Cell membrane permeable fluorescent perylene bisimide derivatives for cell lysosome imaging

Article abstract of DOI:10.1039/c6ra20444a

We have developed acid activated fluorescent probes based on amphiphilic perylene bisimide with morpholine groups on the bay (Lyso-APBI). Incorporating one morpholine group, Lyso-APBI-1 showed an acid activated fluorescence increase of 70-fold upon pH values lowering from 8.0 to 4.0. Lyso-APBI-2, with two morpholine groups on the bay of PBI, had a red-shifted emission and a 190-fold fluorescence enhancement within same pH variation range. These Lyso-APBI probes have excellent membrane permeability, low cytotoxicity, and can rapidly accumulate and specifically activate in cell lysosomes. The double morpholine moieties make Lyso-APBI probes have higher acid activation ratio and better cell lysosome specificity. Moreover, long-time cell imaging proved the relatively high photostability of Lyso-APBI probes in a harsh physiological environment.

Full text of DOI:10.1039/c6ra20444a

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Cell Membrane Permeable Fluorescence Perylene
Bisimide Derivatives for Cell Lysosome Imaging
Cite this: DOI:
10.1039/x0xx00000x
‡
b
Shuchen Zhang,
a Wenfeng Duan,‡ Yanan Xi,b Tao Yang, a,b and Baoxiang Gao,*a,b
We have developed acid activated fluorescent probes based on amphiphilic perylene bisimide
with morpholine groups on the bay (Lyso-APBI . Incorporating one morpholine group, Lyso-
APBI-1 showed acid activated fluorescence increase of 70ꢀfold upon pH values lowering from
8.0 to 4.0. Lyso-APBI-2 two morpholine groups on the bay of PBI, had a redꢀshifted emission
and a 190ꢀfold fluorescence enhancement within same pH variation range. These Lyso-APBI
probes have excellent membrane permeability, low cytotoxicity, can rapidly accumulate and
specifically activate in cell lysosomes. The double morpholine moieties make Lyso-APBI
probes have better cell lysosome specificity. Moreover, longꢀtime cell imaging proved the
relatively high photostability of Lyso-APBI probes in harsh physiological environment.
)
Received 00th January 2012,
Accepted 00th January 2012
,
DOI: 10.1039/x0xx00000x
www.rsc.rg/
perylene bisimide probes with morpholine moieties and
Introduction
polyethylene glycol (PEG) groups
(Lyso-APBI), and
Fluorescence molecular imaging plays a major role in living
tissues and cells research since it is noninvasive, rapid, highly
sensitive and inexpensive.1ꢀ3 With appropriate fluorescent
probes, many biological species in living cells can be targeted
and visualized.4ꢀ5 Several characteristics of a successful
fluorescent probe in liveꢀcell imaging include water solubility,
brightness, photostability, low cytotoxicity and liveꢀcell
investigate the live cell lysosome imaging (Chart 1). The Lyso-
APBI probes in this study are specifically designed to contain
two pairs of threeꢀunit PEG groups on the imides of perylene
bisimides, making the probe water solubility and liveꢀcell
permeability.15 The designed probe also contain a typical
lysosomeꢀtargeting moiety of morpholine on the bay.16 We
anticipated that the photoinduced electron transfer (PET) from
the morpholine units to perylene bisimide dyes would quench
the fluorescence in neutral conditions. After the nitrogen is
protonated in acidic solution, the probe would show enhanced
fluorescence emission due to the blocked PET. 17 The synthetic
approach for Lyso-APBI is outlined in Scheme 1.
permeability.6ꢀ7
Furthermore,
the
longꢀwavelength
excitation/emission fluorophores are favorable for in vivo
bioimaging because of minimum photoꢀdamage to biological
samples, deep tissue penetration, and minimum interference
from background autoꢀfluorescence by biomolecules in the
living
systems.8ꢀ9
However,
the
longꢀwavelength
excitation/emission fluorophores tend to be larger molecules
with extended πꢀconjugation, and they are often poor water
solubility and impermeable to cells.10ꢀ11 Therefore, a pressing
need exists to develop fluorescent dyes satisfying the multiple
criteria of longꢀwavelength excitation/emission, photoꢀ
chemistry stability, high selectivity, and liveꢀcell permeability.
N
O
O
N
O
O
N
O
O
O
O
O
O
O
O
O
O
O
O
O
Lyso-APBI-1
N
O
Perylene bisimide derivatives (PBI) are chromophores that
display longꢀwavelength excitation/emission, high fluorescence
quantum yields and photostability.12ꢀ13 The high stability is a
crucial advantage for longꢀterm sensitive fluorescence
applications.14 PBI probes for Fluorescence lifetime imaging of
intracellular pH have been designed by introducing amino
groups.26 Our group is interested in the development of water
soluble and biocompatible perylene dyes for liveꢀcell
imaging.15 Here, we report the synthesis of amphiphilic
O
N
O
O
N
O
O
O
O
O
O
O
O
O
O
O
O
O
Lyso-APBI-2
O
N
Chart 1 Chemical structures of Lyso-APBI probes
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N
O
O
4
O
Br
O
O
O
O
O
O
i
N
O
B
O
NH2
O
NH
B
+
O
O
N
O
O
N
O
Br
1
R
R
O
N
O
O
N
O
R
R
Lyso-APBI-1
Br
Br
O
O
O
O
O
O
O
O
O
O
O
O
5
ii
iii
iv
O
O
+
O
O
O
+
+
1
Br
4
N
O
O
O
O
Br
R
N
O
N
O
R
2
3
O
O
N
O
Br
R
N
O
R
6
O
O
O
O
O
O
R=
O
N
Lyso-APBI-2
Scheme 1 ( ) MeCN, K2CO3, NaI, N2, overnight; ( ) concentrated sulfuric acid, r.t, 12h; I2, 85°C, Br2, 85°C, 10h; ( ) compound 1, HAc, N-Methyl pyrrolidone, 85°C, N2, 1h; ( )
compound 2, 2M K2CO3, Pd(PPh3)4, toluene, ethanol, N2, 80 °C, 15 h.
APBI-2, two benzene rings on the bay, had a redꢀshifted
maximum absorption to 551 nm, suggesting the Lyso-APBI
Results and discussion
with the introduction of benzene units have increased πꢀ
conjugation.
Lyso-APBI probes were synthesized according to Scheme 1.
PEG 1 18
bromoperylene tetracarboxylic acid bisanhydride
were prepared according to published procedures. The
,
2, 3
19
morpholine reacted with 4ꢀ(Bromomethyl)benzeneboronic acid
pinacol ester to afford the morpholine benzeneboronic acid
0.5
-4
-4
0.6
0.5
0.4
0.3
0.2
0.1
0.0
1.0*10 mol/L
1.0*10 mol/L
A
B
-5
-5
1.0*10 mol/L
1.0*10 mol/L
0.4
0.3
0.2
0.1
0.0
-6
-6
1.0*10 mol/L
1.0*10 mol/L
ester
was obtained via a coupling reaction between PEG
bromoperylene tetracarboxylic acid bisanhydride or
APBI probes were synthesized by Suzuki coupling between
compounds (or ) and morpholine compound . The Lyso-
4
. The amphiphilic perylene bisimide compounds
5
and
and
Lyso-
6
Lyso-APBI-1 in THF
Lyso-APBI-2 in THF
1
2
3.
350
400
450
500
550
600
650
350
400
450
500
550
600
650
Wavelength(nm)
Wavelength(nm)
5
6
4
APBI probes were fully characterized by HꢀNMR
spectroscopy, CꢀNMR spectroscopy and mass spectrometry.
The details of the synthetic compounds can be found in the ESI.
-4
-4
1.0*10 mol/L
1.0*10 mol/L
C
D
0.3
0.2
0.1
0.0
0.3
0.2
0.1
0.0
-5
-5
1.0*10 mol/L
1.0*10 mol/L
-6
-6
1.0*10 mol/L
1.0*10 mol/L
Lyso-APBI-1 in H
2
O
Lyso-APBI-2 in H
O
2
These Lyso-APBI probes show amphiphilic properties with
high solubility in common organic solvents such as toluene,
dichloromethane, tetrahydrofuran, and good water solubility.
The solubility of Lyso-APBI-1 in water is 164g/L. With two
morpholine moieties on the bay of PBI, the water solubility of
Lyso-APBI-2 was improved to 235g/L. The optical properties
of Lyso-APBI probes in aqueous solution were investigated
ultraviolet–visible (UVꢀvis) absorption spectroscopy (Figure 1)
and fluorescence spectroscopy. In amphiphilic tetrahydrofuran
solvent, the maximum absorption of Lyso-APBI-1 appeared at
532 nm along with higher vibronic transition at 488 nm. Lyso-
350 400 450 500 550 600 650
Wavelength(nm)
350 400 450 500 550 600 650 700
Wavelength(nm)
Figure 1 Concentration-dependent UV-Vis absorption spectroscopy of Lyso-APBI in
aqueous solution.
In aqueous solution, Lyso-APBI probes showed aggregation
behaviour, which was investigated vis concentrationꢀdependent
UVꢀVis absorption spectroscopy. In
a lowꢀconcentration
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aqueous solution (1.0 × 10ꢀ6 mol/L), the absorption bands of In pH 8.0 condition, the fluorescence quantum yield of Lyso-
Lyso-APBI probes showed maximum absorption at 538 nm APBI-2 is 0.0015, which lower than that of Lyso-APBI-1
(for Lyso-APBI-1), and 555nm (for Lyso-APBI-2). When the (0.0025). It is attributed to the double quench from two morpholine
aqueous solution concentration increased from 1.0 × 10ꢀ6 mol/L moieties of Lyso-APBI-2 in basic condition. However, the double
to 1.0 × 10ꢀ4 mol/L, a blue shift of the absorption maximum was protonation of nitrogen of Lyso-APBI-2 in acid condition not
observed (Lyso-APBI-1 from 538nm to 515nm; Lyso-APBI-2 only blocked PET, but the dication was also able to suppress the
from 555nm to 536nm), implying the formation of Hꢀtype aggregation of the PBI more effectively.22 Accordingly, Lyso-
cofacial πꢀπ stacking in
solution.20
a
highꢀconcentration aqueous APBI-2 had higher fluorescence quantum yield (0.28) than Lyso-
APBI-1 (0.18) in acid condition. On the basis of the above, the acid
activation ratio of LysoꢀAPBI-2 is 2.7ꢀfold larger than that of
400
1000
LysoꢀAPBI-1. Furthermore, the Lyso-APBI probes showed also
A
B
800
600
400
200
0
300
good reversibility between pH 4.0 and pH 8.0 (Figure 2E and 2F),
which is attributed to the protonation/deprotonation of the
morpholine group. The pKa value of Lyso-APBI-1 and Lyso-APBI-
2 was calculated to be 5.7±0.2 and 5.4±0.2, respectively. This
indicates that the fluorescence response of Lyso-APBI matches well
with the physiological pH range (pH 3.8–5.0) of lysosomes,23
making it promise as red fluorescent probe for lysosome imaging.
pH=2.0
pH=9.3
pH=2.0
200
pH=9.3
100
0
500
600
700
800
500
600
700
800
Wavelength(nm)
Wavelength(nm)
1200
1000
800
600
400
200
0
D
C
400
300
200
100
0
1000
800
600
400
200
400
300
200
100
0
4.4
4.8
5.2
pH
5.6
6.0
4.8
5.2
5.6
pH
6.0
6.4
1
2
3
4
5
6
7
pH
8
9
10 11 12 13 14
1
2
3
4
5
6
7
pH
8
9 10 11 12 13 14
1000
pH=4.0
pH=4.0
F
E
400
300
200
100
0
800
600
400
200
0
Figure 3 In vitro viability of HeLa cells treated with Lyso-APBI-1 (A) and Lyso-
APBI-2 (B) for 24 h and 48 h.
To demonstrate the potential utility of Lyso-APBI probes for
cellular imaging, their cytotoxicity was assessed using MTT
cellꢀviability assay. We have reported that the perylene
bisimide probes with PEG groups show very low cytotoxicity.15
Incorporating morpholine moieties on the bay of PBI, Lyso-
APBI probes have slight increase in cytotoxicity but still
remain low cytotoxicity (Figure S2). The cell viability was over
85% even though 20ꢁM Lyso-APBI was added for 48 h
(Figure 3), indicating that Lyso-APBI probes have a low
cytotoxicity. We ascribed the exceptionally low cytotoxicity to
PEG chains which protect the dyes from interacting
nonspecifically with the extracellular proteins and triggering
immunogenicity and antigenicity inside the cells.24
pH=8.0
pH=8.0
4
0
1
2
3
4
0
1
2
3
Number of Cycle
Number of Cycle
Figure 2 the optical features of Lyso-APBI (10μM) at different pH values.
fluorescence spectra of Lyso-APBI-1 (A) and Lyso-APBI-2 (B) recorded at
different pH values; Plot of fluorescence intensity of Lyso-APBI-1 (C) and Lyso-
APBI-2 (D)vs pH; the fluorescence intensity of Lyso-APBI-1 (E) and Lyso-APBI-2 (F)
between pH 4.0 and pH 8.0.
The pHꢀdependent fluorescence properties of Lyso-APBI probes
in aqueous solution were shown in Figure 2. The fluorescence
intensity of Lyso-APBI probes was too small to be distinguished
from the baseline. In contrast, these Lyso-APBI probes showed
relatively high fluorescence increase as the pH range is lowered
(Figure 2A, 2B). Lyso-APBI-1, the maximum emission wavelength
at 607 nm, showed a fluorescence increase of about 70ꢀfold upon pH
values lowering from 8.0 to 4.0. Lyso-APBI-2, two morpholine
moieties on the bay of PBI, had a redꢀshifted emission maximum at
624 nm and a 190ꢀfold fluorescence enhancement from pH 8.0 to 4.0.
Compared with other acid activated probes, a noteworthy aspect of
Lyso-APBI probes is that these probes have large Stokes shifts in
aqueous solution (Lyso-APBI-1 for 97nm, Lyso-APBI-2 for 73
nm. Table S1). Large Stokes shifts can reduce selfꢀabsorption of the
higher energy part of their emission spectra and eliminate
measurement interference by excitation light and scattered light.21
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Figure 4 Confocal microscopy images of HeLa cells treated with Lyso-APBI: the
bright-field (A) and confocal fluorescence (B) images of HeLa cells co-incubated
with Lyso-APBI-1 and fluorescence intensities of five points in cells (C) the bright-
field (D) and confocal fluorescence (E) images of HeLa cells co-incubated with
Lyso-APBI-2 and fluorescence intensities of five points in cells (F).
To evaluate the feasibility of imaging in living cells using
Lyso-APBI probes, they were employed to stain HeLa cells
for the CLSM images (Figure 4). A few fluorescent dots with
low brightness are observed in the cells stained by Lyso-APBI-
1
(Figure 4B), while, there were highly fluorescent dots in cells
stained by Lyso-APBI-2 (Figure 4E). These results indicate
that Lyso-APBI probes have good cellꢀmembrane permeability.
The quantified fluorescence intensities of multiꢀpoints in cells
stained with Lyso-APBI-1 and Lyso-APBI-2 were shown in
Figure 4C and 4F. The Lyso-APBI-2 showed twoꢀtimes higher
mean fluorescence intensity than Lyso-APBI-1 in cells.
Figure
5
Lysosome-targeting properties of Lyso-APBI in HeLa cells. A)
Colocalization images of HeLa cells stained with Hoechst 33342 (blue channel),
LysoTracker green (green channel), Lyso-APBI-1 (red channel), Overlay of three
channels and the correlation of intensities. B) Colocalization images of HeLa cells
stained with Hoechst 33342 (blue channel), LysoTracker green (green channel),
Lyso-APBI-2 (red channel), Overlay of three channels and the Intensity profile of
region of interest (ROI) cross cell.
In order to examine the lysosomes imaging ability of Lyso-
APBI probes, HeLa cells were coꢀstained with commercially
available lysosomeꢀspecial LysoTracker green probe and Lyso-
APBI probes. As shown in Figure 5, both Lyso-APBI probes
and LysoTracker green display strong localized fluorescence
within lysosomes. The fluorescence images of Lyso-APBI
(red) and LysoTracker green (green) can be merged rather well
(yellow), and the intensity profiles of the linear regions of
interest across HeLa cells stained with Lyso-APBI and
LysoTracker display good overlap. These results indicate that
Lyso-APBI probes can specifically target the lysosomes of
living cells. The coꢀlocalization of Lyso-APBI with the
LysoTracker green was quantified by correlation analysis. The
Pearson coefficient of coꢀlocalization for Lyso-APBI-1 with
LysoTracker green is 0.76, while that for Lyso-APBI-2 is 0.90,
indicating that the Lysosome specificity of Lyso-APBI-2 is
better than that of Lyso-APBI-1. We attribute the high
lysosome specificity to the two acid activated morpholine
moieties in Lyso-APBI-2. Furthermore, the accumulation rate
of Lyso-APBI-2 in cell lysosome was investigated by in situ
realꢀtime CLSM imaging and compared with commercially
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available lysosome probes such as LysoTracker green and
LysoTracker red. After 10 min of cells incubation with 1.0 ꢁM
This work was supported by the National Natural Science
Foundation of China (21274036), the Program for New Century
Excellent Talents in University (NCETꢀ12ꢀ0684), Training
Program for Innovative Research Team and Leading talent in
Hebei Province University (LJRC024). the Program for
Innovative talent in Hebei Province University China
(GCC2014054)
of Lyso-APBI-2
, the fluorescence signal from cells clearly
appeared (Figure 6). In contrast, cell incubation with 1.0 ꢁM of
LysoTracker green or LysoTracker red, fluorescent dots in cells
gradually formed with a lag time of 10 min. It clearly
indicates that the accumulation rate of LysoꢀAPBIꢀ2 in cell
lysosome is faster than that of LysoTracker green and
LysoTracker red.
Experimental
Materials and instrumentation
All the chemicals used in synthesis are analytical pure and were
used as received. UV/Vis spectra were recorded on a Shimadzu
WVꢀ2550 spectrophotometer. Fluorescence spectra were
recorded
on
a
Shimadzu
RFꢀ5301
fluorescence
spectrophotometer. The HꢀNMR spectra and CꢀNMR spectra
were recorded at 20 on 600 MHz or 150 MHz NMR
spectrometer (Bruker). Mass spectra were carried out with
Agilent LC/MSD XCT Trap. CLSM images were obtained
using Olympus confocal laser scanning microscopy (Olympus
Fluoview FV1000)
Cell culture and imaging
Figure 6 Real-time Confocal microscopy images of HeLa cells incubated with
Lyso-APBI-2, LysoTracker green, and LysoTracker red.
HeLa cells were cultured in Dulbecco’s modified Eagle’s
medium (high glucose), supplemented with 10% (v/v) fetal
bovine serum, penicillin (100 units/mL) and streptomycin (100
ꢁg/mL) at 37 in a 5% CO2/95% air incubator in a humidified
atmosphere, and culture media were replaced with fresh
media two to three days. For fluorescence imaging, HeLa cells
were grown in DMEM on a 35 mm glass bottom culture dishes
for at least 24h to enable adherence to the bottom.
The cells were loaded with Lyso-APBI according to a
following procedure. Briefly, the 50 ꢁl of 100 ꢁM PBS solution
of Lyso-APBI was added to the dish (final concentration of
Lyso-APBI is 5 ꢁM), and then the cells were incubated for 30
Furthermore, we examined the feasibility of applying Lyso-
APBI for tracking the pH changes in lysosomes. Treating HeLa
cells with chloroquine to induce an increase in the lysosomal
pH,25 the fluorescence signal in the area of the lysosomes
became weak (Figure. S3). The results suggest that probe Lyso-
APBI could be used for tracking pH changes in lysosomes.
Furthermore, changes in the fluorescence images of HeLa cells
stained by Lyso-APBI under continuous 559 nm laser
scanning were monitored to evaluate the photostability of Lyso-
APBI in cells. After scanning for 3 min, the fluorescence
signal from cells remained almost unchanged. In contrast, the
fluorescence signal from cells stained with the commercially
available LysoTracker probes decreased significantly within
same scanning time (Figure S4). These results prove the
relatively high photostability of Lyso-APBI in harsh
physiological environment.
min at 37
℃. Afterward, the cells were washed several times
with PBS for removing Lyso-APBI
.
Notes and references
a
Key Laboratory of Medicinal Chemistry and Molecular Diagnosis
(Hebei University), Ministry of Education, Baoding, 071002, China.
b
Key Laboratory of Analytical Science and Technology of Hebei
Province, College of Chemistry and Environmental Science, Hebei
University, Baoding, 071002, China. Fax: +86 3125077374, Eꢀmail:
bxgao@hbu.edu.cn.
Conclusions
In summary, we incorporated the morpholine and PEG moieties
into perylene bisimide dyes and successfully synthesized Lyso-
APBI probes. PEG chains imparted these probes amphiphilicity,
excellent membrane permeability, low cytotoxicity. With
†
Electronic Supplementary Information (ESI) available: See
DOI: 10.1039/b000000x/
‡ These authors contributed equally to this work and should be
morpholine moieties on the bay of perylene bisimide, Lyso- considered coꢀfirst authors
APBI probes can specifically activate in cell lysosomes, and
track the pH changes. It is worthy of note that the double
1
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lysosome specificity.
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Acknowledgments
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DOI: 10.1039/C6RA20444A
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The cellular uptake of Lyso-APBI probes is improved by PEG chains, and the
double morpholine moieties make Lyso-APBI probes have higher acid activation
ratio and better cell lysosome specificity