10.1002/chem.201800249
Chemistry - A European Journal
COMMUNICATION
design. To confirm that NNH (and also NOH) preserved this key
feature, we examined the performance of these probes in
detecting endogenous and exogenous HClO with a two-photon
microscopy, using the same experimental conditions as in the
one-photon imaging). As shown in Figure S8, under the excitation
at 800 nm, both NNH- and NOH-labeled cells showed no signals
until the addition of NaClO or LPS+PMA, which triggered bright
green fluorescence after a 20-min incubation. Clearly, NNH
possessed the desirable two-photon absorbance feature in the
imaging of exogenous and endogenous HClO in the cultured
macrophages.
We finally examined the efficacy of NNH in imaging HClO in the
live tissue in a CCl4-induced liver injury model. Liver injury is a
common clinical issue related to a wide range of diseases; but it
is difficult to predict in practice.[14] One most established model of
liver injury was induced by CCl4 in mice, in which CCl4 can be
metabolized by P450 in the hepatocytes to produce abundant
ROS, leading to severe oxidative liver injury.[15] So, it would be
(Figure 5c and d). Second, NNH demonstrated notable
photostability against photo-bleaching throughout our experiment.
To quantitatively evaluate this, we performed a continuous
scanning of the NNH-labeled liver slices under laser irradiation.
Only 3% of the fluorescence lost after 100 scans, which
highlighted the property of NNH in resisting photo-bleaching
(Figure 5e and S10). These are perhaps the two most sought-
after advantages, among many others, of a fluorescent probe
designed for live tissue imaging.
In summary, we report the design of NNH, a water-soluble
fluorescent probe for real-time tracking of HClO in live tissue and
cells, with multiple desirable features including two-photon
absorbance, anti-bleaching capability, efficient cellular uptake
and no cytotoxicity. The key innovation in designing NNH is a
radical, but simple, change in the position of chemical modification
to a fluorophore. Compared with conventional approach to modify
1,8-naphthalimide derivatives at 4’ position, we modified it at 3’
position to introduce a hydrazone, thereby retaining the hydroxy
interesting if NNH could track the production of HClO in this model, group and the optical features of the compound – while
which might further provide useful information. We generated this
model by intraperitoneal injection of 6-8 weeks mice with CCl4
(50%, in olive oil).[16] After 24 h, we isolated liver tissues and
injected NNH (20 μL, 5 mM) or NDMTC (20 μL, 5 mM, as control
probe), and confirmed the successful establishment of liver injury
in the two treatment groups by determination of MPO activity
(Figure 5b). Then, we observed that the group injected with NNH
showed strong fluorescence while that with NDMTC emitted no
signal (Figure 5a). This result suggested that NNH was capable
of detecting endogenous HClO in the zone with liver injury, but,
again, the conventional, water-insoluble product of NDMTC was
incapable to do so.
substantially improving the water solubility of the product. Serial
follow-up tests highlighted that this improvement was crucial for
the performance of the probe in the living system. As the first
hydrazone-based probe for similar purposes, NNH may serve as
an imaging tool for uncovering the roles of HClO in broad
pathological processes.
Acknowledgements
We thank funding supports from Science and Technology
Development Fund, Macao SAR (FDCT 080/2016/A2,
126/2016/A3), University of Macau (MYRG2016-00031-ICMS-
QRCM) and National Science Foundation of China (51503232).
2.0
1.5
1.0
0.5
0.0
Keywords: water-soluble • two-photon • fluorescent probe •
endogenous hypochlorous acid • live tissue
NDMTC
Control
NNH
[1]
[2]
P. S. Hole, R. L. Darley, A. Tonks, Blood 2011, 117, 5816-5826.
a) A. Daugherty, J. L. Dunn, D. L. Rateri, J. W. Heinecke, J. Clin. Invest.
1994, 94, 437; b) P. Wei, W. Yuan, F. Xue, W. Zhou, R. Li, D. Zhang, T.
Yi, Chem. Sci. 2018. 9, 495-501.
[3]
[4]
G. S. He, L.-S. Tan, Q. Zheng, P. N. Prasad, Chem. Rev. 2008, 108,
1245-1330; b) Q. Xu, C. Heo, G. Kim, H. Lee, H. Kim, J. Yoon, Angew.
Chem. Int. Edit. 2015, 54 4890-4894; c) H. Kim, B. Cho, Chem. Rev.
2015, 115, 5014-5055.
a) Y. Feng, S. Li, D. Li, Q. Wang, P. Ning, M. Chen, X. Tian, X. Wang,
Sens. Actuators B: Chem. 2018, 254, 282-290; b) Q. Xu, C. H. Heo, J. A.
Kim, H. S. Lee, Y. Hu, D. Kim, K. M. K. Swamy, G. Kim, S.-J. Nam, H. M.
Kim, Anal. Chem. 2016, 88, 6615-6620; c) B. Zhu, P. Li, W. Shu, X. Wang,
C. Liu, Y. Wang, Z. Wang, Y. Wang, B. Tang, Anal. Chem. 2016, 88,
12532-12538.
Figure 5 Two-photon imaging of HClO in CCl4-induced liver injury model of mice.
(a) Diagram of CCl4-induced liver injury in mice and liver tissue fluorescence
images with NNH and NDMTC; (b) MPO activity assay of CCl4-induced liver
injury model; (c) 3D mapping of CCl4-induced liver tissue under 800 nm
excitation; (d) Depth scan (15-150 μm) in the CCl4-induced liver tissue under
800 nm excitation. Scale bar was 50 μm; (e) Photostability of NNH acquired
under continuous excitation (800 nm). Scale bar was 20 μm
[5]
[6]
X. Liu, Q. Qiao, W. Tian, W. Liu, J. Chen, M. J. Lang, Z. Xu, J. Am. Chem.
Soc. 2016, 138, 6960-6963.
a) Y. Tang, X. Kong, A. Xu, B. Dong, W. Lin, Angew. Chem. Int. Edit.
2016, 55, 3356-3359; b) X.-X. Zhang, H. Wu, P. Li, Z.-J. Qu, M.-Q. Tan,
K.-L. Han, Chem. Commun. 2016, 52, 8283-8286; c) Y. Li, X. Xie, X. e.
Yang, M. Li, X. Jiao, Y. Sun, X. Wang, B. Tang, Chem. Sci. 2017, 8,
4006-4011; d) Z.-R. Dai, L. Feng, Q. Jin, H. Cheng, Y. Li, J. Ning, Y. Yu,
G.-B. Ge, J.-N. Cui, L. Yang, Chem. Sci. 2017, 8, 2795-2803; e) Y. Kim,
We noticed two remarkable features of NNH in this live-tissue
imaging. First, its two-photon fluorescence enabled us to observe
clear fluorescent signals at different horizontal plane of a
complete liver – without the need of slicing. Under two-photon
excitation (800 nm), the penetration reached as deep as 150 μm
This article is protected by copyright. All rights reserved.