A Series of Zn(II) Terpyridine-Based Nitrate Complexes as Two-Photon Fluorescent Probe for Identifying Apoptotic and Living Cells via Subcellular Immigration

Article abstract of DOI:10.1021/acs.inorgchem.8b00620

Two-photon active probe to label apoptotic cells plays a significant role in biological systems. However, discrimination of live/apoptotic cells at subcellular level under microscopy remains unachieved. Here, three novel Zn(II) terpyridine-based nitrate c

Full text of DOI:10.1021/acs.inorgchem.8b00620

Article
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Cite This: Inorg. Chem. XXXX, XXX, XXX−XXX
A Series of Zn(II) Terpyridine-Based Nitrate Complexes as Two-
Photon Fluorescent Probe for Identifying Apoptotic and Living Cells
via Subcellular Immigration
Dandan Liu,† Mingzhu Zhang,† Wei Du,† Lei Hu,† Fei Li,† Xiaohe Tian,*,‡ Aidong Wang,⊥
Qiong Zhang,† Zhongping Zhang,§ Jieying Wu,† and Yupeng Tian*,†
‡
†Department of Chemistry, Key Laboratory of Functional Inorganic Material Chemistry of Anhui Province and School of Life
Science, Anhui University, Hefei 230039, P. R. China
⊥School of Chemistry and Chemical Engineering, Huangshan College, Huangshan, P. R. China
§School of Chemistry and Chemical Engineering, Anhui University, Hefei 230601, P. R. China
S
* Supporting Information
ABSTRACT: Two-photon active probe to label apoptotic cells plays a
significant role in biological systems. However, discrimination of live/
apoptotic cells at subcellular level under microscopy remains
unachieved. Here, three novel Zn(II) terpyridine-based nitrate
complexes (C1−C3) containing different pull/push units were
designed. The structures of the ligands and their corresponding Zn(II)
complexes were confirmed by single-crystal X-ray diffraction analysis.
On the basis of the comprehensive comparison, C3 had a suitable two-
photon absorption cross section in the near-infrared wavelength and
good biocompatibility. Under two-photon confocal microscopy and
transmission electron microscopy, it is found that C3 could target
mitochondria in living cells but immigrate into the nucleolus during the
apoptotic process. This dual-functional probe (C3) not only offers a
valuable image tool but also acts as an indicator for cell mortality at
subcellular level in a real-time manner.
photon probes has suffered from low penetration and photon
damage as well as the autofluorescence, which were obstacles
to their extensive use for imaging deep tissues and living
bodies. Two-photon absorption (2PM) has been reported for
deep penetration and avoiding autofluorescence of back-
ground, which is a promising approach to selectively detect
apoptosis.11−14 Therefore, apoptosis-targeting 2PA probes
containing optimized biocompatibility would be the ideal
candidates for imaging apoptosis.
Recently, the use of metal complexes became an attractive
approach for 2PM imaging. The metal complexes possessed
unique merits:15−17 (1) energy-level process, which promote
the electron transfer to enhance nonlinear optical (NLO)
activity after coordinating with metal ions; (2) deep
penetration and rapid response, which were important factors
in bioimaging applications; (3) moreover, intense fluorescence
and electron dense cores rendered them as dual-function
imaging for 2PM microscopy and transmission electron
microscopy (TEM).18 The merits of metal complexes were
not only that they possessed a stronger NLO effect but also
that they had potential bioimaging applications. Thereinto,
INTRODUCTION
■
Apoptosis, as a kind of programmed cell death, is vital for
modulating various processes in cells. Signaling of such process
occurred in different pathways, which, for example, include
viscosity changes and DNA damage.1−4 Apoptosis exhibits
several biochemical changes including cell lysis, the loss of
mitochondrial electrochemical potential, and nuclear mem-
brane damage that together result in various human conditions,
such as immunodeficiency, ischemic damage, cancer, and
neurodegenerative disease.5−8 Thus, real-time monitoring
apoptosis might play a great important role in clinical
diagnosis. Numerous fluorescent probes have been designed
and synthesized for detection of apoptosis. Tan and co-workers
utilized nanoparticle-based sensors to detect apoptotic cells
due to the breaking of plasma membrane permeability and
mitochondrial membrane permeability in early apoptosis.9
However, these nanoparticle-based sensors result in long
synthesis process, large molecular weight, and low water
solubility. He and co-workers have demonstrated an approach
for the study of detection apoptosis upon induction of one-
photon fluorescent probe;10 for that, the organic cationic
compound could accumulate in mitochondria with normal
MMP, and label the nucleolus in MMP vanishing cells due to
its static interaction with DNA. However, the use of one-
Received: March 14, 2018
© XXXX American Chemical Society
A
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−
Figure 1. (a) The structures for all the compounds. (b) Crystal structures of C1−C3 (H and NO3 were omitted, thermal ellipsoids at 50%
probability).
C2. Yield: 0.50 g (77%). IR (KBr, cm−1): 3061 (w), 2937 (w),
zinc complex with considerable two-photon activity has drawn
increasing attention.19−23 Because of their unique biocompat-
2839 (w), 1589 (s), 1522 (s), 1474 (s), 1360 (s), 1263 (s), 1151 (m),
1
1017 (s), 796 (m), 763.80 (w), 603 (w). H NMR (400 MHz, d6-
DMSO, ppm): δ 3.95 (s, 6H), 4.05 (s, 6H), 7.33 (d, 2H, J = 8.6 Hz),
ibility and less cytotoxicity, fluorescent zinc complexes could
be ideal to monitor apoptosis, while offering stable signal and
7.51 (m, 4H), 7.94 (m, 6H), 8.05 (d, 2H, J = 8.5 Hz), 8.29 (t, 4H, J =
less invasiveness toward living cells. However, Zn2+ fluorescent
7.5 Hz), 9.15 (d, 4H, J = 8.1 Hz), 9.31 (s, 4H); 13C NMR (100 MHz,
probes with both 2PA activity and apoptosis specificity are
scarcely reported according to our knowledge.
d6-DMSO, ppm): δ 55.98, 111.79, 120.35, 121.66, 123.49, 127.68,
141.23, 147.77, 149.32, 151.62, 155.33. MS (MALDI-TOF): m/z,
−
calcd: 928.26. Found: 401.10 [M-2NO3 ]2+/2.
Spurred on by this, Zn-terpyridine complexes (C1−C3)
obtained through self-assembly relied on the following merits
(Figure 1a): (1) Different terminal substituents in the
complexes can adjust the pull/push electronic capability of
the molecule and achieve enhanced 2PA properties; (2) The
metal Zn(II) was chosen as the central atom owing to its low
cytotoxicity and cationic nature, which can make the
complexes accumulate in the negatively charged mitochondria;
(3) Nitrate ions in the zinc complexes further increased their
solubility in water. As expected, Zn(II) complex C3 possessed
excellent 2PA properties in the near-infrared (NIR) region and
was applied for bioimaging application. 2PM imaging showed
that C3 could be accumulated in negatively charged
mitochondria24,25 and immigrated to the nucleolus in
apoptotic cells due to increased nuclear membrane perme-
ability. All these results suggest that C3 as a dual probe could
be a promising candidate for targeting apoptosis in 2PM
imaging.
C3. Yield: 0.54 g (81%). IR (KBr, cm−1): 3060 (w), 2969 (w),
2927 (w), 1588 (s), 1531 (m), 1472 (m), 1353 (s), 1212 (m), 1155
1
(m), 1016 (m), 790 (m), 682 (m). H NMR (400 MHz, d6-DMSO,
ppm): δ 1.19(m, 12H), 3.54 (q, 8H, J = 6.9 Hz), 6.92 (t, 4H, J = 14.6
Hz), 7.48 (dd, 4H, J = 5.5, 7.1 Hz), 7.91 (d, 4H, J = 4.8 Hz), 8.25 (m,
4H), 8.36 (m, 4H), 9.03 (d, 4H, J = 8.1 Hz), 9.22(d, 4H, J = 27.1
Hz). 13C NMR (100 MHz, d6-DMSO, ppm): δ 12.48, 44.71, 111.75,
118.07, 122.73, 12 7.55, 129.73, 141.35, 146.84, 150.00. MS
−
(MALDI-TOF): m/z, calcd: 950.38. Found: 412.17 [M-2NO3 ]2+/2.
RESULTS AND DISCUSSION
■
Crystal Structure Description. Structures of Ligands. As
shown in Figure S1, the single-crystal structures of L2 and L3
have been reported in the literature,26,27 the ligands L1 and L3
crystallized in monoclinic system with P21/c space group; L2
̅
belonged to triclinic system with P1. The dihedral angle
between P1 and P2 was 6.31° in L1, 7.43° in L2, and 5.04° in
L3. The excellent planarity suggested that L3 was conducive to
a better delocalized π-electron system.28
EXPERIMENTAL SECTION
■
Structures of Zn(II) Complexes. Crystals of C1 and C2
belonged to triclinic with P1 space groups, and for C3, it was
̅
The synthetic procedures of the terpyridine ligands (L1−L3) and the
relevant characterization data were listed in Supporting Information.
Synthesis of C1. L1 (0.46 g, 1.40 mmol) was dissolved in ethanol
(50 mL), and Zn(NO3)2·6H2O (0.21 g, 0.70 mmol) was added and
then refluxed for 3 h. After it cooled to 25 °C it was filtered. The
residue was purified by ethanol to give the product as a white solid
(0.44 g, 74%). IR (KBr, cm−1): 1614 (s), 1599 (s), 1516 (m), 1475
(m), 1432 (m), 1381 (s), 1228 (s), 1165 (s), 1015 (m), 844 (m), 793
monoclinic system with P21/c space group (Table S1).
Compared with the free ligands, the complexes displayed the
expected octahedral geometry with two terpyridine ligands and
one central Zn atom.29 Because of the steric demand of the
ligands, the N−Zn−N angles in the complexes were within the
75.01−76.01° range, deviating from the idealized values of 90°
(Table S2). Furthermore, the dihedral angle between P4 and
P3 followed the order of C1 (12.44°) > C3 (11.55°) > C2
(8.00°), revealing that there is a distinct substitute change in
the terminal substitutent for the terpyridine moiety of the
complexes with subtle effect on their dihedral angles (Figure
1b). As expected, the structural characteristics had a great
impact on the optical performance and 2PA response.
1
(m), 571 (m). H NMR (400 MHz, deuterated dimethyl sulfoxide
(d6-DMSO), ppm): δ 7.51 (m, 4H), 7.65 (t, 4H, J = 8.6 Hz), 7.96 (d,
4H, J = 4.4, 8.0 Hz), 8.30 (t, 4H, J = 7.6 Hz), 8.53 (m, 4H), 9.15 (d,
4H, J = 8.0 Hz), 9.40 (s, 4H). 13C NMR (100 MHz, d6-DMSO,
ppm): δ 116.47, 120.95, 123.52, 127.69, 130.66, 131.99, 141.31,
147.73, 149.46, 153.87, 163.00. Mass spectrometry (MS; matrix-
assisted laser desorption ionization time-of-flight (MALDI-TOF)):
−
m/z, calcd: 844.11. Found: 359.25 [M-2NO3 ]2+/2.
B
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Figure 2. UV−Vis absorption (a) and normalized fluorescence (b) spectra for C1−C3 in benzene (c = 10 μM).
Optical Properties. The UV−vis absorption spectra of the
complexes were presented in Figure 2a, and the complexes
present three absorption bands. The absorption bands at 280−
295 nm could be assigned to the π−π* transition.30 The
absorption bands located at 327−356 nm were attributed to
the intramolecular charge transfer (ICT) transition, and the
absorption band at 417 nm was assigned to the metal-to-ligand
charge transfer (MLCT), which was further proved by time-
dependent density functional theory (TD-DFT).
The low-energy bands of C1 and C2 were calculated at 328
and 341 nm (corresponding to the highest occupied molecular
orbital (HOMO) → lowest unoccupied molecular orbital
(LUMO+1) and HOMO−1 → LUMO, respectively), which
might be ascribed to the ICT and mixed with the ligand-to-
ligand charge transfer (LLCT) transition (Figure S3). The
high-energy bands (280−290 nm) were assigned to the
πterpyridine−π*terpyridine transitions (HOMO → LUMO in C1,
HOMO−1 → LUMO+1 in C2). In addition, the C3 presented
a little different transition features (Figure S3c), the high-
energy band (λmax = 294 nm) was assigned mainly to the
πterpyridine−π*terpyridine transition due to the HOMO−1 →
LUMO+1. And the moderate bands at 327 and 356 nm
(corresponding to the HOMO−1 → LUMO+2 and HOMO
→ LUMO+2, respectively), correspond to the ICT mixed with
the LLCT transition. Moreover, the low-energy peak at 417
nm was attributed to the MLCT transition (HOMO−2 →
LUMO). In general, the TD-DFT calculations could offer
reasonable explanation for their experimental absorption
bands.
The maximal absorbance bands and the fluorescence peaks
of the complexes in benzene presented a remarkable red shift
in the sequence of C1 < C2 < C3 (Figure 2), which could be
attributed to the electron-pushing strength of the end group in
the complexes correspondingly: N(CH2CH3)2 > OCH3 > F.
Meanwhile, TD-DFT of the Zn(II) complexes at low-energy
peaks were 3.72 eV (C1), 3.58 eV (C2), and 2.97 eV (C3),
respectively, giving an approving explanation on the red-shift
phenomenon from C1 to C3.
Nonlinear Optical Properties (NLO). Given that there was a
weak fluorescent emission for C2 and C3 in dimethyl sulfoxide
(DMSO; Figure S4), the third-order nonlinear optical
properties of the compounds were measured by open-aperture
Z-scan technique.31,32 An obvious enhancement of σ values
was observed from ligands to the Zn(II) complexes (Figure 3),
which was attributed to the extension of the conjugation
length. Besides, the order of the maximum σ values of the
Figure 3. Z-Scan data for L1−L3 and C1−C3 (c = 10 μM) in
DMSO. The solid curve is the theoretical fitting, and the dots are the
experimental data.
complexes C1−C3 is C1 (709 GM) < C3 (1068 GM) < C2
(1260 GM). It can be explained that the planarity of the
complexes will be different due to the coordination with the
different ligands and that the good planarity will benefit to
enhance the ability of charge transfer, thus increasing the two-
photon absorption properties. This conclusion is highly in
agreement with the results from both crystal structures and
Lippert−Mataga plots (Table S3 and Figures 1 and S5).
Subcellular Location of C3 in Living Cells and Its
Immigration during Apoptosis. As depicted in Figure S6,
the low toxicity in living cells enables the compounds (L2, L3,
C2, C3) as prominent fluorescent probes. Since C3 had
excellent comprehensive photophysical optical properties, as
well as a larger σ value in the longer excitation wavelength,
which increased the penetration depth, C3 was subsequently
used to test the capability of cellular location. As shown in
Figure S7, C3 exhibited fibrillar luminescence in the cytosol,
and no luminescence was observed in the nucleolus. To further
determine the initial subcellular location of C3, the
colocalization experiments with C3 and various organelle
markers (Mito-tracker, ER-tracker, Lyso-tracker, and Nuc-red)
were performed (Figure 4a). 2PM images strongly suggested
that C3 targeted the intracellular mitochondria (Pearson
correlation coefficient Rr = 0.86), which was also confirmed via
processed fluorescence intensity profiles (Figure 4b). The
affinity of C3 toward mitochondria was not unexpected, since
its cationic nature might render the binding with subcellular
compartment with high membrane potential.
C
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Figure 4. (a) Determination of subcellular localization of C3 under 2PM. HeLa cells were incubated with C3 (λex = 720 nm, λem = 450−480 nm)
for 20 min and coincubated with Mito-tracker (MitoTracker Red FM, λex = 580 nm, λem = 590−620 nm) for 20 min, ER-tracker (ER-Tracker red,
λex = 588 nm, λem = 600−625 nm) for 20 min, Lyso-tracker (LysoTracker Red, λex = 576 nm, λem = 580−610 nm) for 20 min, and Nuc-red
(NucRedLive647, λex = 647 nm, λem = 650−670 nm) for 10 min, respectively. The scale bars represent 20 μm. (b) The fluorescence intensity
profiles of C3 and trackers.
Intriguingly, at a starvation pretreated group (serum-free
condition) from Figure 5a, while the cells partially underwent
apoptosis, stronger signals in the whole nucleus were found
within the apoptotic cells, which were extremely swollen and
misshaped in bright-field micrographs. Since apoptosis was
accompanied by increased permeability of the nuclear
membrane and disappearance of mitochondrial membrane
potential, it might cause the C3 migration from mitochondria
to nucleus and “turn-on” the fluorescence upon nuclear acid
binding.
To further confirm that C3 accumulated in mitochondria
when the cells were alive and immigrated to nucleolus during
apoptosis, apoptotic cells triggered by classic inducer was
performed.33 As shown in Figure S8, Live/Dead assays with
Syto9 (a commercial living cells dye) and propidium iodide
(PI, a commercial cells dye) again confirmed that C3 targeted
the mitochondria in live cells. However, as displayed in Figure
5b, the fluorescence of C3 in drug-induced cells (5-Fu, cis-
platinum) was well-colocalized with that of PI, which
demonstrated that C3 exhibited excellent nucleolus-targeting
abilities that are attributable to the increased permeability of
the nuclear membrane during apoptosis.34 The results verified
our design idea and implied the distribution of C3 relocated
from mitochondria to nucleolus during apoptosis. Further-
more, the targeting capability of C3 in Triton-x100 treated
cells and the fixed HeLa cells were well-consistent with the
drug-induced cells,35 further demonstrating that disrupted
mitochondrial membrane potential and increased nuclear
membrane permeability leads to the transfer of C3 from
mitochondria to nucleolus. During Triton-permeabilization
and classic paraformaldehyde fixation (Figure S9) the whole
cells was immobilized, and C3 displayed stronger signal than
that in apoptotic cells. It is noteworthy that the bright circular
signal within apoptotic cell nucleus might correspond to
nucleolus, which mainly consists of negatively charged RNA.
This implied that C3 in apoptotic cells may target nuclear
RNA. As RNA also abundantly exists in mitochondria and
nucleolus, it also might imply that the binding substance of C3
in living and fixed cells is mitochondrial and nucleolar RNA.
In addition to 2PM results, transmission electron micros-
copy (TEM) image of C3 was performed to confirm cellular
distribution. As shown in Figure 6a, C3 in nontreated cells
displayed abundant cylinder-like structures in the cell cytosol,
highly corresponding to the mitochondria. In contrast, HeLa
cells incubated with C3 and pretreated with 5-Fu presented
reduced contrast in cytosolic regions with a large number of
vacuoles, a classic symbol of early apoptosis, while significantly
greater contrast was detected in the nucleolus (Figure 6b).
This observation strongly supported our previous hypothesis
that the intracellular immigration of C3 is from mitochondria
to RNA-rich nucleolus.
It is interesting to note that C3 stained mitochondrial
displayed a significant contrast within inner-membrane matrix
(Figure 6c), while compared to mitochondria treated solely
with osmium tetroxide (Figure 6d), a membrane electron
microscopic agent, indicated much less contrast in inner-
membrane matrix (Figure 6e). This again suggested C3 is
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Figure 5. (a) HeLa cells starvation treated for 12 h and then stained with C3. White arrows identify the apoptocic cells in the culture. (b)
Treatment with the apoptotic stimulants 5-Fu (5-fluorruracil), cis-platinum, and Triton-x100 then stained C3 and costain PI (λex = 493 nm, λem
=
620−640 nm). Scale bar = 20 μm, and the relative fluorescence intensity of Cyt (cytoplasmic) and Nuc (nucleus) in HeLa cells after apoptosis-
induced treatment.
Figure 6. (a) TEM micrograph of live HeLa cells incubated with C3. (b) 5-Fu induced apoptotic cells incubated with C3. (c) Enlarged TEM
micrograph from selected regions from (a) showing inner-membrane matrix. (e) Untreated HeLa cells solely stained with osmium tetroxide clearly
showed bilayer mitochondria and less contrasted inner-membrane matrix. The scale bar represents 5 μm. Abbreviations: mt = mitochondria, n =
nuclear, no = nucleolus, nm = nuclear membrane, im = inner-membrane matrix, v = vesicles.
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Figure 7. (a) The real-time monitoring images of HeLa cells intracellular fluorescence of C3 after treatment with H2O2. (b) Plot of fluorescence
intensity obtained from (a). (c) The real-time monitoring images of HeLa cells intracellular fluorescence of C3 after treatment with CCCP. Scale
bar = 20 μm.
Figure 8. (a) Fluorescence spectral changes of 5 μM C3 with 20 mM RNA. (b) Z-Scan data for 5 μM C3 with 20 mM RNA. (c) 1H NMR titration
change for C3 + RNA in D2O. (d) Models obtained after molecular modeling for the interaction of C3 with RNA fragment.
actually capable of penetrating mitochondrial bilayer mem-
brane and targeting matrix RNA.
cell morphology was observed and that mitochondrial signal
was successfully obtained. After the extension of H2O2
treatment time, the cells showed shrinking morphologic
changes as well as an attenuated fluorescence of mitochondria
and an increased fluorescence of nucleolus (Figure 7a). After
∼360 s, the maximum fluorescence intensity was observed in
nucleolus. In general, the nuclear membrane permeability was
increased with the extension of the treatment time by H2O2,
resulting in more C3 probes being transported to the
The Real-Time Imaging of C3 Subcellular Immigrating.
Taking advantage of the applicability to real-time imaging, C3
was applied to investigate intracellular distribution during
apoptosis induced by H2O2. The live HeLa cells were initially
stained with C3, and the fluorescence intensities were collected
(Figure 7 and Supporting Information movie). 2PM imaging
results showed that, before adding H2O2, no obvious change in
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nucleolus. At the same time, these significant changes in
intracellular fluorescence with H2O2 time indeed matched well
with the average fluorescence intensity of the linear region
across the cells (Figure 7b). The sharp difference in
fluorescence responses between the live and apoptotic cells
highlighted the specificity and sensitivity of the C3 probe in
detecting cell mortality in a real-time manner. Similar results
were observed with CCCP-treated (to decrease mitochondrial
membrane potential) apoptosis process (Figure 7c); with the
extension of CCCP treatment time, C3 could no longer
accumulate in mitochondria, yet integrated nuclear membrane
prevented its entry, resulting in the decrease of the
fluorescence in mitochondria.
discriminate live and apoptotic cells from organelles differ-
entiated fluorescence.
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.inorg-
chem.8b00620.
Crystal data, photophysical properties, and character-
ization data of all the compounds (PDF)
Related fluorescence intensities and HeLa cells stained
with C3 (AVI)
Mechanism on Targeting and Localization Properties. To
explain the mechanism that C3 targeted nucleolus in apoptotic
cells, the interaction of C3 with RNA (nuclear acids) was
preliminarily performed by single-photon fluorescent and two-
photon fluorescent spectra. Such interactions will be
strengthened upon the specific binding to the complementary
target RNA (Figure 8), thus resulting in a “turn-on” effect.
Accession Codes
CCDC 1534933, 1568681, 1570231, and 1587882 contain the
supplementary crystallographic data for this paper. These data
can be obtained free of charge via www.ccdc.cam.ac.uk/
data_request/cif, or by emailing data_request@ccdc.cam.ac.
uk, or by contacting The Cambridge Crystallographic Data
Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44
1223 336033.
1
To confirm that C3 was internalized with RNA, H NMR
titration experiments were performed (Figure 8c). The protons
Ha (Ha, Ha′, Ha″, and Ha′′′) and Hb (Hb, Hb′, Hb′′, and
Hb′′′) of the pyridine ring were upfield-shifted, and the signal
intensity gradually weakened after titrating RNA, which
suggested the H around C−C bond provided the possible
binding sites for RNA. In combination with the optical
properties, C3 and RNA interactions might affect the
electronic distribution in the molecule.
Further molecular docking explained the mechanism of C3
binding to RNA. Evidently, C3 could insert duplex RNA
(Figure 8d), which is consistent with the fluorescence titration
experiments. It also indicated that the hydrogen bonds
between C3 and RNA are derived from terpyridine groups
and RNA base pairs.
AUTHOR INFORMATION
Corresponding Authors
*E-mail: yptian@ahu.edu.cn. (Y.-P.T.)
*E-mail: xiaohe.t@ahu.edu.cn. (X.-H.T.)
■
ORCID
Dandan Liu: 0000-0002-1584-9299
Wei Du: 0000-0002-7959-6227
Fei Li: 0000-0001-9822-7526
Xiaohe Tian: 0000-0002-2294-3945
Zhongping Zhang: 0000-0002-0991-7611
Author Contributions
All of these explorations suggested that probe C3 primarily
served as a selective two-photon probe for apoptotic cells. It
was confirmed that C3 showed a significant emission
enhancement with RNA addition. Such result is critical, since
mitochondrial RNA-based metal probes were rarely reported.
It is known that RNA including tRNA (tRNA), mRNA
(mRNA), and rRNA (rRNA) can be found in cytosol,
mitochondria, and nucleolus. C3 thus offered a tool to
investigate their dynamic change during apoptotic process,
with high biocompability and photon stability. Related studies
were undertaken and will be reported in further publication.
D.D.L. and M.Z.Z. contributed equally to this work and should
be considered as cofirst authors.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by a grant from the National Natural
Science Foundation of China (51432001, 51672002,
21602003, 51772002, and 21501001), Anhui Provincial
Natural Science Foundation of China (1708085MC68), and
Anhui Univ. Doctor Startup Fund (J01001962), Returnees
innovation and entrepreneurship key support program,
Postdoctoral Science Foundation of Anhui Province
(2016B092), China Postdoctoral Science Foundation
(2017M612050). The Natural Science Foundation of Anhui
Province (1508085MB34). Focus on returned overseas scholar
of Ministry of Education of China, the Higher Education
Revitalization Plan Talent Project (2013).
CONCLUSIONS
■
In conclusion, three Zn(II) complexes with 2PA were
synthesized and confirmed through single-crystal X-ray
diffraction analysis. With slight modification of the terminal
substituent, these zinc complexes had obvious changes in the
photophysical performance. The moderate 2PA activity as well
as high cell viability of the Zn(II) complex make C3 a
promising tool for bioimaging applications. 2PM and TEM
images established that C3 initially localized to the
mitochondria and then moved to the nucleolus during
apoptosis, possibly binding with RNA. The success of C3
should be attributed to the aborative adjusting of the chemical
structure of organic cationic complexes. Furthermore, the real-
time studies using 2PM demonstrated that C3 was able to act
as an indicator for cell mortality at subcellular level. These
results make C3 of interest as the basis to design a probe to
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