Induced self-assembly and disassembly of water-soluble alkynylplatinum(ii) terpyridyl complexes with switchable near-infrared (NIR) emission modulated by metal-metal interactions over physiological pH: Demonstration of pH-responsive NIR luminescent probes in cell-imaging studies

Article abstract of DOI:10.1039/c3sc50196e

Water-soluble alkynylplatinum(ii) terpyridine complexes, [Pt{tpy(C ₆H₄CH₂NMe₃-4)-4′}(C≡C- Ar)](OTf)₂ [Ar = C₆H₃-(OH)₂-3,5 (1), C₆H₄OH-4 (2), C₆H₃-(OMe) ₂-3,5 (3)], have been synthesized and characterized. The photophysical and electrochemical properties of the complexes have been studied. Complex 1 has been found to undergo aggregation at low pHs, leading to metal-metal and/or π-π interactions and the emergence of a triplet metal-metal-to-ligand charge transfer (³MMLCT) emission in the near-infrared (NIR) region, the intensity of which has been enhanced 1350-fold over that at physiological pH. Such 'switchable' NIR emission of complex 1 was employed in cell-imaging experiments. The pH response of the ³MMLCT emission of complex 1 in cellular compartments has been studied using experiments with fixed Madin-Darby canine kidney (MDCK) cells, while live cell-imaging experiments revealed that complex 1 could function as a NIR luminescent probe for the tracking of the location of acidic organelles such as lysosomes.

Full text of DOI:10.1039/c3sc50196e

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EDGE ARTICLE
Induced self-assembly and disassembly of water-soluble
alkynylplatinum(II) terpyridyl complexes with
“switchable” near-infrared (NIR) emission modulated by
metal–metal interactions over physiological pH:
demonstration of pH-responsive NIR luminescent
probes in cell-imaging studies†
Cite this: Chem. Sci., 2013, 4, 2453
Clive Yik-Sham Chung,a Steve Po-Yam Li,b Man-Wai Louie,b Kenneth Kam-Wing Lob
a
*
and Vivian Wing-Wah Yam
Water-soluble alkynylplatinum(II) terpyridine complexes, [Pt{tpy(C6H4CH2NMe3-4)-40}(C^C–Ar)](OTf)2
[Ar ¼ C6H3–(OH)2-3,5 (1), C6H4OH-4 (2), C6H3–(OMe)2-3,5 (3)], have been synthesized and characterized.
The photophysical and electrochemical properties of the complexes have been studied. Complex 1 has
been found to undergo aggregation at low pHs, leading to metal–metal and/or p–p interactions and
the emergence of a triplet metal-metal-to-ligand charge transfer (3MMLCT) emission in the near-
infrared (NIR) region, the intensity of which has been enhanced 1350-fold over that at physiological pH.
Such ‘switchable’ NIR emission of complex 1 was employed in cell-imaging experiments. The pH
response of the 3MMLCT emission of complex 1 in cellular compartments has been studied using
experiments with fixed Madin–Darby canine kidney (MDCK) cells, while live cell-imaging experiments
revealed that complex 1 could function as a NIR luminescent probe for the tracking of the location of
acidic organelles such as lysosomes.
Received 22nd January 2013
Accepted 11th March 2013
DOI: 10.1039/c3sc50196e
www.rsc.org/chemicalscience
complexes in aqueous media induced by the addition of
synthetic polyelectrolytes4b,c,5a,d,g as well as single-stranded
Introduction
nucleic acids5c, f and aptamers,5f which in turn led to remarkable
UV-vis and near-infrared (NIR) emission spectral changes. Such
signicant spectral changes have been exploited in the detec-
tion of important biomolecules,4b,c,5c, f–h their conformational
changes5c, f and their enzymatic activities.5g Of the single-
stranded DNAs, the pH modulation of cytosine to i-motifs under
acidic conditions has also led to changes in the self-assembly of
the platinum(II) complexes.5c In addition, the self-assembly of
platinum(II) terpyridyl-based metallosupramolecular diblock
copolymers mediated by pH-responsive micellization that could
induce UV-vis and NIR emission spectral changes at acidic pH
has also been reported.5e
Intracellular pH plays a crucial role in many biological
processes.9 A small deviation of the pH values may lead to
cardiopulmonary and neurological diseases such as Alzheimer's
disease,10 or may be associated with the formation of solid
tumours.11 Therefore, the development of a method for the
continuous monitoring of pH with high selectivity and sensitivity
isofgreatimportance. LuminescentpHsensorsare advantageous
over other optical sensors due to their higher sensitivity and
convenience as well as the non-invasive nature of uorescence
microscopy.12 Probes that show NIR emission are particularly
Square-planar platinum(II) polypyridine complexes have attrac-
ted much attention due to their rich spectroscopic properties.1–8
A particular class of platinum(II) complexes with long-standing
interest is the alkynylplatinum(II) terpyridine complexes.4,5 The
presence of d8–d8 metal–metal interactions and p–p stacking of
the terpyridine ligands in these complexes has led to interesting
spectroscopic and luminescence properties.4b–d,5 Recently, our
group has reported the self-assembly of terpyridylplatinum(II)
aInstitute of Molecular Functional Materials (Areas of Excellence Scheme, University
Grants Committee, Hong Kong) and Department of Chemistry, The University of
Hong Kong, Pokfulam Road, Hong Kong, P. R. China. E-mail: wwyam@hku.hk; Fax:
+852 2857 1586; Tel: +852 2859 2153
bDepartment of Biology and Chemistry, City University of Hong Kong, Tat Chee Avenue,
Kowloon, Hong Kong, P. R. China
† Electronic supplementary information (ESI) available: Electronic absorption
spectra of aqueous solutions (50 mM NaCl) of complexes 1,
2 and 3.
Dimerization plot for monomer–dimer equilibrium for complex 1.
a
Concentration-dependent UV-vis absorption spectra of complex 1 at pH 10.
Changes in absorbance of complex 1 at 650 nm with seven cycles of pH
switching. Emission spectra of complex 3 at 25 ꢀC at pH 4, 7 and 10. Relative
emission intensity of complex 2 in an aqueous solution (50 mM NaCl) at
755 nm against pH. Emission spectra of complex 1 (20 mM) in aqueous solution
(50 mM NaCl) at different pHs at 25 ꢀC. See DOI: 10.1039/c3sc50196e
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important as low-energy radiation can increase optical trans- absorptions are ascribed to the intraligand (IL) p / p* transi-
parency and lessen tissue autouorescence.13 Nowadays, the tions of the alkynyl and the terpyridine ligands, whereas the
working principle of the NIR luminescent pH sensors is conned lower-energy absorptions are tentatively assigned as the admix-
to the following strategies: turning on of the luminescence by tures of dp(Pt) / p*(tpy) metal-to-ligand charge transfer
blocking photoinduced electron transfer (PET) upon protonation; (MLCT) and alkynyl-to-terpyridine ligand-to-ligand charge
reduction of the effective conjugation length of the cyanine dyes transfer (LLCT) transitions. In addition, upon light excitation,
upon protonation/deprotonation; or aggregation of organic dyes/ complexes 1, 2 and 3 in acetonitrile–methanol solution mixture
platinum(II) complexes.5e,14 The exploration of a combination of (5 : 1, v/v) are found to emit at 582, 624 and 654 nm respectively,
the above strategies is anticipated to lead to the development of while in dimethylformamide solution complex 3 shows an
sensors with more signicant emission spectral changes in the emission centred at 634 nm (Table 1). Taken into account the
NIR region and thus with higher sensitivity.
microsecond lifetime and the large Stokes shi of the emission,
In view of the fact that the aggregation of terpyr- it is likely that the emission originates from a triplet metal-to-
idylplatinum(II) complexes would lead to signicant emission ligand charge transfer (3MLCT) (3[dp(Pt) / p*(tpy)]) excited
spectral changes in the NIR region under different stimuli,4b–d,5 state with mixing of a triplet ligand-to-ligand charge transfer
we are interested in designing water-soluble alkynylplatinum(II) (3LLCT) (3[p(C^C–R) / p*(tpy)]) character, typical of alkynyl-
terpyridine complexes that would undergo aggregation and platinum(II) terpyridine complexes.4,5 Complexes 1–3 in the solid
deaggregation as well as PET over changes in pH which are in state show low-energy emissions ranging from 716 to 759 nm at
the physiological range, resulting in drastic emission spectral 298 K, and the emissions are found to show signicant red shis
changes in the NIR region. Together with the interest in the upon cooling to 77 K (Table 1). It is likely that these low-energy
search for a new class of NIR luminescent probes with emission solid-state emissions of the complexes are derived from triplet
intensity resistant to self-quenching upon aggregation, in metal-metal-to-ligand charge transfer (3MMLCT) excited states
contrast to those commonly found in the reported pH-respon- resulting from the formation of Pt/Pt and/or p–p interactions
sive NIR luminescent probes,14 herein we report the synthesis in the solid lattice.4,6c Lattice contraction of the complexes upon
and investigation of the spectral changes of the water-soluble cooling to 77 K would result in stronger Pt/Pt and p–p inter-
3
alkynylplatinum(II) terpyridine complexes shown in Scheme 1 actions, leading to the shi of the MMLCT emission to lower
with pH, as well as their in vitro pH response and potential energies.4,6c
application in biological imaging.
The electrochemical data of complexes 1–3 in dime-
thylformamide with 0.1 M Bu4NPF6 as supporting electrolyte
n
have been summarized in Table S1.† The cyclic voltammograms
of complexes 1–3 display two quasi-reversible reduction couples
at E1/2 ¼ ꢁ0.77 to ꢁ0.83 V and ꢁ1.33 to ꢁ1.34 V vs. SCE and an
irreversible oxidation wave at Epa ¼ +0.84 to +1.29 V vs. SCE,
which are tentatively assigned as the successive one-electron
reductions of the terpyridine ligands of the complexes, and the
metal-centred/alkynyl ligand-centred oxidation respectively.3a,6c,8
In addition, all the complexes show an irreversible reduction
wave at Epc ¼ ꢁ1.23 to ꢁ1.25 V vs. SCE (Table S1†), which is likely
attributed to the 4-{(trimethylammonium)methyl}benzene-
based reduction of the substituted terpyridine ligand.17 For
complexes 1 and 2 which contain phenolic proton(s), irreversible
oxidation waves at Epa ¼ +0.97 and +0.73 V vs. SCE, respectively,
were found in their cyclic voltammograms recorded in dime-
thylformamide solution, while the solutions of complexes 1 and
2 in dimethylformamide in the presence of nBu4NOH show
irreversible oxidation waves at Epa ¼ +0.61 and +0.55 V vs. SCE
respectively. It is likely that these irreversible oxidation waves are
derived from the oxidations of the phenolic moieties, typical of
those reported in the literature.18 The shi of the potentials for
the irreversible oxidation waves to less positive values in the
presence of nBu4NOH can be rationalized by the deprotonation
of the phenolic proton(s).
Results and discussion
Synthesis, characterization, photophysical and electro-
chemical studies of the complexes
Water-soluble alkynylplatinum(II) terpyridine complexes 1–3
were synthesized by dehydrohalogenation reactions of
[Pt{tpy(C6H4CH2NMe3-4)-40}Cl](OTf)2
2,4c,15
with the correspond-
ing functionalized alkynes using modications of a literature
procedure for [Pt(tpy)(C^CC6H5)](OTf).3a,16 The complexes were
puried by recrystallization via the slow diffusion of diethyl
ether vapour into methanol–acetonitrile solution mixtures of
the complexes. All the complexes have been successfully char-
1
acterized by H and 13C{1H} NMR, IR, FAB-MS and elemental
analysis. Details of the synthesis and characterization data are
included in the ESI.†
The electronic absorption spectra of complexes 1–3 in dime-
thylformamide solution reveal intense electronic absorptions at
274–354 nm and lower-energy absorptions at ca. 408–496 nm
(Table 1; Fig. S1 in ESI†). According to previous spectroscopic
work on alkynylplatinum(II) terpyridine complexes,4,5 the intense
Aggregation studies of the complexes in aqueous solutions at
different pHs
The 1H NMR spectra of complex 1 in D2O are shown in Fig. 1. At
Scheme 1 Chemical structures of complexes 1–3.
2454 | Chem. Sci., 2013, 4, 2453–2462
pH 4, the terpyridine proton signals are found to be poorly
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Table 1 Photophysical data of complexes 1–3
Electronic absorption, l/nm (3/dm3
molꢁ1 cmꢁ1
Complex
Medium (T/K)
)
Emission, lem/nm (s0/ms)a
b
1
DMF (298)
274 (44 870), 290 (44 870), 314
(29 210), 326 (23 050), 340 (18 200),
352 (15 140), 422 (6660), 472 (5840)
272 (30 300), 290 (33 480), 309
(18 380), 328 (11 170), 342 (8910),
419 (3990), 465 (3670), 512 sh (1000)
262 (29 490), 293 (24 720), 330
(14 810), 382 (7400), 457 (5630), 587
(2160), 663 (600)
262 (28 740), 297 (23 130), 330
(14 500), 353 (10 980), 457 (5410),
587 (1970), 663 (610)
230 (28 740), 276 (22 440), 323
(15 000), 351 (10 930), 471 (4990),
628 (1650)
—
CH3CN–CH3OH (5 : 1, v/v) (298)
pH 7 aqueous solutionc (298)
pH 3 aqueous solutionc (298)
pH 10 aqueous solutionc (298)
582 (0.2)
788 (0.1)
795 (0.1)
b
—
Solid (298)
Solid (77)
DMF (298)
737 (<0.1)
758 (0.2)
—
b
2
274 (66 330), 290 (60 210), 310
(36 820), 336 (21 190), 354 (14 420),
382 (6820), 408 (6750), 496 (5940)
274 (20 790), 310 (9790), 331 (5890),
345 (4520), 408 (1760), 480 (1620)
261 (27 150), 297 (21 230), 324
(12 180), 334 (10 520), 484 (3630),
580 (1670)
261 (27 620), 298 (21 100), 325
(12 370), 387 (4450), 484 (3690), 580
(1720)
285 (26 240), 305 (19 360), 333
(12 450), 406 (3650), 529 (2450), 700
(910)
CH3CN–CH3OH (5 : 1, v/v) (298)
624 (0.2)
750 (0.1)
pH 7 aqueous solutionc (298)
pH 3 aqueous solutionc (298)
pH 10 aqueous solutionc (298)
755 (0.1)
b
—
Solid (298)
Solid (77)
DMF (298)
716 (0.1)
755 (0.2)
634 (0.1)
3
278 (56 040), 290 (53 870), 310
(31 590), 348 (14 310), 426 (6130),
464 (5720)
CH3CN–CH3OH (5 : 1, v/v) (298)
pH 7 aqueous solutionc (298)
pH 3 aqueous solutionc (298)
pH 10 aqueous solutionc (298)
264 (26 020), 270 (26 300), 290
(27 600), 309 (16 590), 330 (10 390),
344 (8520), 453 (3380), 505 sh (960)
269 (23 140), 287 (19 440), 298
(16 470), 306 (14 040), 336 (8240),
347 (6910), 456 (3680), 575 (1220)
269 (23 250), 287 (19 690), 298
(16 660), 306 (14 250), 336 (8380),
347 (7050), 456 (3710), 575 (1220)
269 (23 350), 287 (19 750), 298
(16 700), 306 (14 300), 336 (8450),
347 (7050), 456 (3710), 575 (1220)
654 (0.1)
788 (<0.1)
788 (<0.1)
788 (<0.1)
Solid (298)
Solid (77)
759 (0.1)
791 (0.3)
a
Emission lifetimes were recorded with ꢂ 10% uncertainty. b Non-emissive. c Concentration of the complexes in aqueous solution (50 mM NaCl)
was 200 mM.
resolved at 20 ꢀC, but become much better resolved at elevated terpyridine proton signals are found to be better resolved at
temperatures (Fig. 1a). With reference to the previous study of elevated temperatures, suggesting that deaggregation has
alkynylplatinum(II) terpyridine complexes,5b it is likely that in occurred. Interestingly, the terpyridine proton signals at pH 10
ꢀ
aqueous solution at pH 4 complex 1 would aggregate at 20 C are found to be less broadened compared to those at pH 4
through metal–metal and/or p–p interactions and would (Fig. 1b), indicating that in aqueous solution complex 1 shows a
undergo deaggregation at higher temperatures. Similarly, for higher degree of aggregation at pH 4 than at pH 10. It is
the 1H NMR spectra of complex 1 in D2O at pH 10 (Fig. 1b), the conceivable that deprotonation of the phenolic protons of
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Fig. 2 Dynamic light scattering (DLS) experiments of complex 1 (200 mM) in
aqueous solution (50 mM NaCl) at 25 ꢀC at pH 4 (red) and pH 10 (green).
spectrum of complex 1 in aqueous solution (50 mM NaCl) at
pH 4 shows an intense absorption band at 465 nm and a lower-
energy absorption feature at 600 nm and is shown in Fig. S2.†
According to previous spectroscopic work on alkynylplatinu-
m(II) terpyridine complexes4,5 and the corresponding studies in
dimethylformamide solution, the absorption band at 465 nm is
tentatively assigned as admixtures of dp(Pt) / p*(tpy) MLCT
and alkynyl-to-terpyridine LLCT transitions. The lower-energy
Fig. 1 (a) 1H NMR spectra of complex 1 (200 mM) in D2O with 50 mM NaCl at pH
4 at different temperatures. (b) 1H NMR spectra of complex 1 (200 mM) in D2O
with 50 mM NaCl at pH 4 and pH 10 at different temperatures. The pH values are
adjusted by the addition of 0.02 M HCl and 0.02 M NaOH in D2O.
complex 1 at high pH leads to an increase in the hydrophilicity absorption at 600 nm, which is absent in dimethylformamide
of the complex and hence its deaggregation in aqueous solution, is found to show a decrease in absorbance upon an
medium. This is supported by the 1H NMR spectra of complex 2, increase in temperature (Fig. S3a†). On the other hand, a
which has one less phenolic proton than complex 1. The ter- decrease in temperature would lead to the growth of the
pyridine proton signals of complex 2 in D2O are also less absorption at 600 nm and a drop in the MLCT absorption,
broadened at higher temperatures and pH. This suggests that in which is reversible for at least six repeated cycles (Fig. S3b†). As
aqueous solution complex 2 would show a lower degree of supported by 1H NMR and DLS studies (vide supra), the
aggregation at high temperatures, typical of alkynylplatinum(II) absorption centred at 600 nm should originate from metal-
terpyridine complexes,5b and that deprotonation of the phenolic metal-to-ligand charge transfer (MMLCT) transitions, associ-
proton at high pH would lead to an increase in the hydrophi- ated with metal–metal and/or p–p interactions. The aggrega-
licity of complex 2 and its deaggregation in aqueous medium.
tion of complex 1 in aqueous solution (50 mM NaCl) at pH 4 is
The aggregation process of complex 1 in aqueous solution at supported by concentration-dependent UV-vis absorption
different pHs has also been studied by dynamic light scattering studies, where the low-energy absorption tail at 670 nm does
(DLS) experiments (Fig. 2). A number-averaged hydrodynamic not follow Beer's law (Fig. 3). This suggests that ground-state
diameter (Dh) of ca. 1.8 mm was found in pH 4 aqueous solution of aggregation of the complex molecules occurred in the pH 4
complex 1, while the DLS experiment of complex 1 at pH solution at high concentrations of complex 1, resulting in Pt/
10 revealed a smaller value (ca. 1.3 mm) with lower signal inten- Pt and/or p–p interactions and hence the MMLCT absorption.
sity. Moreover, an even smaller particle size (Dh of ca. 0.3 mm) was With reference to the dimerization model of platinum(II) ter-
observed in the pH 10 solution of complex 1 (Fig. 2). Based on the pyridine complexes reported in the literature,2a the dimeriza-
nding of large particles in mm range in both the pH 4 and 10 tion equilibrium constants of complex 1 in pH 4 aqueous
aqueous solutions, complex 1 would undergo aggregation with solution due to metal–metal and p–p interactions, KMM and
the formation of Pt/Pt and/or p–p interactions in the aqueous Kpp, are estimated to be 3.84 ꢃ 105 and 1.65 ꢃ 105 M respec-
solutions. However, the degree of aggregation of complex 1 at pH tively from the dimerization plot (Fig. S4†).
10 is probably lower than that at pH 4, as suggestedby the smaller
The electronic absorption spectrum of complex 1 at pH 10
Dh value and the lower intensity of the light scattering signal of (Fig. S2†) shows an absorption band at 480 nm and a broad
the large aggregates (Dh of ca. 1.3 mm) and the presence of smaller absorption tail at 650 nm. The absorption at 480 nm is similarly
particles in the solution, indicating the unfavourable formation assigned to MLCT transitions, while the absorption tail at
of aggregates of complex 1.
650 nm, which is more intense at temperatures lower than 20 ꢀC
The ground-state aggregation/deaggregation of complex 1 in but weaker at higher temperatures (Fig. S5†), should originate
aqueous solution at different pHs has been further investigated from MMLCT transitions through metal–metal and/or p–p
by UV-vis absorption measurements. The electronic absorption interactions. Interestingly, the MMLCT absorption at pH 10 is
2456 | Chem. Sci., 2013, 4, 2453–2462
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aqueous solution (50 mM NaCl) of complex 3, with methoxy
substituent on the alkynyl ligand instead of phenolic protons in
complexes 1 and 2, at 457 and 580 nm (Fig. S11 and S12†) are
both found to be unchanged at different pHs (Fig. S11†). This
suggests that the aggregation and deaggregation processes of
complexes 1 and 2 are modulated by the acid-base behaviour of
their phenolic protons. Deprotonation of the phenolic protons
in complexes 1 and 2 would occur at higher pH, which would
make the complexes more hydrophilic in nature, leading to the
deaggregation of the complexes in aqueous medium.
NIR emission spectral changes of the complexes in aqueous
solutions over physiological pH
The emission spectra of complex 1 in aqueous solution (50 mM
NaCl) at various pH values have been recorded by excitation at
480 nm (Fig. 4). At pH 4, an NIR emission band centred at
795 nm is observed, which is found to diminish at higher
temperature (Fig. 5). Although non-radiative decay would be
more efficient at higher temperature, such a drastic decrease in
Fig. 3 Concentration-dependent UV-vis absorption spectra of complex 1 in
aqueous solution (50 mM NaCl) at pH 4. Apparent absorbance refers to the
absorbance value corrected to 1 cm path length equivalence. Inset shows the plot
of apparent absorbance at 670 nm against concentration of complex 1.
red-shied compared to that at pH 4, which should not be due
to stronger aggregation at pH 10 as suggested from the 1H NMR
spectra of complex 1 in D2O at pH 4 and 10 (Fig. 1b), the DLS
experiments of complex 1 in pH 10 aqueous solution (Fig. 2) and
the smaller changes in the MMLCT absorption with tempera-
ture at pH 10 (Fig. S3a and S5†). Instead, it is likely that the red
shi is due to deprotonation of the phenolic protons of complex
1 at pH 10, that will lead to a stronger electron-donating effect of
the alkynyl ligand, and hence a rise of the energy of the dp(Pt)
orbital. Similar shis of the absorption band to the red with an
increase in pH have been reported previously.4a The smaller
extent of aggregation of complex 1 in pH 10 aqueous solution is
further supported by the results from concentration-dependent
electronic absorption study of the complex in pH 10 aqueous
solution, which shows a low-energy absorption tail at 750 nm
that follows Beer's law (Fig. S6†). This may probably be due to
the small association constant of the complex molecules in
aqueous solution at pH 10. It is worth noting that the aggre-
gation and deaggregation of complex 1 in aqueous solution
have been found to be reversible over at least seven cycles of pH
switching between 4 and 10, as monitored by the changes in
MMLCT absorption of complex 1 at 650 nm (Fig. S7†). For the
UV-vis absorption spectrum of complex 1 at pH 7 (Fig. S2†), the
absorption bands at 465 and 600 nm are assigned as MLCT/
LLCT and MMLCT transitions, respectively, similar to that at
pH 4. The weaker MMLCT absorption also indicates that
complex 1 is less aggregated at pH 7 than at pH 4.
ꢀ
the emission at 795 nm (the emission at 80 C was ca. 18% of
that at 15 ꢀC) cannot simply be accounted for by enhanced non-
radiative decay alone. Together with the observation of the
emission in the NIR region, the emission is most likely attrib-
uted to the aggregation of the complex 1 molecules through
With reference to the UV-vis absorption spectra of complex 1
at different pHs and at different temperatures (Fig. S2–S5†) and
the 1H NMR spectra of the terpyridine protons of complex 2 in
D2O, the electronic absorption features of complex 2 in aqueous
solution (50 mM NaCl) at both pH 4 and 7 centred at 488 and
585 nm as well as those at pH 10 centred at 498 and 600 nm are
similarly assigned as the respective MLCT/LLCT and MMLCT
transitions (Fig. S8–S10†). The red shi of the MLCT/LLCT and
MMLCT absorptions at pH 10 is also ascribed to the deproto-
nation of the phenolic proton in complex 2. On the other hand,
Fig. 4 (a) Emission spectra of complex 1 (200 mM) in aqueous solution (50 mM
NaCl) at various pH values at 25 ꢀC. lex ¼ 480 nm. (b) A plot of relative emission
the MLCT/LLCT and MMLCT absorption characteristics of an intensity of complex 1 at 795 nm versus pH.
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metal–metal and/or p–p interactions, where the dz2 orbitals on the zero–zero transition energy, E0–0, determined to be 1.84 eV
the platinum atoms of the complex molecules would overlap from the intersection of the electronic absorption and emission
with each other. This results in the formation of ds and ds* spectra of complex 1 in aqueous solution (50 mM NaCl), the
3
orbitals, leading to the MMLCT emission. At higher tempera- Gibbs free energy change of the PET from the phenolic moiety
ture, deaggregation probably occurs, and hence a signicant of the alkynyl ligand, DG, is estimated to be ꢁ0.06 and ꢁ0.42 eV
decrease in the low-energy emission is found.
in neutral and basic solution respectively. This indicates that
The emission intensity is relatively unchanged from pH 3.5 PET would be energetically more favourable to occur in basic
to 5.6. However, upon an increase in pH above 5.6, the NIR solution, probably due to the deprotonation of the phenolic
emission shows a signicant drop in intensity and becomes groups on the alkynyl ligand of complex 1 which becomes more
completely “turned-off” at or above pH 7.6. In view of the electron-rich. With a combination of aggregation/deaggregation
aggregation/deaggregation of complex 1 at different pHs as of platinum(II) complex moieties and PET, the changes in the
suggested from the 1H NMR, DLS and UV-vis absorption studies NIR emission intensity of complex 1 with pH is found to be ca.
(Fig. 1–3 and S1–S6†), the decrease in the NIR emission intensity 1350-fold, which is the highest ever reported value to the best of
has been ascribed to the deprotonation of the phenolic protons our knowledge. Such drastic changes would allow the NIR
of complex 1, whose pKa* is determined to be 6.27 from the emission of complex 1 to be “off” at physiological pH (7.0–7.4)
emission spectral changes with pH (Fig. 4b).19 It is likely that the but to be “on” upon small changes of pH below 7.0, for example,
deprotonation leads to an increase in the hydrophilicity of in tumour cells.11 Furthermore, although the NIR emission of
complex 1, causing deaggregation of the complex, and hence a complex 1 originates from aggregation of the complex moieties,
3
drop in its MMLCT emission intensity. This has further been the emission is relatively insensitive to the temperature below
ꢀ
supported by a lack of pH-dependence of the NIR emission of an 50 C (Fig. 5). This is probably due to the formation of strong
aqueous solution of the control complex, 3, at 788 nm aggregates of complex 1 in aqueous solution, requiring higher
(Fig. S13†). On the other hand, the NIR emission of an aqueous temperature to provide sufficient energy for the deaggregation.
solution of complex 2 centred at 755 nm is found to diminish at As a result, a small change in physiological temperature would
higher pH (Fig. S14†), similar to the spectral changes of complex not have signicant interference on the detection of pH by the
1. However, the spectral changes of complex 2 are much smaller NIR emission spectral responses of complex 1.
than those of complex 1, possibly due to the presence of only
one phenolic proton in complex 2. Therefore, the change in
hydrophilicity of complex 2 aer deprotonation (pKa* deter-
mined to be 7.71 from the emission spectral changes with pH,
Cell-imaging experiments using complex 1 as a pH-responsive
NIR luminescent probe
Fig. S14†) is not as signicant as that in complex 1, leading to a With the promising pH response and the attractive NIR emit-
smaller decrease in the 3MMLCT emission intensity at ting feature in solution, we were prompted to investigate the
higher pH.
potential application of complex 1 as a pH-responsive NIR
In addition, the complete “turning-off” of the NIR emission luminescent probe in cell-imaging experiments. Since the
of complex 1 at high pH, which is not commonly observed in the 3MMLCT emission of complex 1 arising from the aggregation of
reported deaggregation of platinum(II) terpyridine complexes,5b,d,e complex moieties could be altered by the presence of proteins
may suggest the presence of an additional quenching pathway. and biomolecules as shown by previous studies of alkynylpla-
Based on the electrochemical data of complex 1 (Table S1†) and tinum(II) terpyridine complexes with biomolecules in sol-
ution,4b,c,5c, f, g,h the effect of pH on the 3MMLCT emission in cells
was rst studied by using Madin–Darby canine kidney (MDCK)
cells xed with methanol. Methanol xation can result in
precipitation of proteins and removal of lipids from cells,20 and
thus any observable change in 3MMLCT emission intensity
would not be due to the binding of complex 1 to proteins or
other biomolecules. Moreover, as xed cells have permeable
membranes,20 any difference in the emission intensity should
not be ascribed to different concentrations of complex 1 in
cellular compartments. Therefore, xed-cell experiments with
incubation of buffer solutions at different pHs can reveal the pH
3
response of MMLCT emission of complex 1 in cells. Confocal
microscopy images of xed MDCK cells incubated with 20 mM of
complex 121 in serum- and phenol red-free Dulbecco's modied
Eagle's medium (DMEM) at 37 ꢀC for 1 h,22 followed by further
incubation in pH 5.72 MES buffer solutions at room tempera-
ture for 10 min, revealed a strong NIR emission band at lem
¼
750 ꢂ 50 nm (Fig. 6a). As xed cells have permeable
membranes,20 the intracellular pH should be equal to the pH
value of the buffer solution, suggesting that the xed cells
Fig. 5 Emission spectral changes of complex 1 (200 mM) in aqueous solution
(50 mM NaCl) at pH 4 with temperature. lex ¼ 452 nm. Inset: a plot of relative
emission intensity at 795 nm versus temperature.
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incubated with complex 1 at an intracellular pH of 5.72 would
show strong NIR emission. On the other hand, incubation of the
xed MDCK cells with 20 mM of complex 1 in serum- and phenol
red-free DMEM at 37 ꢀC for 1 h, followed by further incubation
in pH 6.75 and 7.80 MES buffer solutions, respectively, at room
temperature for 10 min, resulted in weak NIR emission (Fig. 6d
and g), indicating that the observation of NIR emission is
closely associated with the intracellular pH environment. A
more detailed study on the NIR emission of the xed MDCK
cells incubated with 20 mM of complex 1 followed by the incu-
bation with buffer solutions at different pH values has been
performed (Fig. 7). It has been found that the NIR emission
intensity of the xed MDCK cells starts to decrease when the
intracellular pH increases above 5.5, with the most signicant
diminution from pH 6 to 7, which is similar to the pH response
of the NIR emission of complex 1 in solution described above.
In addition, the NIR emission intensity of the xed MDCK cells
showed a sigmoidal relationship with the intracellular pH, with
a pKa* value determined to be 6.30, which is in good agreement
with the pKa* value of complex 1 (6.27) deduced from the
Fig. 7 Emission intensity changes of fixed MDCK cells incubated with 20 mM of
complex 1 at 37 ꢀC for 1 h followed by incubation with buffer solutions of known
pH at room temperature for 10 min. The emission of fixed MDCK cells was
measured by confocal microscopy with lex ¼ 488 nm and lem ¼ 750 ꢂ 50 nm.
emission spectral changes in solution at different pHs. As a (vide supra). Although the changes in the NIR emission intensity
result, the NIR emission should be ascribed to the 3MMLCT of complex 1 in the xed cells (ca. 118-fold) are smaller than
emission of complex 1 inside the xed MDCK cells. Upon an those in solution (ca. 1350-fold), probably due to the lower
increase in intracellular pH, deprotonation of the phenolic concentration of complex 1 in the cell-imaging experiments, the
protons in the alkynyl ligands of complex 1 would lead to an NIR emission of complex 1 in the cellular compartments still
increase in the hydrophilicity of the complex, resulting in both have signicant changes in confocal microscopy (Fig. 6a, d and
3
deaggregation and PET quenching to “turn off” the MMLCT g) over pH 6 to 7, which lies in the pH range that is highly
emission, which is similar to that observed in solution relevant to many cellular events and diseases such as tumour
formation.9–11
The observation of pH-responsive 3MMLCT emission of
complex 1 in xed MDCK cells prompted us to further study the
potential application of complex 1 as a pH-responsive NIR
luminescent probe for imaging acidic cellular compartments,
such as lysosomes, in living cells. Confocal microscopy images
of live MDCK cells incubated with 20 mM of complex 1 in serum-
ꢀ
and phenol red-free DMEM at 37 C for 1 h revealed a strong
NIR emission at lem ¼ 750 ꢂ 50 nm in vesicular distribution
(Fig. 8a–d). The MDCK cells remained viable aer the incuba-
tion as revealed by (1) their morphology in the differential
interference contrast (DIC) images and (2) the 3-(4,5-dimethyl-2-
thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay result
(ca. 97% viability as compared to untreated MDCK cells). The
NIR emission showed good co-localization with the green
emission of LysoSensor Green DND-189 (as indicated by the
yellow spots in the merged image), with a Mander's co-locali-
zation coefficient of 95%, where the Mander's co-localization
coefficient is a measure of the fraction of NIR emission of
complex 1 co-localizing with the green emission of DND-189.23
On the other hand, MDCK cells incubated with the control
complex 3, which exhibits pH-insensitive NIR emission upon
aggregation (Fig. S14†), in serum- and phenol red-free DMEM
for 1 h showed NIR emission, but in a rather diffused manner
Fig. 6 Confocal microscopy images of fixed MDCK cells incubated with 20 mM of
with a lower degree of co-localization with DND-189 (Fig. 8e–h;
complex 1 for 1 h followed by incubation with buffer solutions of pH 5.72 (a–c),
Mander's co-localization coefficient ¼ 57%). As DND-189 is
6.75 (d–f) and 7.80 (g–i) respectively at room temperature for 10 min. lex ¼ 488
known to accumulate and show stronger green emission in
nm and lem ¼ 750 ꢂ 50 nm. Luminescence (a, d and g), bright-field (b, e and h)
acidic organelles such as lysosomes, a high degree of co-local-
and overlaid (c, f and i) microscopy images are shown in the corresponding
figures.
ization with the strong NIR emission of complex 1 suggests that
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Fig. 8 Confocal microscopy images of live MDCK cells incubated with 20 mM of complex 1 for 1 h (a–d), and the microscopy images of live MDCK cells incubated with
20 mM of complex 3 for 1 h (e–h). lex ¼ 488 nm. Co-staining with 1 mM of DND-189 in serum- and phenol red-free DMEM was carried out 15 min prior to the imaging
experiments. Luminescence images at lem ¼ 750 ꢂ 50 nm (a and e), differential interference contrast (DIC) images (b and f), luminescence images at lem ¼ 525 ꢂ 25 nm
from the co-stained DND-189 (c and g) and the merged images of the emission at lem ¼ 750 ꢂ 50 nm and 525 ꢂ 25 nm (d and h) are shown in the corresponding
figures.
the complex emission is enhanced by the low pH in the envi- ligands, leading to an increase in the hydrophilicity of the
ronment in a way that is similar to the observed pH-dependent complexes and hence deaggregation of the complex moieties.
emission in solution and in xed cells. Therefore, our results Due to this deaggregation, together with the occurrence of PET,
showed that the emission intensity of complex 1 can be utilized the NIR emission of complex 1 is completely “turned-off” at pH
to distinguish acidic lysosomes from cytoplasm and other above 7.6. Cell-imaging experiments with complex 1 have
cellular organelles, where only weak NIR emission was demonstrated its potential as an NIR luminescent probe for the
observed, probably due to the deaggregation of the complex tracking of cellular compartments with deviations from physi-
moieties as well as PET quenching of the 3MMLCT emission at ological pH, such as lysosomes. Also, the complex may be
higher pH. On the other hand, it is possible that complex 3 can further exploited for probing intracellular events that involve
label cellular organelles other than acidic lysosomes as its NIR pH changes.
emission is not sensitive to changes in pH, and hence a more
diffused NIR emission that shows a lower degree of co-locali-
zation with DND-189 is found in the confocal microscopy
Acknowledgements
images (Fig. 8e and h). This further supports that the different
NIR emission intensity of complex 1 in MDCK cells is mainly
due to the different pH values of the cellular compartments.
With the observable changes in the NIR emission intensity in
the cells, complex 1 is a promising candidate for the tracking of
acidic organelles, such as lysosomes, or cellular events that
involve changes over pH in the physiological range.
V.W.-W.Y. acknowledges support from the University Grants
Committee Areas of Excellence Scheme (AoE/P-03/08), the
General Research Fund (GRF) (HKU 7064/11P) and Collabora-
tive Research Fund (CRF) (HKUST2/CRF/10) from the Research
Grants Council of Hong Kong Special Administrative Region,
China. C.Y.-S.C acknowledges the receipt of a Postgraduate
Studentship administered by The University of Hong Kong.
Dr A. Y.-Y. Tam is acknowledged for his helpful discussions.
Conclusions
Notes and references
In conclusion, water-soluble alkynylplatinum(II) terpyridine
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3MMLCT NIR emission through metal–metal and/or p–p
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