
(40) Lo, K. K.-W.; Lee, T. K.-M.; Zhang, K. Y. Inorg. Chim. Acta
2006,359,1845−1854.
(41) Westerfield,M.TheZebrafishBook:AGuide forthe Laboratory
Use of Zebrafish (Danio rerio); University of Oregon Press: Eugene,
OR,2007.
(42)Demas,J.N.;Crosby,G.A.J.Phys.Chem.1971,75,991−1024.
(43) Wallace, L.; Rillema, D. P.Inorg. Chem. 1993,32,3836−3843.
(44) Lahnsteiner, F. Theriogenology 2008,69,384−396.
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