BODIPY-based fluorescent probes for mitochondria-targeted cell imaging with superior brightness, low cytotoxicity and high photostability

Article abstract of DOI:10.1016/j.dyepig.2017.03.009

Two mitochondria-targeted fluorescent probes (TPP- and TEA-BODIPY) were rationally designed and easily synthesized. These probes can specifically stain mitochondria in living cells with superior brightness, low cytotoxicity and high photostability, thus a

Full text of DOI:10.1016/j.dyepig.2017.03.009

Accepted Manuscript
BODIPY-based fluorescent probes for mitochondria-targeted cell imaging with
superior brightness, low cytotoxicity and high photostability
Tao Gao, Huan He, Rong Huang, Mai Zheng, Fang-Fang Wang, Yan-Jun Hu, Feng-
Lei Jiang, Yi Liu
PII:
S0143-7208(16)31466-8
10.1016/j.dyepig.2017.03.009
DYPI 5835
DOI:
Reference:
To appear in: Dyes and Pigments
Received Date: 23 December 2016
Revised Date: 23 February 2017
Accepted Date: 3 March 2017
Please cite this article as: Gao T, He H, Huang R, Zheng M, Wang F-F, Hu Y-J, Jiang F-L, Liu Y,
BODIPY-based fluorescent probes for mitochondria-targeted cell imaging with superior brightness, low
cytotoxicity and high photostability, Dyes and Pigments (2017), doi: 10.1016/j.dyepig.2017.03.009.
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Graphical abstract
Two BODIPY-based fluorescent probes can specifically stain mitochondria with
superior brightness, low cytotoxicity and high photostability.

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Short communication
BODIPY-based fluorescent probes for mitochondria-targeted cell
imaging with superior brightness, low cytotoxicity and high
photostability
Tao Gao,abc† Huan He,a† Rong Huang,a Mai Zheng,a Fang-Fang Wang,a Yan-Jun Hu,c
Feng-Lei Jiang*a and Yi Liu*a
a State Key Laboratory of Virology & Key Laboratory of Analytical Chemistry
for Biology and Medicine (Ministry of Education), College of Chemistry and
Molecular Sciences, Wuhan University, Wuhan 430072, P. R. China. E-mail:
fljiang@whu.edu.cn; yiliuchem@whu.edu.cn; Tel: +86 27 68756667.
b School of Nuclear Technology and Chemistry & Biology, Hubei University of
Science and Technology, Xianning 437100, P. R. China.
c
Hubei Key Laboratory of Pollutant Analysis & Reuse Technology, Hubei
Normal University, Huangshi 435002, P. R. China.
†These authors contributed equally to this work.

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Abstract:
Two mitochondria-targeted fluorescent probes (TPP- and TEA-BODIPY) were
rationally designed and easily synthesized. These probes can specifically stain
mitochondria in living cells with superior brightness, low cytotoxicity and high
photostability, thus are quite promising as mitochondrial tracking probes.
Keywords: Fluorescent probeꢀmitochondriaꢀimagingꢀphotostability

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1. Introduction
Mitochondria, the important organelles in almost all eukaryotic cells, play a crucial
role in a number of vital cellular processes, including energy generation, central
metabolism, redox signalling, calcium modulation and apoptotic pathways [1-3].
Recent discoveries showed that mitochondrial morphology was closely associated
with cell functions [4-7]. When cells are impaired, the size, number and structure of
mitochondria often change. For instance, mitochondrial swelling is often observed in
damaged cells as a common phenomenon [8,9]. It was also pointed out that
mitochondrial dysfunction could induce mitophagy, which led to the change of cell
function and several neurodegenerative disorders, including Parkinson’s disease and
Alzheimer’s disease [10,11].
On account of the importance of mitochondria, fluorescent dyes targeting
mitochondria have attracted broad interests because of their capability of observing
mitochondrial morphology so as to provide useful information for biochemical
cellular processes and even early diagnosis of diseases [12,13]. As is well known, a
lot of organic dyes have been used in the imaging of mitochondria, such as
Rhodamine [14-16], F16 [17,18], MitoTracker@RedCMXRos (MTR) and
MitoTracker@Green FM (MTG). However, most of these fluorescent dyes suffer
from severe drawbacks including difficult modification of molecular structure, poor
photostability and weak biocompatibility which limit their applications in living cell
studies. So, it is still highly necessary and urgent to design mitochondria-targeted
fluorescent probes with high affinity and selectivity. Recently, boron-dipyrromethene
(BODIPY) derivatives are regarded as one of the most popular family of fluorescent
dyes in the field of bioimaging owing to their advantages, e. g. large fluorescence
quantum yield (QY), easy modification, sharp fluorescence emission, high extinction
coefficient and excellent photostability [19-21]. Previous mitochondria-targeted
fluorescent dyes mainly utilized delocalized lipophilic cations (DLC) as cargo groups
in consideration of the negative mitochondrial membrane potential [22,23]. The most
widely used mitochondria-targeted delivery moiety nowadays is cationic
triphenylphosphine (TPP+). However, reports about mitochondria-targeted capability

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of a simpler cationic triethylamine (TEA+) are sparse. In this paper, we report two
novel mitochondria-targeted fluorescent probes, namely TPP-BODIPY and
TEA-BODIPY, by combining the cargo TPP+ and TEA+ with the fluorophore
BODIPY by a flexible carbon chain, respectively. By this way, the cationic groups can
guide BODIPY into mitochondria, while BODIPY still maintains its attractive
properties.
2. Results and discussion
2.1. Synthesis
The target products were synthesized in three steps (Scheme 1) [24]. First, the
reaction of 4-hydroxybenzaldehyde with 1,4-dibromobutane in the presence of K2CO3
as a base gave 1 in 86% yield. Second, the aldehyde 1 was reacted with 2,
4-dimethylpyrrole using trifluoroacetic acid (TFA) as a catalyst, followed by the
oxidation with 2, 3-dichloro-5, 6-dicyano-1,4-benzoquinone (DDQ) and subsequent
complexation with BF3.OEt2 in the presence of triethylamine (Et3N), which afforded a
highly emissive fluorophore (2) in 22% yield. Finally, reflux of 2 with
triphenylphosphine (TPP) or triethylamine (TEA) in toluene gave the title product 3
(TPP-BODIPY) and 4 (TEA-BODIPY) in 46% and 37% yield, respectively. All the
products were well characterized by NMR (Fig.S1) and mass spectrometry (Fig.S2).
The mass spectroscopic data showed main peaks at m/z 496.35 and 657.40, which
were attributed to M+ for TPP-BODIPY and TEA-BODIPY, respectively, with
deviation less than 1% from the theoretical values. TEA-BODIPY could be
absolutely dissolved in water, while TPP-BODIPY could be readily dissolved in
water by dilution from a stock solution in dimethyl sulphoxide (DMSO). In all
experiments, the solutions contained less than 1% (v/v) DMSO, which was usually
allowed in biology-related experiments.

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Br
O
OH
Et3N, BF3-OEt2
CH2Cl2, r.t.
K2CO3
TFA, DDQ
CH2Cl2, r.t.
Br
+
+
Br
DMF, r.t.
N
H
CHO
1
CHO
F N
B
F N
P
O
Br
Ph3P or Et3N
toluene, reflux
F N
B
F N
(3, TPP-BODIPY)
Br
O
F N
B
F N
Br
2
N
O
(4, TEA-BODIPY)
Scheme 1. Synthetic route of TPP-BODIPY and TEA-BODIPY.
Fig. 1 Absorption and emission spectra of TPP-BOBIPY (a) and TEA-BOBIPY (b)
with a concentration of 5 µM in water. The insets are the photographs of
TPP-BOBIPY (a) and TEA-BOBIPY (b) under ambient (left) and UV light (365 nm,
right), respectively.
Table 1 Photophysical properties of TPP-BODIPY and TEA-BODIPY (a
Absorption maxima. b Emission maxima. c Molar extinction coefficient).
a
b
λabs
λem
MECc
Compound (solvent)
QY
(nm)
496
496
496
497
(nm)
509
509
508
510
(M-1cm-1)
6.95×104
7.88×104
6.22×104
7.41×104
TPP-BODIPY (H2O)
TPP-BODIPY (CH3CN)
TEA-BODIPY(H2O)
TEA-BODIPY(CH3CN)
0.49
0.75
0.69
0.75

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2.2. Photophysical properties
Recent literatures indicated that the microenvironment of mitochondria changes
during many cellular processes involved in spatial arrangement and protein
composition [25,26]. Thus the in vivo imaging capacity of mitochondria-targeted
fluorescent dyes is closely related to their stability in surrounding environments. Here,
the optical properties of TPP-BODIPY and TEA-BODIPY in commonly used polar
solvents (water and acetonitrile) were studied (Fig. 1 and Table 1). The absorption
and emission maxima of TPP-BODIPY are centred at 496 nm and 509 nm
respectively in water (Fig. 1a). And we can find that the absorption and emission
maxima of TPP-BODIPY in acetonitrile are consistent with those in water (Fig. S3a
in ESI). Moreover, there is a smaller peak at ~ 400 nm which is contributed by
triphenylphosphine (Fig. 1a). The results also show that the polarity of different
solvents has almost no impact on the optical properties of TEA-BODIPY (Fig. 1b
and Fig. S3b). These results indicate the probes can be applied in wide conditions.
Owing to the non-conjugation between the fluorescent BODIPY core and the cargo
group (TPP+ or TEA+) via a flexible carbon chain, there is no variation in the
absorption and emission maxima upon replacement of cargo group TPP+ by TEA+, i.e.
absorption maximum ~ 496 nm and emission maximum ~509 nm. In addition, the
fluorescence intensity of TEA-BODIPY is significantly stronger than that of
TPP-BODIPY both in water and acetonitrile (Fig. 1 and Fig. S3). Besides, the molar
extinction coefficients for both probes in water and acetonitrile are in the range of 6 –
8 ×104 M-1 cm-1, demonstrating BODIPY is an ideal candidate as a light absorbing
material in broad application fields.
BODIPY is a commonly used fluorophore, but it encounters some troubles when
applied in biological system due to its poor water solubility [27,28]. To solve this
problem, many researchers modified BODIPY with some water-soluble groups
[29-31]. However, the QY of BODIPY would inevitably decrease during the
modification [32-35]. In this work, the two probes have very good water solubility.
More importantly, the QY of the two probes in water were 0.49 and 0.69 (Fig. S4,
using Rhodamine123 as reference [36]), respectively, which are apparently larger than

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those of the BODIPY-based water-soluble probes reported previously [27-35].
Additionally, the larger QY of TEA-BODIPY than that of TPP-BODIPY indicated
the former might be more excellent than the latter for living cell imaging. Overall, the
excellent water solubility and fluorescence properties demonstrated the tremendous
application prospect of these two probes.
2.3. Stability of the probes under different pH
To our knowledge, mitochondria present weak alkaline environment in living cells
[37-39]. Eligible mitochondria-targeted fluorescent probes must be able to work in a
mitochondrial physiology environment. Experimental results show that the two probes
exhibit high fluorescence intensity in the pH range from 2 – 10 (Fig. 2), so they are
suitable for both acid and basic conditions.
1.0
TPP-BODIPY
TEA-BODIPY
0.5
0.0
1
2
3
4
5
6
pH
7
8
9 10 11
Fig. 2 pH dependent fluorescence intensity of TPP-BOBIPY (5 µM) and
TEA-BOBIPY (5 µM).
2.4. Cytotoxicity
Low cytotoxicity is one of the most critical requirements for fluorescent probes.
The cytotoxicity of TPP-BODIPY and TEA-BODIPY were evaluated using a
standard MTT [3-(4, 5-dimethylthiazol)-2, 5-diphenyltetrazolium bromide] method.
Two cell lines, cervical carcinoma (HeLa) and gastric cancer (SGC-7901) cell lines,
were incubated with TPP-BODIPY and TEA-BODIPY at a concentration range
from 0 to 200 µM for 48 h, and the cytotoxic effects of the two probes are shown in
Fig. 3 and Fig. S5.

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Fig. 3 Cell viability of Hela cells treated with different concentrations of
TPP-BODIPY (a) and TEA-BODIPY (b) for 48 h.
It clearly shows that those two fluorescent probes exhibit no obvious toxicity
towards carcinoma and normal cell lines at low concentrations. For instance, over half
of the Hela cells are still alive after treatment with 100 µM of TPP-BODIPY for 48 h
(Fig. 3a). In fact, cells for bioimaging were exposed to a quite low concentration (0.5
µM) of the probes. Notably, TEA-BODIPY shows much less cytotoxicity than
TPP-BODIPY even at relatively high concentration (200 µM, Fig. 3b). The results
suggest that TEA-BODIPY may be more suitable for imaging of living cells than
TPP-BODIPY in consideration of the quite low cytotoxicity and high QY (0.69).
2.5. Confocal microscopic imaging
To investigate the imaging performance of TPP-BODIPY and TEA-BODIPY in
HeLa cells, the confocal microscopic imaging experiment were conducted by using a
Nikon A1 Confocal laser scanning microscope. As shown in Fig. 4, TPP-BODIPY
and TEA-BODIPY display bright green fluorescence in HeLa cells, owing to their
really high quantum yieldsꢁand give no fluorescence in the red channel (Fig. S6).
From co-localization images of the fluorescent probes with Mito Tracker Red (MTR),
it can be observed that the green fluorescence of TPP-BODIPY and TEA-BODIPY
is perfectly overlapped with the red fluorescence of MTR (Figs. 4c and 4g). The
Pearson correlation coefficients, which are used to quantify the overlap between the
fluorescence of MTR and that of TPP-BODIPY or TEA-BODIPY, are calculated as
0.87 and 0.90. As depicted in Figs. 4d and 4h, the changes in the fluorescence
intensity profiles of linear regions of interest (ROIs) are synchronous for

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TPP-BODIPY (or TEA-BODIPY) and MTR, indicating that TPP-BODIPY and
TEA-BODIPY can selectively accumulate in mitochondria of living cells. Moreover,
the fluorescent images of cells incubated with TEA-BODIPY are brighter than those
of TPP-BODIPY, which could be ascribed to the higher QY of TEA-BODIPY (0.69)
than TPP-BODIPY (0.49).
Fig. 4 Fluorescent images of HeLa cells stained with 0.5 µM TPP-BODIPY (a) or
0.5 µM TEA-BODIPY (e) for 4 h, 0.5 µM MTR for 15 min (b, f). Panel c and g are
the overlap of (a) and (b), (e) and (f), respectively. Panel d and h are the fluorescence
intensity profiles of ROIs across HeLa cells. Green lines represent the intensity of the
probe and red lines represent the intensity of MTR. Excitation wavelength: 488 nm
for TPP-BODIPY and TEA-BODIPY, and 543 nm for MTR.
2.6. Cellular uptake
The cellular uptake of the mitochondria-targeted fluorescent probes by HeLa cells
was examined by flow cytometry. HeLa cells were incubated with 0.5 µM
TPP-BODIPY or 0.5 µM TEA-BODIPY for different times, and then the uptake
content was surveyed with the intrinsic fluorescence of probes. The uptake rates for
both probes are quite fast, and over 80% probes are internalized within 1 h (Fig. 5).
Additionally, the fluorescence intensity for TEA-BODIPY is higher than that of
TPP-BODIPY as observed by flow cytometry, which is consistent with their quantum
yields. With this merit, the staining with TEA-BODIPY will necessitate a shorter
incubation time and improved efficiency of the mitochondrial imaging.

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Fig. 5 Flow cytometric analysis of the cellular uptake of TPP-BODIPY (0.5 µM) and
TEA-BODIPY (0.5 µM) by HeLa cells.
2.7. Photostability
Photostability of fluorescent probe is one of the major concerns when applied for
real-time imaging of living cells [40,41]. The photostability of TPP-BODIPY and
TEA-BODIPY were measured by a Carl Zeiss LSM 710 laser confocal microscope
with an excitation energy of 65 µW. There is almost no loss of fluorescence signal of
TPP-BODIPY after irradiation for 218 s (Figs. 6a and S7). TEA-BODIPY shows
good photostability (Figs. 6b and S8) with small loss of signal due to the minor
shrinkage of the target cell (Fig. 6b), which results in less collection of fluorescence
signal, rather than the photobleaching of TEA-BODIPY. In contrast, the fluorescence
image of commercial MTR cannot be observed after irradiation for only 136 s (Figs.
6c and S9), and the fluorescence signal decreases by more than 80% after irradiation
(Fig. 6d). It demonstrates that MTR possesses poor stability under the irradiation, as
reported by previous study [42,43]. So, TPP-BODIPY and TEA-BODIPY are
expected to have better performances than MTR for long-time tracking in living cells
because of their excellent photostability.

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(d)‐
Fig. 6 Photobleaching experiments of 0.5 µM TPP-BODIPY (a), 0.5 µM
TEA-BODIPY (b) and 0.5 µM MTR (c) in HeLa cells with irradiation for 1, 5, 9 and
13 times. The right column (d) shows the loss of fluorescence signal (%) with
increasing irradiation times.
3. Conclusion
In conclusion, two fluorescent probes, namely TPP-BODIPY and TEA-BODIPY,
were successfully synthesized and applied to mitochondria-targeted imaging in living
cells. They possessed many advantages, including superior brightness, high
photostability, low cytotoxicity and fast internalization, thus they are quite promising
as candidates for mitochondria-specific imaging. In addition, compared with the
commonly used mitochondria-targeted functional group (e.g. TPP+), TEA+ has smaller
chemical structure, better water solubility and biocompatibility, it may provide new
strategies for the future design and fabrication of highly efficient
mitochondria-targeted fluorescent probes.
Acknowledgments
The authors gratefully acknowledge the financial support from National Natural
Science Foundation of China (21573168, 21303126), National Science Fund for
Distinguished Young Scholars of China (21225313), Natural Science Foundation of
Hubei Province (2014CFA003), Foundation of Hubei Key Laboratory of Pollutant
Analysis & Reuse Technology (PA150102), and Science and Technology Research

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Foundation of Hubei Provincial Department of Education (Q20162802).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the online version, at
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