A fluorescent light-up probe with AIE characteristics for specific mitochondrial imaging to identify differentiating brown adipose cells

Article abstract of DOI:10.1039/c4cc00452c

We report the design and synthesis of a specific mitochondrial fluorescent probe AIE-MitoGreen-1 with AIE characteristics to monitor the mitochondrial morphology changes and identify the differentiation process of living brown adipose cells. The probe AIE-MitoGreen-1 has significant advantages such as high cell-permeability, good mitochondrial retention, low background fluorescence, large Stokes shift, and low toxicity. This journal is the Partner Organisations 2014.

Full text of DOI:10.1039/c4cc00452c

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A fluorescent light-up probe with AIE
characteristics for specific mitochondrial imaging
to identify differentiating brown adipose cells†
Cite this: Chem. Commun., 2014,
0, 8312
5
Received 19th January 2014,
Accepted 9th June 2014
a
b
cd
e
Meng Gao,‡ Choon Kiat Sim,‡ Chris Wai Tung Leung, Qinglian Hu,
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bf
cd
ae
Guangxue Feng, Feng Xu,* Ben Zhong Tang* and Bin Liu*
DOI: 10.1039/c4cc00452c
www.rsc.org/chemcomm
4
We report the design and synthesis of a specific mitochondrial against specific proteins, or transfection of DNA constructs
5
fluorescent probe AIE-MitoGreen-1 with AIE characteristics to encoding fluorescent proteins. Therefore, it is highly desirable
monitor the mitochondrial morphology changes and identify to develop a simple and minimally invasive imaging method for
the differentiation process of living brown adipose cells. The probe observing the differentiation process of brown adipose cells.
AIE-MitoGreen-1 has significant advantages such as high cell-
A key characteristic of brown adipose cells during differentiation
permeability, good mitochondrial retention, low background is that their mitochondrial content and activity will increase to
fluorescence, large Stokes shift, and low toxicity.
accommodate the larger metabolic demands. By observing the
changes in the number, morphology and subcellular localizations
There are two types of adipose cells in human: white adipose cells, of mitochondria through fluorescence imaging, a non-invasive
which can store energy as triglycerides, and brown adipose cells, method to identify the differentiation stages of brown adipose
1a
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which can dissipate energy as heat. Recently, there has been cells could be provided. Although a variety of fluorescent dyes have
increasing interest in understanding the differentiation process of been developed for mitochondrial imaging, such as rhodamines,
brown adipose cells, which are critical for the treatment of obesity rosamines, carbocyanines, styryl dyes, BODIPY and thiol-reactive
related diseases, such as type 2 diabetes, atherosclerosis, and Mitotracker dyes, they usually have large p-planar structures giving
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cardiovascular disease. Compared with positron emission rise to small Stokes shifts (less than 40 nm), and their fluorescence
2
tomography and magnetic resonance imaging methods,
would undergo self-quenching by high-concentration accumulation
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fluorescence imaging gives unique advantages such as high in mitochondria. To tackle this challenge, a novel fluorescent probe
sensitivity, spatial and temporal detection abilities. However, AIE-MitoGreen-1 has been designed on the basis of the salicylada-
traditional fluorescence imaging methods for observing the zine fluorophore and positively charged pyridinium groups for
differentiation process of brown adipose cells usually require specific mitochondrial imaging (Scheme 1). The salicyladazine
3
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lengthy processes: fixation, immunofluorescence with antibodies fluorophore uses two novel emission mechanisms: aggregation-
induced emission (AIE) via restriction of intramolecular rotation
10
around the N–N bond and excited-state intramolecular proton
transfer (ESIPT) via intramolecular hydrogen bonds. As a result of
these two mechanisms, they are non-emissive in dilute solutions
but are highly emissive as nanoaggregates, which can be well
retained in mitochondria without self-quenching effects and also
produce a large Stokes shift (4150 nm).
a
b
c
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Institute of Materials Research and Engineering (A*STAR), 3 Research Link,
Singapore 117602
Singapore Institute for Clinical Sciences, Brenner Centre for Molecular Medicine
(
A*STAR), 30 Medical Drive, Singapore 117609. E-mail: Xu_Feng@sics.a-star.edu.sg
Department of Chemistry, Division of Biomedical Engineering, The Hong Kong
University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong,
China. E-mail: tangbenz@ust.hk
d
SCUT-HKUST Joint Research Laboratory, Guangdong Innovative Research Team,
State Key Laboratory of Luminescent Materials & Devices,
South China University of Technology, Guangzhou 510640, China
Department of Chemical and Biomolecular Engineering, National University of
Singapore, 4 Engineering Drive, Singapore 117576. E-mail: cheliub@nus.edu.sg
Department of Biochemistry, Yong Loo Lin School of Medicine,
National University of Singapore, Singapore 117597
e
f
†
Electronic supplementary information (ESI) available: General experimental
procedures and characterization data for all compounds. See DOI: 10.1039/
c4cc00452c
Scheme 1 Design of fluorescent mitochondrial probe AIE-MitoGreen-1
‡
These authors contributed equally to this work.
with ‘‘AIE + ESIPT’’ characteristics.
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Scheme 2 Synthetic route for AIE-MitoGreen-1.
Fig. 2 Cytotoxicity experiments of AIE-MitoGreen-1 and MT at various
concentrations (0, 2.5, 5, 10 mM) in brown preadipose cells for 24 h.
Fig. 1 (A) Normalized UV spectra of AIE-MitoGreen-1 in DMSO solution
(black); PL spectra of AIE-MitoGreen-1 at the solid state (red) and in DMSO
solution (blue); (B) plot of relative PL intensity (I/I , 532 nm) versus the
0
solvent composition of the ethanol–hexane mixture of AIE-MitoGreen-1.
Concentration: 10 mM; excitation wavelength: 356 nm.
The synthetic procedure for AIE-MitoGreen-1 is shown in Fig. 3 Fluorescence images of brown preadipose cells stained with
.0 mM AIE-MitoGreen-1 and 50 nM MT: (a) image from AIE-MitoGreen-1.
b) Image from MT. (c) Merged image of a and b. (d) Wash-free image from
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Scheme 2. The reaction of 2,4-dihydroxybenzaldehyde 3 with
,6-dibromohexane first generated compound 3 in 67% yield,
which further reacted with hydrazine hydrate to afford the
(
1
AIE-MitoGreen-1 after 20 min. (e) Wash-free image from AIE-MitoGreen-1
after 24 h. (f) Wash-free image of MT after 20 min. Scale bar = 20 mm.
salicyladazine compound 4 in 85% yield. Compound 4 further Excitation and emission wavelength: 405 nm and 500–550 nm for AIE-
reacted with pyridine to afford the desired product AIE-MitoGreen-1 MitoGreen-1; 560 nm and 581–688 nm for MT.
in 93% yield. Their structures were confirmed with NMR and mass
spectroscopies (see ESI† for details).
Fig. 1 shows the photophysical properties of AIE-MitoGreen-1. (MT) for 24 hours, indicating that AIE-MitoGreen-1 is much less
An absorption maximum was seen at 356 nm in DMSO solution cytotoxic than MT.
while an emission maximum was seen at 532 nm (Fig. 1A). This
AIE-MitoGreen-1 was assessed for its ability to specifically
emission was strong at the solid state, but it was almost zero in localize in mitochondria of live brown preadipose cells using a
DMSO solution, suggesting that the emission is induced by confocal fluorescence microscope. After incubation with 5 mM
aggregation. Moreover, the near-absence of overlap between AIE-MitoGreen-1 for 20 min (Fig. 3a), the tubular and reticular
the absorption and the emission spectra, shown by a large structures of mitochondria were clearly visible. The costaining
Stokes shift of 176 nm, suggests minimal self-absorption, a experiment with MT showed an excellent overlap in the merged
quality that is highly desirable for fluorescence imaging. The picture (Fig. 3b and c), suggesting that AIE-MitoGreen-1 stains
photoluminescence (PL) of AIE-MitoGreen-1 was also tested mitochondria specifically. The fact that compound 3 was not
in ethanol–hexane mixtures with different hexane fractions to able to stain the brown preadipose cells suggested that the
fine-tune solvent polarity and solute aggregation (Fig. S1, ESI†). pyridinium groups were essential to achieve cellular intake
As hexane was added to ethanol from 0 to 90 vol% to reduce and specific mitochondria imaging. To further explore whether
polarity, the fluorescence signal of AIE-MitoGreen-1 remains low AIE-MitoGreen-1 can be used for direct in situ imaging of
(Fig. 1B). However, 99 vol% of hexane produced a 20-fold enhance- mitochondria, brown preadipose cells were incubated with
ment of emission, suggesting an AIE effect caused by the formation AIE-MitoGreen-1 for 20 min and imaged directly without wash-
of AIE-MitoGreen-1 aggregates with reduced solubility but not by ing. The fluorescence signals of AIE-MitoGreen-1 were confined
the change of solvent polarity.
in mitochondria with a negligible background signal from the
The cytotoxicity of AIE-MitoGreen-1 was evaluated by the culture medium (Fig. 3d), supporting the hypothesis that
widely used MTT assay. The samples were incubated with 0, 2.5, aggregated AIE-MitoGreen-1 in the mitochondria gives higher
5.0 and 10 mM AIE-MitoGreen-1 for 24 hours, and the results fluorescence than the unaggregated forms in the medium.
showed that cell viabilities were close to 100% (Fig. 2). In contrast, This specific staining of mitochondria remained even after
only less than 10% of the cells were viable after incubation with incubation for 24 hours (Fig. 3e). In contrast, using the com-
5.0 mM of a commercial mitochondrial dye, MitoTracker red FM mercial MT probe without washing, a higher background signal
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Fig. 4 Differentiating brown adipose cells (a–d, bright field) stained with
AIE-MitoGreen-1 (e–h, 5 mM) and MT (i–l, 50 nM), and their merged
images (m–p). Scale bar = 20 mm. These images were obtained using a
confocal microscope.
Fig. 5 HeLa cells (a–e, bright field) stained with AIE-MitoGreen-1
(
3
f–j, 5 mM) and MT (k–o, 50 nM), and their merged images (p–t). Scale bar =
0 mm. These images were obtained using a fluorescence microscope.
was observed (Fig. 3f). These results clearly demonstrate that
AIE-MitoGreen-1 is suitable for specific and long-time tracing
of mitochondria with a high signal-to-background ratio and
retention ability.
AIE-MitoGreen-1 is a highly suitable imaging agent for directly
Differentiation of brown preadipose cells was carried out visualizing the mitochondria morphology and localization, and
1
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with the previously described method. The overall cellular it is able to identify the differentiation stages of brown adipose
morphology changed from a fibroblastic shape to a spherical cells. The photostability of AIE-MitoGreen-1 was also tested through
shape (Fig. 4a–d). To visualize the mitochondria number and continuous scanning using a confocal microscope, and the signal
morphology changes during the differentiation process, 5 mM loss of AIE-MitoGreen-1 is less than 10% in B5 min upon constant
AIE-MitoGreen-1 was added to the differentiation media at laser exposure (Fig. S3, ESI†). As the pH in mitochondria is in the
day 1, 3, 5, and 7. The changes in mitochondrial morphology range of 7–8, the physical stability of AIE-MitoGreen-1 was tested in
and locations were shown by the confocal fluorescence images buffer solutions with pH of 7 and 8, and no obvious changes in
(Fig. 4e–h): at day 1 and 3, the mitochondria had a tubular their UV absorption were observed after 24 hours, indicating the
and reticular morphology; at day 5 and 7 of differentiation, good stability of the probe (Fig. S4, ESI†).
mitochondria appeared as punctate morphology. The specific
In summary, we report a novel fluorescent probe AIE-MitoGreen-1
imaging of AIE-MitoGreen-1 for mitochondria during the differen- with AIE characteristics for specific mitochondrial imaging. It is easy
tiation process was also confirmed by the co-staining experiment to synthesize, and has significant advantages such as high cell
with MT (Fig. 4i–p). Pearson’s correlation coefficient (R ; from +1 to permeability, a low background signal, good cellular retention
r
À1), indicating the degree of linear dependence between two and low toxicity, enabling the real-time continuous monitoring of
variables, is used to quantify the staining region overlap between mitochondria morphology without washing steps. This probe has
AIE-MitoGreen-1 and MT. Fluorescent signals of the two dyes been used for identifying the differentiation stages of brown adipose
collected from two different channels are well overlapped with cells in a quick and easy manner, showing that the mitochondria
Rr = 0.80 (day 1), 0.85 (day 3), 0.86 (day 5), and 0.81 (day 7). The undergo significant changes from a reticular, tubular organization to
adipogenic differentiation of the cells was further confirmed by a punctate morphology during the process. Fine tuning of the
Oil-Red-O lipid staining, and the generation of lipid droplets targeting ligands will yield more specific probes for monitoring
could be clearly seen from day 3, a characteristic feature for various biological processes.
successful adipogenic differentiation (Fig. S2, ESI†).
We thank the Singapore National Research Foundation
To further demonstrate that the mitochondrial network changes (R-279-000-390-281), the SMART (R279-000-378-592), Institute
are caused by the differentiation process, undifferentiating Hela of Materials Research and Engineering (IMRE/12-8P1103), intra-
cells were used as a control to show that their mitochondria were mural funding from Singapore Institute for Clinical Sciences
always presented as a reticular network (Fig. 5). The morphology (A*STAR), the Research Grants Council of Hong Kong (HKUST2/
of mitochondria was observed by adding 5 mM AIE-MitoGreen-1 CRF/10 and N_HKUST620/11) and Guangdong Innovative Research
to the culture media from day 1 to 5, respectively. There was Team Program (201101C0105067115) for financial support. We
almost no change in the mitochondria morphology throughout are also grateful to Dr Yu-Hua Tseng and Kai Ge for the brown
the whole process. Taken together, these results have shown that preadipose cell line WT-1.
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