cells [2-6]. However, free zinc ions participate in numerous crucial physiological and pathological processes. In cells, the
concentrations of zinc ions is regulated by three main protein families: metallothioneins (MTs), zinc transport proteins (ZnT), and
Zrt/Irt-like proteins (ZIPs) [7,8]. MTs, which function as binding proteins and Zn2+ donors, not only bind Zn2+ with high affinity but
can also release self-binding Zn2+ under the induction of nitrosation [9-11]. ZnT and ZIPs are two kinds of important zinc transporters,
which are responsible for the input and output of intracellular zinc ions, respectively [12]. ZnT8, one of the ZnT families, is responsible
for the transport of zinc in β cells, thus would affect the production, secretion and metabolism of insulin [13]. Scott et al. had reported
that the concentration of zinc in diabetic cadavers is only half of that in non-diabetic cadavers [14]. Moreover, loss of zinc homeostasis
has been associated with several neurological diseases, including Alzheimer’s disease, Parkinson disease, amyotrophic lateral sclerosis,
and prostate cancer [15,16]. Notably, prostate cancer is the leading cause of cancer death in men, and causes no symptoms in its early
curable stage. Considerable literatures indicate that the level of zinc ion in healthy prostate is ten times higher than other soft tissues.
Moreover, the zinc content in peripheral zone of prostate would drastically reduce in the early stage of malignant prostate and the
decreasing of zinc content precedes cancerization [17-21]. Based on these, the development of a powerful tool for the real time
monitoring of Zn2+ distribution, uptake, and trafficking in living system is highly demanded.
Fluorescent techniques, which have been widely used in imaging various kinds of analytes in living cells and tissues, have some
unique advantages, such as non-invasive, higher sensitivity, and excellent temporal-spatial resolution [22-26]. For a recent example,
Xiao et al. designed a FRET based probe to research pH changes in lysosomes of macrophages during inflammation [27]. It is known
that naphthalimide has been applied in the construction of various fluorescent probes [28]. To date, a wide variety of naphthalimide-
based fluorescent probes and their biological applications have been developed as an efficiency labelling approach. For example, Guo
et al. reported a highly selective and sensitive fluorescent chemosensor for Hg2+ [29]. Wang et al. developed a pH-resistant Zn2+ sensor
derived from 4-aminonaphthalimide [6]. Cui et al. reported a ratiometric fluorescent probe for direct detection of N-Acetyltransferase
[30]. However, the naphthalimide-based fluorescent probes are still not widely used in intracellular labelling and imaging owing to its
solvatochromism, and poor fluorophore brightness in aqueous solution [31]. By fusing a twisted water-soluble heterocycle onto
naphthalimide scaffold, we have constructed a novel nonplanar and rigid fluorophore structures, which not only enhanced the rigidity,
but also confirmed the nonplanarity of the fluorophore, and thus decreased the aggregation of the fluorogenic molecules [32]. Inspired
by this work, herein, we designed a highly sensitive fluorescent probe for tracking intracellular zinc ions and direct imaging of
prostatic tissue in mice by using this novel naphthalimide-based fluorophore and N,N-bis(2-pyridylmethyl) ethylenediamine (BPEN) as
the receptor for zinc ions.
Probe ZnDN can be readily synthesized as we previously reported [6,32] (Scheme S1 in Supporting information). Based on the
fluorescent recognition mechanism of photo-induced electron transfer (PET), BPEN was introduced into the imide moiety of the
naphthalimide fluorophore as the receptor of Zn2+.
We first conducted the pH-titration of the probe ZnDN and its Zn(II) complex. The pH value of the test system was adjusted by HCl
and NaOH solution. As shown in Fig. S4 (Supporting information), probe ZnDN emits very weak fluorescence at pH 4.0-9.0, which
can be attributed to the PET processes from the aniline nitrogen to the fluorophore. While ZnDN-Zn(II) complex exhibits the enhanced
fluorescence and is not subject to interference induced by pH changes in the range of pH 4.0-9.0. Therefore, the subsequent
experiments will be carried out in the PBS buffer (10-2 mol/L, pH 7.4).
The fluorescence titration of Zn2+ was then examined in PBS buffer (10-2 mol/L, pH 7.4). With addition of 10-3-10-2 mol/L (0-1
equiv.) Zn2+, the fluorescence intensity at 525 nm increased gradually, and reached the maximum with the addition of 1 equiv. Zn2+
(Fig. S5A in Supporting information). Upon addition of Zn2+, the biggest intensity could reach 17 times and fluorescence quantum
yield enhanced almost 10 times from 0.08 to 0.8. According to Job’s plot (Fig. S6 in Supporting information), ZnDN formed the 1:1
complex with Zn2+, and the association constant of the complex was 7.04 × 108 L/mol. The detection limit of ZnDN for Zn2+ was
calculated to be 4.0 × 10-9 mol/L based on LOD = 3σ/s.
The selectivity and competitiveness of ZnDN toward different metal ions (Zn2+, Ca2+, K+, Mn2+, Na+, Ag+, Cr3+, Co2+, Fe3+, Pb2+, Ni2+
with perchlorate anions) were further investigated in PBS buffer (10-2 mol/L, pH 7.4). Upon addition of 3 equiv. different metal ions,
negligible changes were observed in the fluorescence emission spectra (Fig. S8 in Supporting information). However, the fluorescence
intensity was significantly enhanced after Zn2+ was added. Even in the presence of 3 equiv. of other metal ions, the fluorescence
intensity exhibited similar enhancement when Zn2+ was added. Therefore, probe ZnDN should have the potential in imaging
intracellular zinc ions.
The biocompatibility and subcellular localization ability of probe ZnDN were evaluated before living cell imaging. MTT assay was
used to determine the cytotoxicity of ZnDN in MCF-7 cells and A549 cells. The cells viability was above 80% with the concentration
of ZnDN ranging from 0 to 2×10-5 mol/L after 24 h incubation (Fig. S9 in Supporting information), which indicated that ZnDN had
low toxicity and could be used for imaging in living cells. Then we investigated the subcellular location of ZnDN in A549 cells with
Lyso Tracker® Red DND-99 L-7528, a commercial lysosome marker. As shown in Fig. 1, the fluorescence imaging of ZnDN (Fig.
1B) was overlapped well with Lyso Tracker® Red DND-99 L-7528 (Fig. 1A), with the Pearson’s correlation coefficient at 0.94 (Fig.
1D), which indicated that ZnDN was mainly localized in lysosome. Since this novel fluorescent skeleton had an application in two-
photon microscope (TPM) imaging [32], we also investigated the TMP imaging ability of ZnDN. The fluorescence signal of ZnDN
with one-photo excitation (Fig. 1J) showed excellent overlap with that of two-photo excitation (Fig. 1I), indicating that ZnDN could be
used for TPM imaging of zinc ions in living cells.
Wang, Yuyu
