Halides tuning the subcellular-targeting in two-photon emissive complexes: Via different uptake mechanisms

Article abstract of DOI:10.1039/c7cc03640j

We reported a simple and universal strategy by tuning halides (Cl, Br and I) in terpyridine-Zn(ii) complexes to achieve different subcellular organelle targeting (nucleolus, nucleus and intracellular membrane systems, respectively) via different cellular

Full text of DOI:10.1039/c7cc03640j

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Reactivity differences of Sc3N@C2n (2n 68 and 80). Synthesis of the
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Halides Tuning Subcellular-Targeting in Two-Photon Emissive
Complexes via Different Uptake Mechanisms
Xiaohe Tian,‡[a,b] Yingzhong Zhu,‡[b]Qiong Zhang,[b] Ruilong Zhang,*[b] Jieying Wu,[b] and Yupeng
Tian*[b,c]
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
element ion, Zn(II) with d10 electronic configuration, was
usually used to synthesize metal-organic complexes with low
toxicities and strong emissions.[26-29] In present work, we
We reported a simple and universal strategy by tuning halides (Cl,
Br and I) in terpyridine-Zn(II) complexes to achieve different
subcellular organelles targeting (nucleolus, nucleus and
intracellular membrane system, respectively) via different cellular
uptake mechanisms, resulting from halides triggering different
polymorphies of these complexes.
reported that three terpyridine-ZnX2 (X = Cl, Br and I)
complexes can localize in distinct subcellular organelles via
different cell entry mechanisms, achieving to visualize
Nucleolus, nucleus, endoplasmic reticulum (ER) and other subcellular organelles simply.
subcellular organelles are of universal interesting, as each Firstly, the ligand, diethyl 3-(4-([2,2':6',2''-terpyridin]-4'-
subcellular compartment plays a key role in cell dividing, yl)phenyl)pentanedioate
(DTPP) and its three complexes
protein translation/transportation and otherwise.[1] Direct in DTPP-ZnCl2
,
DTPP-ZnBr2 and DTPP-ZnI2 (Scheme 1 and S1)
cellulo visualization of these subcellular structures by the aid have been synthesized, and characterized by 1H NMR, 13C NMR,
of fluorescent probes is very convenient as a powerful MS and single-crystal XRD (Fig. S1-S7). Subsequently, their
research method.[2-5] Within numerous fluorescent probes fluorescent properties have been investigated in Fig. 1. With
reported, we can change the location of fluorophores in living the excitation at 385 nm, DTPP exhibited strong fluorescence
cells through the alteration of the targeting moieties, such as at 480 nm. Different from the ligand DTPP, its three complexes
triphenylphosphonium could accumulate in mitochondria,[6-8] underwent a redshift with the emission at around 540 nm,
and morpholine could specially target acidic lysosomes.[9-11] since the ligand binding to Zn(II) led to more extensive π-
However, such methods were time consuming and costly, and conjugated system and stronger intramolecular charge
brought obstacles for further modification. Consequently, transfer, which were certified by computer-aided calculations
seeking a simple way to tune localizations intracellularly (Fig. S8). In the two-photon fluorescence studies, it was found
through the functionalization of fluorescent dyes is urgently that the ligand DTPP could not emit two-photon fluorescence.
required.
Metal-organic coordination is a widely used method to assign photon emissions of the three complexes were apparently
advantages to organic compound by the introductions of metal enhanced than that of DTPP as the reasons pointed above[30]
Interestingly, after coordinating with Zn(II) halides, the two-
.
ions and counter-anions, including tunable properties and It should be noted that ligand DTPP without two-photon
fluorescent enhancement.[12-14] In general, lanthanide ions (e.g. emission can be effectively avoid the interference from the
Eu(III) and Tb(III)),[15-17] group VIII elements ions (e.g. Ir(III), disassociation of complexes under two-photon fluorescent
Ru(II) and Pt(II))[18-20] and other transition metal ions with d0, d5 imaging. On the basis of the optical spectra of three halides
and d10 electronic conformations[21-23] were usually applied in complexes, we selected 850 nm as excitation wavelength, and
preparing cooridinations with strong one- and two-photon 550 - 600 nm as collected range (Fig. S9) for two-photon
fluorescence. Whereas most of these ions were not essential biological imaging after evaluating their cytotoxicity (Fig. S10,
elements with relatively high toxicity, which would bring out MTT assay).
large invasive effects and unexpected reactions in biological
applications.[24, 25] To avoid these interferences, an essential
X
X
Zn
N
N
N
N
N
N
N
ZnX
2
X = Cl, Br, I
X = Cl DTPP-ZnCl
2
X = Br DTPP-ZnBr
2
X = I DTPP-ZnI
2
EtOOC
N
EtOOC
DTPP
COOEt
COOEt
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Scheme 1. The structures of ligand DTPP and its three zinc halide and DAPI. (c) Two-photon fluorescent images of dividing HepG2 cells
complexes DTPP-ZnX2 (X = Cl, Br and I). treated with DTPP-ZnBr2 and DAPI. Scale bars = 20 m.
µ
b)
a)
1200
800
400
0
100
75
50
25
0
DTPP
DTPP-ZnCl
DTPP-ZnBr
DTPP-ZnI
DTPP
DTPP-ZnCl
DTPP-ZnBr
DTPP-ZnI
To precisely determine the subcellular locations of these
complexes, HepG2 cells incubated individually with DTPP-ZnCl2
DTPP-ZnBr2 and DTPP-ZnI2 were co-stained with the
commercial organelle probes Syto9 for intracellular RNA
(cytosolic and nuclear RNA), 4’,6-diamidino-2-phenylindole
(DAPI) for nuclear DNA, and lipophilic endoplasmic reticulum
tracker (ER tracker), respectively (Fig. 2). In Fig. 2a, DTPP-ZnCl2
displayed a high signal overlap with Syto9 in the nuclear region
but not in cytosolic region, and its Pearson’s coefficient (Rr)
was 0.5286, suggesting that DTPP-ZnCl2 is mainly located
within nucleoli. Meanwhile, the incubation of HepG2 cells with
DTPP-ZnBr2 displayed perfect overlap with DNA-specific DAPI
(Rr=0.8352), which can evident that intracellular binding
sources of DTPP-ZnBr2 is nucleic DNA. In contrast to DTPP-
2
2
2
2
,
2
2
450 500 550 600 650 700 750
450 500 550 600 650 700
Wavelength (nm)
Wavelength (nm)
Fig.1 One- (a) and two-photon excited at 850 nm (b)
fluorescence spectra of DTPP DTPP-ZnCl2 DTPP-ZnBr2 and
DTPP-ZnI2
,
,
.
Subsequently, live cells staining using the three complexes
were performed. HepG2 cells (human liver hepatocellular
carcinoma) as a model were individually incubated with each
of the complexes (1 μM) for 60 min, and then probed under
two-photon confocal laser scanning microscopy. The three
complexes displayed intense in cellulo fluorescence after
incubation (Fig. 2 and S11). Intriguingly, the locations of the
three complexes in living cells were completely different.
While DTPP-ZnCl2 and DTPP-ZnBr2 clearly located at nuclear
region, signals from DTPP-ZnI2 apparently exclude from cell
nuclear but a generalized cytosolic staining pattern was
observed.
ZnCl2 and DTPP-ZnBr2, DTPP-ZnI2 exhibited higher level of
overlapping with ER tracker, and its Pearson’s coefficient was
Rr = 0.6921 (Fig. 2a). The relatively lower Pearson’s coefficient
implied that DTPP-ZnI2 also could be internalized with other
membrane-rich organelles, such as mitochondria and Golgi
apparatus.
More importantly, as shown in magnified images (Fig. 2b), it is
apparently that as DTPP-ZnCl2 preferentially binds to nucleoli
over other nuclear substances, since no emission from DAPI
was detected from DTPP-ZnCl2 Labelled nucleolar
region(indicated by white circle). Moreover, during cell
dividing in a pre-metaphase cell, for instance, when the
nuclear membrane disappeared and nucleoli disassembled (Fig.
2c), the luminescent signal of DTPP-ZnBr2 remained highly
colocalized with DAPI, which again strongly implied that the
binding sources of DTPP-ZnBr2 in living cells nuclear is DNA (Fig.
S13), which also supported by molecular docking results (Fig.
S14). The initial two-photon confocal studies indicated the high
affinity of these complexes with their correspondence
subcellular compartments over another intracellular species.
To further confirm the intracellular distributions of the three
complexes, transmission electron microscopy (TEM) tests were
Fig. 2 (a) Two-photon fluorescent images of HepG2 cells treated
Fig. 3 (a) TEM images of HepG2 treated with OsO4 solely as control
experiments. (b-d) TEM images of HepG2 treated with DTPP-ZnCl2,
DTPP-ZnBr2 and DTPP-ZnI2, respectively. Abbreviations: n = nucleus,
with DTPP-ZnX2 (X
= Cl, Br and I) and the corresponding
colocalization dyes. For DTPP-ZnCl2, DTPP-ZnCl2 and DTPP-ZnI2,
Syto9, DAPI and ER dyes were used, respectively. (b) Two-photon
fluorescent images of HepG2 cells treated with DTPP-ZnCl2, Syto9
mt
= mitochondria, pm = plasma membrane, nm = nuclear
membrane, no=nucleolu, v = vesicles, er = endoplasmic reticulum,
2
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rer = rought endoplasmic reticulum, chr = chromosomes. Scale bar= 5 μm.
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Fig. 4 (a) Treatment with the cell entry inhibitors show uptake of DTPP-ZnCl2 (upper), DTPP-ZnBr2 (central) and DTPP-ZnI2 (lower)
by HepG2 cells, while propidium iodide (PI) indicated the cell mortality after treatments. (b) Relative fluorescence intensities of
cells treated with DTPP-ZnCl2 (left), DTPP-ZnBr2 (middle) and DTPP-ZnI2 (right). Scale bars = 50 μm.
[31]
performed using these three complexes as contrast agents
To seek the reasonable explanation for this intriguing
DTPP-
(Fig. 3). We firstly used osmium tetroxide (OsO4) incubating phenomenon, cell uptake mechanisms of DTPP-ZnCl2
,
cells as control experiments to enhance the membrane ZnBr2 and DTPP-ZnI2 were investigated under different
contrast. As shown in left column of Fig. 3a, positive control incubation temperatures (0 and 25 oC) and via inhibition
cells were stained solely with OsO4, a phospholipid contrast studies using well-documented endocytosis and active
agent that widely used in TEM, and their intracellular transport inhibitors.[32, 33] According to the results as shown in
membrane structures including mitochondria, vesicles, and Fig. 4a and 4b, we proposed the uptake mechanisms: the three
rough endoplasmic reticulum, plasma membrane and nuclear complexes entered cells via different energy-dependent
membrane were clearly observed in magnified picture. When uptake mechanisms; where DTPP-ZnCl2 via classic endocytosis,
HepG2 cells incubated with DTPP-ZnCl2 without OsO4, DTPP-ZnBr2 via active transport, and DTPP-ZnI2 via a non-
intracellular membrane compartments presented negligible endocytotic/non-active transport, but energy dependent
signal, whereas two representative micrographs showed much pathway. As a result, complexes DTPP-ZnCl2, DTPP-ZnBr2 and
better contrast in cell nucleolus due to the accumulation of DTPP-ZnI2 penetrated through the plasma/nuclear membrane
DTPP-ZnCl2 (Fig. 3b). In the case of DTPP-ZnBr2, it was found and accumulated to a great extent within nucleoli, nucleus and
that nuclear region beard much higher concentration in an other intercellular membrane system, respectively. It is
interphase cell, and DTPP-ZnBr2 clearly localizes within noteworthy that endocytotic mechanism might also partially
chromatin (Fig. 3c, left). It was also interesting to note that in a involved in taking up DTPP-ZnBr2 in living cells, as nacodazole,
pre-metaphase cell, once the nuclear membrane disappeared, 2-deoxy-D-glucose and colchicine obviously influenced the
consistent DTPP-ZnBr2 signal was observed from DNA dense intracellular diffusion and caused unspecific cytosolic
chromosomes (Fig. 3c, right) and displayed typical fluorescence. Above proposed cell entry mechanisms were
chromosomal quaternary tubular structure. On the contrary, further supported using pre-fixed and permeabilized cells.
when HepG2 cells were incubated with DTPP-ZnI2, much less Once membrane proteins lost their functions due to the
nuclear uptake occurred with a clear presentation of the fixation, these complexes displayed non-specificity towards
intracellular membrane compartments including ER, nucleoli, nucleus and endoplasmic reticulum (Fig. S16 and S17),
mitochondria, vesicles and nuclear membrane. The above TEM and
a generalized whole cell-staining pattern was also
studies are in well agreement with the initial cell staining observed. These results indicated that the coordinated halide
results exploited under confocal microscopy; further anions significantly influence the cell entry pathway, leading to
strengthen those complexes DTPP-ZnCl2, DTPP-ZnBr2 and different localizations in living cells.
DTPP-ZnI2 targeted different subcellular compartments, It is noted that the three complexes may be not very stable in
although the three complexes share similar structural apart actual biological environments, since there are a large number
from the coordinate halide moiety.
of bio-molecules in living cells, including thiols, amino acids,
various anions and others species. In this study, we mixed the
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This work was supported by a grant for the National Natural
Science Foundation of China (21602003, 51372003, 21271004,
51432001, and 51271003, 21501001), Anhui University Doctor
Startup Fund (J01001962). The work of molecular modeling
calculations on Discovery Studio (Dassault Systemes BIOVIA,
Discovery Studio Modeling Environment, Release 4.5, San
Diego: Dassault Systemes, 2015) was contributed by Professor
Aidong Wang from Huangshan University, China.
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