Luminescent oligo(ethylene glycol)-functionalized cyclometalated platinum(ii) complexes: Cellular characterization and mitochondria-specific localization

Article abstract of DOI:10.1039/c3cc47150k

A readily tunable series of non-planar oligo(ethylene glycol)-substituted phosphorescent Pt(ii) complexes has been investigated as live cell imaging agents; suitable structural modifications can give good cellular uptake, traceable mitochondria-specific localization and potent cytotoxic characteristics towards HeLa cells.

Full text of DOI:10.1039/c3cc47150k

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Luminescent oligo(ethylene glycol)-functionalized
cyclometalated platinum(II) complexes: cellular
characterization and mitochondria-specific
localization†
Cite this: Chem. Commun., 2014,
50, 1711
Received 18th September 2013,
Accepted 4th December 2013
Zhengqing Guo, Wah-Leung Tong and Michael C. W. Chan*
DOI: 10.1039/c3cc47150k
www.rsc.org/chemcomm
A readily tunable series of non-planar oligo(ethylene glycol)-substituted distribution within the cytoplasm (with partial localization in the
phosphorescent Pt(II) complexes has been investigated as live cell perinuclear region)8 or staining of the plasma membrane.9
imaging agents; suitable structural modifications can give good cellular
Mitochondria are the primary cellular producers of ATP and
uptake, traceable mitochondria-specific localization and potent cyto- play important roles in the cell cycle, regulation of metabolism, and
toxic characteristics towards HeLa cells.
signaling and programmed cell death (apoptosis).10 Hence, mito-
chondria are attractive targets for the development of drug leads
The design of molecular optical imaging probes that exhibit with superior characteristics and reduced side effects, and the
traceable cellular localization is a challenging endeavor. Transition integration of a luminescence reporting element would facilitate
metal–organic1 (and lanthanide2) complexes offer great promise as visualization and controllable targeting. Organic fluorophores that
diagnostic and therapeutic agents due to their modularity and display selective mitochondrial staining are well-established and
adaptability, well-defined structure and coordination geometry, typically rely upon cationic triphenylphosphonium or ammonium
availability of potential binding sites, and the possibility of functionalities.11 Recently, reports on mitochondria-specific
visible-light and phosphorescent signaling from tunable, diverse phosphorescent and metal–organic probes [Re(I),12 Ru(II),13
excited states (without interference from organic/background Ir(III),14 Pt(II);4 Eu(III)15] have begun to emerge.
fluorescence).3 However, although a rational relationship between
We are currently engaged in the development of functionalized
molecular structure and cellular uptake/localization may be anti- phosphorescent complexes featuring environmentally responsive
cipated and would be highly beneficial for application studies, Pt(II) moieties, with the aim of creating molecular assemblies with
this level of understanding remains unrealized.
unusual photophysical and sensing characteristics that allow the
Previous studies concerning luminescent platinum(II) bioprobes reporting of molecular-level perturbations and events.16,17 Herein,
have largely employed the ‘‘planar lipophilic cation’’ design strategy. we describe the synthesis and spectroscopic properties of a readily
Two related Pt(II) systems, supported by cyclometalated [6-aryl-2,20- tunable family of oligo(ethylene glycol)-substituted cyclometalated
bipyridine and mono-(N-heterocyclic carbene)]4 or [2-arylpyridine and Pt(II) complexes, and investigate their applications as live cell
bis-(NHC)]5 ligand sets, were reported to display contrasting cellular imaging agents. We demonstrate that suitable structural modifi-
localization, namely to mitochondria and the endoplasmic reticulum cations can confer mitochondria-specific localization and potent
(ER) respectively, although both classes show cytotoxic behavior. The cytotoxic characteristics to these non-planar cationic probes.
application of a PPh3-containing cationic cyclometalated Pt(II) lumino-
A versatile synthetic route was devised for the Pt(II) complexes
phore as a bioimaging agent was investigated;6 this complex was (Scheme 1; see the ESI† for details); the oligo(ethylene glycol) chain
expected to target the mitochondria (see below), but nucleolus- was incorporated to improve biocompatibility, while the 4-phenyl
specific localization was observed. Similar preferential staining of moiety at the central pyridine ring was designed to augment emis-
the nucleolus was detected for the neutral tridentate cyclometalated sion efficiency.18 For example, a modified Krohnke-type reaction was
¨
[2,6-di(20-pyridyl)phenyl]PtCl complex, which displays low cytotoxi- performed to generate 4-phenyl-6-phenol-2,20-bipyridine, and sub-
city.7 On the other hand, reports on anionic Pt(II) probes have revealed stitution yielded the key 4-phenyl-6-aryl-2,20-bipyridine precursor
bearing a tosylate-terminated tetra(ethylene glycol) substituent, which
readily undergoes substitution with phenols and secondary amines
to give the desired series of ligands. Subsequent metalation
with K2PtCl4 in refluxing glacial acetic acid, followed by treat-
Department of Biology and Chemistry, City University of Hong Kong, Tat Chee Avenue,
Kowloon, Hong Kong, China. E-mail: mcwchan@cityu.edu.hk
† Electronic supplementary information (ESI) available: Experimental and synthetic
ment with PPh3 in CH3CN/CH3OH (1 : 1) and salt metathesis
using LiClO4, afforded the cationic complexes 3–7 with different
details; characterization and photophysical data; confocal microscopy details and
additional images. See DOI: 10.1039/c3cc47150k
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The intracellular distribution of the Pt(II) probes has been
investigated using laser-scanning confocal microscopy. The micro-
scopy images of HeLa cells incubated with each complex (2.5 mM)
at 37 1C for 1 hour were obtained. Intracellular emissions were
observed upon excitation at 405 nm, confirming that these cationic
oligo(ethylene glycol)-substituted complexes can be internalized by
live cells. Complexes 1–5 were distributed in the cytoplasm with
some degree of punctate staining in the perinuclear region (Fig. S4,
ESI†), but moderately weak emissions were detected in each case
(including for the quinoline derivative 5), which hampered further
subcellular characterization. Upon changing to the morpholine (6)
and piperidine (7; Fig. 2a) analogues, strong emissions with intense
punctate staining of HeLa cells were detected. The emission spectra
of 6 and 7 obtained from HeLa cells display good resemblance to
those in solution (Fig. S5, ESI†), strongly suggesting that the Pt(II)
luminophores remain intact inside the cells. The weaker emissions
of 1–5 should not be ascribed to poor cellular uptake, since the
intracellular amounts of platinum for all complexes fall within a
comparable level of 10ꢀ16 mole (see Table 1).
To evaluate possible organelle-specific localization, the confocal
microscopy images of HeLa cells co-incubated with 7 (and 6) and
various commercially available fluorescent dyes, namely MitoTracker
Green FM (for mitochondria; Fig. 2b for 7), LysoTracker Green
DND-26 (for lysosomes), ER Tracker Green (for ER domains) and
Hoechst 33342 (for DNA), were examined. The confocal images of 7
and MitoTracker Green FM are closely superimposable (Fig. 2c),
indicating that these two dyes are co-localized, which is also the case
for 6 (Fig. S6, ESI†). No significant co-localization was found for 6 or 7
(Fig. S7 and S9, respectively, ESI†) with the lysosome-, ER- and
nucleus-specific dyes. The lack of significant co-localization for 6
and LysoTracker is of interest, bearing in mind the established ability
of alkylmorpholine to target the acidic nature of lysosomes.20 The
complete absence of co-localization for 6 or 7 and the DNA binder
Hoechst 33342 is also intriguing, considering the known propensity of
square planar Pt(II) complexes to accumulate in the nucleus by cross-
linking and/or intercalation,6,7,21 and we attribute this to their non-
planar structures imposed by the 4-phenyl and bulky PPh3 moieties.
The intensity profiles of the linear regions of interest across HeLa
cells stained with 6 (and 7) and MitoTracker Green FM (Fig. S6
and S8, respectively, ESI†) display good overlap. The co-localization
of 6 and 7 with the fluorescent dyes was quantified by correlation
analysis. The Pearson coefficients (Rr) of co-localization for 6 with
MitoTracker, LysoTracker and ER Tracker are 0.80, 0.64 and 0.55,
respectively, while those for 7 are 0.73, 0.58 and 0.38, respectively.
These results indicate that complexes 6 and 7 can preferentially
localize in the mitochondria of HeLa cells. Incubation of HeLa cells
with 6 and 7 at 4 1C resulted in a very weak emission (Fig. S10, ESI†),
indicating that the complexes entered the cells through an energy-
dependent endocytosis pathway. In a time-dependent MTT assay,
minimal decreases of cell survival percentage were recorded after
incubation of HeLa cells with 6 and 7 at 37 1C for 8 hours at a dose
concentration of 2.5 mM (Fig. S11, ESI†). Control experiments have
been undertaken to investigate the intracellular localization of the
corresponding neutral derivatives of 3, 6 and 7 (where PPh3 is
replaced by Cl), but no emission was detected after incubation with
HeLa cells (presumably due to minimal uptake).
Scheme 1 Structures of platinum(II) complexes.
oligo(ethylene glycol)-appended functionalities and lipophilicities (see
below). The parent (1) and ethoxy (2) derivatives have been prepared
for comparative studies. All complexes have been characterized by
NMR spectroscopy, mass spectrometry and elemental analysis. For
example, the 31P{1H} NMR spectrum of 7 displays a singlet signal at
25.5 ppm, flanked by characteristic 195Pt satellites with a 1JP,Pt value of
4000 Hz, which matches the corresponding coupling for the doublet
peak in the 195Pt NMR spectrum at ꢀ4143 ppm (Fig. S1, ESI†).
The photophysical properties of complexes 1–7 have been inves-
tigated by UV-vis absorption and emission spectroscopy (Fig. 1 and
Table S1, ESI†). The visible absorption bands of 2–7 at wavelengths
>400 nm are tentatively assigned to an admixture of p(alkyl-O-aryl)/
d(Pt) - p*(bpy) (1ILCT/1MLCT), following previous studies on
neutral alkoxy congeners17c,19 and in contrast to the d(Pt) -
p*(bpy) (1MLCT) transition of 1.18a All complexes in this study are
luminescent in solution at room temperature. Compared with the
3MLCT emission of 1 (lmax 534 nm in CH3CN), the emission bands
of 2–7 are red-shifted to ca. lmax 580 nm and exhibit minor
solvatochromism (for 3: Fig. S2, ESI†), which is indicative of an
3ILCT excited state, although mixing with 3MLCT is likely.17c,19 As a
representative example, the structure of 3 was optimized by DFT
calculations (Fig. S3, ESI†). The electron densities of the HOMO and
LUMO from the energy-minimized calculated (Gaussian) structure
are shown to be primarily localized on the 6-(alkyl-O)-aryl-2,20-bpy
ligand and to a small extent on the Pt atom (and not on the 4-phenyl
or PPh3 moieties), thus consistent with the above assignment.
Fig. 1 Normalized emission spectra (inset: low-energy UV-vis absorption
bands) of selected complexes (10ꢀ5 M; spectra of 3 and 4 closely matches
5–7 and are omitted for clarity) in CH3CN at 298 K.
1712 | Chem. Commun., 2014, 50, 1711--1714
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Fig. 2 Confocal microscopy images of HeLa cells incubated with (a) 7 (2.5 mM, 1 hour at 37 1C; lex = 405 nm, lem = 580–750 nm) and (b) MitoTracker
Green FM (25 nM, 20 min; lex = 488 nm, lem = 520–560 nm); (c) merged images of (a) and (b). Scale bar = 25.0 mm.
Table 1 Lipophilicity (log Po/w), cellular uptake and cytotoxicity (IC50) of
2–7 towards HeLa cell lines
reported to exhibit pronounced cytotoxic activity, and it is feasible
that the observed toxicity (especially for 6 and 7) also stems from
mitochondrial accumulation29 causing dysfunction or damage lead-
ing to cell death, although alternative pathways should be considered.
In summary, suitable derivatization and variation in lipophili-
city of luminescent non-planar oligo(ethylene glycol)-functionalized
platinum(II) complexes have been shown to give traceable
mitochondria-specific localization in HeLa cells, and preliminary
results have been obtained which provide support for the inter-
pretation regarding imaging studies (i.e. emission quenching
upon uptake; restoration in hydrophobic domains). Future work
will aim to exploit the readily derivatizable nature of this system to
gain greater insight into the factors affecting cellular uptake and
localization, as well as investigate the origin of the cytotoxicity.
The work described in this communication was fully supported
a
Complex
log Po/w
Intracellular amtb/fmol
IC50c/mM
2
3
4
5
6
7
6.1 ꢁ 0.13
5.2 ꢁ 0.12
5.4 ꢁ 0.12
5.9 ꢁ 0.12
5.5 ꢁ 0.12
4.0 ꢁ 0.02
–2.30d
0.63 ꢁ 0.06
0.27 ꢁ 0.09
0.58 ꢁ 0.09
0.71 ꢁ 0.13
0.75 ꢁ 0.09
0.83 ꢁ 0.07
n.d.e
2.4 ꢁ 0.06
9.4 ꢁ 1.6
10.0 ꢁ 1.7
9.2 ꢁ 1.0
8.8 ꢁ 0.6
10.0 ꢁ 0.5
100 ꢁ 4.6
Cisplatin
a
b
See ref. 22. Amount of platinum associated with average HeLa cells
upon incubation with complex (5.0 mM) at 37 1C for 1.5 h; determined
by ICP-AES. For HeLa cells incubated with complex at 37 1C for 24 h.
From ref. 23. Not determined.
c
d
e
Data on lipophilicity, cellular uptake and cytotoxicity have
been collated (Table 1). The lipophilicity of a biological probe is by a grant from the Research Grants Council of the Hong Kong
a critical attribute that strongly influences cellular uptake, Special Administrative Region, China (CityU 100212). We are
intracellular localization and cytotoxicity,13,24 and it has been grateful to Mr Kenneth K.-K. Lau, Michael W.-L. Chiang, and
demonstrated that lipophilic cations accumulate in mitochondria H.-H. Chan for assistance with cellular experiments.
as a result of the negative potential difference across the mito-
chondrial membrane.25 The lipophilicity (log Po/w) of the com-
plexes has been evaluated by reversed-phase HPLC and values of
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should not be discounted; the less intense intracellular emission may
be produced by (weaker) binding at mitochondrial domains that are
less hydrophobic than those targeted by 6 and 7.
1714 | Chem. Commun., 2014, 50, 1711--1714
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