In vitro and in vivo imaging application of a 1,8-naphthalimide-derived Zn2+ fluorescent sensor with nuclear envelope penetrability

Article abstract of DOI:10.1039/c3cc46862c

A newly developed fluorescent sensor, Naph-BPEA, shows a specific turn-on response to Zn²⁺ and can be excited by visible light. The in situ nuclear Zn²⁺ imaging in HeLa and HepG2 cells reveals the nuclear envelope penetrability of the sensor. The specific sensor location in a zebrafish larva was also demonstrated.

Full text of DOI:10.1039/c3cc46862c

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In vitro and in vivo imaging application of a
1,8-naphthalimide-derived Zn2+ fluorescent
sensor with nuclear envelope penetrability†
Changli Zhang,ab Zhipeng Liu,c Yunling Li,a Weijiang He,*a Xiang Gaod and
Zijian Guo*a
Cite this: Chem. Commun., 2013,
49, 11430
Received 7th September 2013,
Accepted 14th October 2013
DOI: 10.1039/c3cc46862c
www.rsc.org/chemcomm
A newly developed fluorescent sensor, Naph-BPEA, shows a spe-
cific turn-on response to Zn2+ and can be excited by visible light.
The in situ nuclear Zn2+ imaging in HeLa and HepG2 cells reveals
the nuclear envelope penetrability of the sensor. The specific
sensor location in a zebrafish larva was also demonstrated.
The subcellular Zn2+ imaging of specific compartments using
sensors with specific intracellular distribution patterns is of great
interest, since many subcellular organs are involved in Zn2+ homeo-
stasis with variable roles.1 For example, the nucleus has been
correlated with intracellular Zn2+ transportation and storage,2 and
the Zn2+ storage protein metallothionein can be re-distributed from
the cytoplasm to the nucleus during phase transition from G1
to S.2a,3 Moreover, many nuclear proteins such as transcription
factors, polymerases, and DNA remodeling factors cannot work
properly without Zn2+.4 Understanding the roles of nuclear Zn2+
demands sensors with nuclear envelope penetrability to realize the
in situ nuclear Zn2+ imaging. However, the Zn2+ sensors with nuclear
envelope penetrability are rare so far, besides the genetically
encoded FRET sensors developed by Palmer and coworkers.2a Small
molecular Zn2+ sensors with nuclear envelope penetrability are also
desirable due to their advantages such as a simple staining proce-
dure, fine reproducibility, and high stability and sensitivity. Newport
Green (diacetate form of cell membrane permeability functioning
via intracellular hydrolysis) and Zinquin (UV excitable) are among
the very few examples displaying an even distribution throughout
the entire cell including the nucleus,2b,5 however Zinquin is unable
to enter the nucleus of numerous cell lines and cannot visualize the
nuclear Zn2+ enhancement induced by Zn2+/pyrithione incubation.3b
Scheme 1 Synthesis of Naph-BPEA and the chemical structures of Naph-BPEA-e,
and NBD-BPEA.
Herein, we report a new turn-on fluorescent Zn2+ sensor with
nucleus envelope penetrability, Naph-BPEA (Scheme 1). With the
visible light excitability and cell membrane permeability, this sensor
is able to realize not only the nuclear Zn2+ imaging but also the
in vivo Zn2+ imaging in a zebrafish larva.
The new sensor was derived from 4-amino-1,8-naphthalimide
(ANaph), an internal charge transfer (ICT) fluorophore with
visible light excitability and medium quantum yield,6 and its
DNA targeting ability was expected to improve the nucleus
localization of Naph-BPEA. In fact, the anion sensors based on
ANaph are able to enter the nucleus.7 The Zn2+ ionophore
N,N0-bis(pyridin-2-ylmethyl)ethane-1,2-diamine (BPEA) was incor-
porated as the ICT donating group of the ANaph fluorophore. Its
2-(2-aminoethoxyl)ethanol moiety was introduced to improve the
aqueous solubility of this sensor. Naph-BPEA was prepared from
acenaphthene via a four step procedure with a total yield of 8.6%
(Scheme 1, for details, please see the ESI†). Its analogue with the
ethyl group replacing its 2-(2-aminoethoxyl)ethanol tail, Naph-
BPEA-e, was also prepared (Scheme S2, ESI†).
a State Key Laboratory of Coordination Chemistry, Coordination Chemistry
Institute, School of Chemistry and Chemical Engineering, Nanjing University,
Nanjing 210093, P. R. China. E-mail: heweij69@nju.edu.cn, zguo@nju.edu.cn;
Fax: +86 25 83314502; Tel: +86 25 83597066
b Department of Chemistry, Nanjing Xiaozhuang College, Nanjing 210017,
P. R. China
c Department of Chemistry, Liaocheng University, Liaocheng 252059, P. R. China
d Animal Model Research Center, Nanjing University, Nanjing 210061, P. R. China
† Electronic supplementary information (ESI) available: Synthesis, spectroscopic
studies, imaging results and experimental details. See DOI: 10.1039/c3cc46862c
Naph-BPEA emits in aqueous HEPES buffer (10 mM, 50 mM
HEPES, 100 mM KNO3, 1% DMSO, pH 7.2) with the emission
and excitation maxima being at 546 and 450 nm, respectively.
c
11430 Chem. Commun., 2013, 49, 11430--11432
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Naph-BPEA emission distinctly, except for Cu2+ and Co2+ that
induce the minor emission quenching. The intracellular Zn2+
sensing behavior of Naph-BPEA will not be interfered by Cd2+
and Pb2+ in the normal case due to the extremely low intra-
cellular level of Cd2+ and Pb2+. Moreover, the presence of cellular
abundant Na+, K+, Ca2+ and Mg2+ (1000 equiv.) does not affect
the Zn2+ sensing behavior of Naph-BPEA. It is proposed that the
Naph-BPEA analogue with a specific azo-crownether or azo-
cryptand as a Zn2+ chelator to combine with the ANaph fluoro-
phore might be helpful in enhancing the sensing selectivity over
Cd2+ and Pb2+, since the radius of Zn2+ is smaller than those of
the two cations. The stable emission of Naph-BPEA in the pH
range from 6.5 to 9.0 indicates the pH-independent fluorescence
of Naph-BPEA in physiological microenvironments (Fig. S3,
ESI†). In addition, the detection limit (3s/slope) of this sensor
was determined to be 57 nM (Fig. S4, ESI†). All these suggest that
this sensor might be a suitable candidate as an imaging agent
for intracellular Zn2+.
Fig. 1 Emission (a, 10 mM, lex, 450 nm) and absorption (b, 100 mM) spectra of
Naph-BPEA in HEPES buffer (50 mM, 100 mM KNO3, pH 7.2) obtained upon
titration with Zn2+ solution (1.2 mM for a, 10 mM for b) at 25 1C. The buffer
contains 1% (for a) or 10% (for b) DMSO (v/v). Inset in (a): the titration profile
based on the emission at 540 nm; inset in (b): the titration profile based on the
absorbance at 400 nm. (c) A histogram of the emission enhancement factor
(based on the emission at 540 nm) of Naph-BPEA (10 mM) induced by different
The intracellular distribution pattern and Zn2+ imaging
ability of Naph-BPEA were investigated in living cells. The
punctated cytosolic fluorescence pattern circling an internal
metal cations in the same buffer of (a). Na+, K+, Ca2+, Mg2+, 1000 equiv., other dim area can be observed in the Naph-BPEA-stained HepG2
cells (Fig. S5, ESI†). When exogenous Zn2+ are introduced into
metal cations, 1 equiv. lex for (a) and (c) is 450 nm.
the cells via ZnSO4–pyrithione incubation (5 mM, 1 : 1), the
entire cell turns fluorescent, and the former punctated region
Upon Zn2+ titration, Naph-BPEA displays a linear emission becomes even brighter. The following treatment by the
enhancement with [Zn2+]total, and the emission enhancement membrane-permeable Zn2+ chelator TPEN (N,N,N0,N0-tetra-
factor is B4 when the [Zn2+]total–[Naph-BPEA] ratio of 1 : 1 is kis(2-pyridylmethyl)ethylenediamine) results in a recovered
attained. Even higher [Zn2+]total does not lead to any further dim image with the punctated fluorescence pattern becoming
emission enhancement (Fig. 1a). UV titration of Naph-BPEA weaker than that before Zn2+ incubation (Fig. S5, ESI†). These
with Zn2+ exhibits an absorption hypsochromic shift from 453 results suggest that Naph-BPEA is an effective Zn2+ imaging
to 442 nm, and the clear isosbestic point at 442 nm implies that agent with cell membrane permeability, and can be distributed
only one zinc coordination process occurs upon Zn2+ titration throughout cells including the nucleus.
(Fig. 1b). The titration profile based on the emission at 540 nm
The nuclear envelope penetrability and nuclear Zn2+ ima-
and that based on the absorbance at 400 nm both suggest a 1 : 1 ging ability of Naph-BPEA were confirmed in the HeLa cells
Zn2+ binding stoichiometry of Naph-BPEA. The new set of co-stained with Naph-BPEA and the nucleus dye Hoechst
signals appearing in the 1H NMR spectrum of Naph-BPEA upon 33 342. The navy blue colour in the Hoechst channel image
Zn2+ titration becomes the only set of signals when the [Zn2+]– (lex, 351 nm; band path 420–470, blue channel) of the stained
[Naph-BPEA] ratio of 1 : 1 is attained (Fig. S1, ESI†). This cells shows the nucleus location (Fig. 2a). Imposing this image
phenomenon also confirms the 1 : 1 Zn2+ binding stoichio- on the Naph-BPEA channel image (lex, 488 nm, band path
metry. Moreover, the Zn2+-induced signal shifts for protons of 500–600 nm, green channel) of the same cells confirms that the
ANaph and BPEA moieties in the 1H NMR spectrum suggest punctated fluorescence pattern in the green channel image
that all the N atoms of the BPEA moiety were directly involved locates in the cytoplasm, while the nucleus turns dim upon
in Zn2+ coordination. It is the direct Zn2+ coordination of the excitation at 488 nm (Fig. 2b and c). Upon Zn2+–pyrithione
terminal amino group of BPEA that induced the blue shift of incubation, the green channel image displays the total fluor-
the ICT absorption band, since this amino group also acts as escent cells (Fig. 2e). Co-localization via overlaying the green
the ICT donating group of the ANaph fluorophore. In fact, Zn2+ channel image on the blue channel image demonstrates the
titration also induces the minor emission blue shift (B10 nm). pale blue nucleus as the co-staining effect of Naph-BPEA and
The dissociation constant of the Zn2+–Naph-BPEA complex was Hoechst 33 342, inferring the coexistence of labile Zn2+ and
estimated to be B4 nM. The quantum yield of the Zn2+–Naph- Naph-BPEA in the nucleus (Fig. 2f). The subsequent TPEN
BPEA complex was determined to be 0.19 with NBD–NHCH3 in treatment resulted in the recovered dim nucleus and the weak
acetonitrile as the reference.8
cytosolic punctated fluorescence pattern (Fig. 2g and h). All
The specific Zn2+-amplified fluorescence of Naph-BPEA these confirm that Naph-BPEA can be distributed throughout
was verified via screening the metal cations of interest the cells including the nucleus. It is the low nuclear labile
(Fig. 1c). Except for Zn2+, only Cd2+ and Pb2+ among the tested Zn2+ level that resulted in the initial dim nucleus, and the labile
cations trigger the emission enhancement, with the respective Zn2+ fluctuation in the nucleus induced by Zn2+–pyrithione
factors of B3- and 1.3-fold. Other tested cations do not affect incubation and TPEN treatment can be visualized clearly using
c
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Fig. 3 Fluorescence images of a Zn2+-fed 5-day-old zebrafish larva obtained
by a fluorescence microscope. (a) The Naph-BPEA-stained larva (5 mM, 28.5 1C,
30 min); (b) the NBD-TPEA-stained larva (5 mM, 28.5 1C, 30 min).
was observed as the larva notochord (Fig. 3a). Such a stream is
not visible if the larva is not Zn2+-fed. Moreover, it cannot be
observed by NBD-TPEA staining even when the larva is Zn2+-fed
(Fig. 3b). The Zn2+ imaging ability in the notochord of the
zebrafish larva implies that Naph-BPEA can be a potential
in vivo imaging agent for Zn2+ involved in the development of
zebrafish.
Fig. 2 Confocal fluorescence images of HeLa cells co-stained with Hoechst
33342 (5 mg mLꢁ1 in PBS, 10 min) and Naph-BPEA (5 mM in PBS, 20 min). (a–c):
images of the co-stained cells; (d–f): images of the co-stained cells followed by
Zn2+–pyrithione incubation (5 mM, 1 : 1, 5 min); (g–i): images of Zn2+-incubated
cells treated further by TPEN (25 mM in PBS, 10 min). (a), (d) and (g): The Hoechst
channel (blue channel, lex, 351 nm, band path 420–470 nm) images; (b), (e) and
(f): the Naph-BPEA channel (green channel, lex, 488 nm, band path 500–600 nm)
images; (c), (f) and (i): overlays of the related blue/green channel images.
In conclusion, a new Zn2+ fluorescent sensor, Naph-BPEA,
was developed from 1,8-naphthalimide. Its nuclear envelope
penetrability and specific Zn2+-amplified fluorescence provide
this sensor the ability to image nuclear Zn2+ in HeLa and
HepG2 cells. The in situ nuclear Zn2+ imaging using this sensor
is esterase-independent and can be accomplished by visible
light excitation. This study also suggests that ANaph incorpora-
tion might be an effective strategy to devise a sensor with
nuclear envelope penetrability.
We thank the National Basic Research Program of China (No.
2011CB935800) and the National Natural Science Foundation of
China (No. 21271100, 21131003, 21021062, and 91213305) for
financial support.
Naph-BPEA. The intracellular distribution pattern of Naph-
BPEA suggests the possibility of simultaneously monitoring
labile Zn2+ levels in the nucleus and cytoplasm.
NBD-BPEA (Scheme 1) is a turn-on Zn2+ sensor developed
similarly via incorporating BPEA as the ICT donating group of
the ICT fluorophore 4-amino-7-nitro-2,1,3-benzoxadiazole
(ANBD).9 Co-localization imaging of HeLa cells co-stained with
Hoechst 33 324 and NBD-BPEA disclosed that Zn2+ imaging via
NBD-BPEA showed only the cytosolic punctated fluorescence
pattern even upon Zn2+–pyrithione incubation (Fig. S6, ESI†).
With the identical Zn2+ chelator BPEA, Naph-BPEA and NBD-
BPEA are expected to have a similar Zn2+ binding ability, and
the dim nucleus upon Zn2+ incubation in the case of NBD-BPEA
implies that there are no sensor molecules distributed in the
nucleus. The different intracellular distribution patterns
between NBD-BPEA and Naph-BPEA suggest that the DNA-
targeting ANaph might be the origin for the nuclear envelope
penetrability of Naph-BPEA. In fact, its analogue Naph-BPEA-e
with the ethyl tail displays also the nuclear envelope penetr-
ability (Scheme 1 and Fig. S7, ESI†). The nucleus penetrability
of Naph-BPEA does not have evident impact on the cell viability,
and the cells appear to show the normal morphology during the
imaging process. The MTT assays disclosed that the cellular
viability of HeLa and HepG2 cells after 48 h of Naph-BPEA
incubation (5 mM) is 94.5 ꢀ 2.0% and 98.5 ꢀ 1.0%, respectively.
The nuclear Zn2+ imaging ability and nuclear envelope
penetrability of Naph-BPEA inspire us to determine its in vivo
Zn2+ imaging ability in a transparent zebrafish larva (5-day-old).
The larva image obtained via Naph-BPEA staining displays the
two zygomorphic fluorescent regions around its ventricle, simi-
lar to the image obtained via staining with a Zn2+ sensor NBD-
TPEA.10 Moreover, the additional fluorescent stream in the tail
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