Co-self-assembled nanoaggregates of BODIPY amphiphiles for dual colour imaging of live cells

Article abstract of DOI:10.1039/c5cc04757a

Co-self-assembled vesicular nanoparticles of two structurally comparable amphiphilic boron-dipyrromethene (BODIPY) dyes with dequenchable dual colour fluorescence were prepared for ratiometric imaging of live cells.

Full text of DOI:10.1039/c5cc04757a

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Co-self-assembled Nanoaggregates of BODIPY
Amphiphiles for Dual Colour Imaging of Live
Cells
Cite this: DOI: 10.1039/x0xx00000x
a,b
b
a
b
b
Gang Fan,b YaoꢀXin Lin, Le Yang, FuꢀPing Gao, YingꢀXi Zhao, Zengꢀ
b
b
a
Received 00th January 2012,
Accepted 00th January 2012
Ying Qiao, Qiong Zhao, YunꢀShan Fan, Zhijian Chen,* and Hao
b
Wang*
DOI: 10.1039/x0xx00000x
www.rsc.org/
Co-self-assembled vesicular nanoparticles of two structurally amphiphiles carrying both hydrophilic and hydrophobic moieties
comparable amphiphilic boron-dipyrromethene (BODIPY) have been reported to form fluorescent nanoparticles and
14
dyes with dequenchable dual colour fluorescence were elongated aggregates in aqueous media. These aggregates
prepared for ratiometric imaging of live cells.
display distinct optical properties from that of monomeric dyes,
14a
e.g. redꢀshifted nearꢀinfrared fluorescence
or aggregationꢀ
15
In recent years, the construction of fluorescence imaging probes
by molecular selfꢀassembly process is of great interest to the
researchers in the interdisciplinary area of supramolecular
induced emission enhancement, which could be beneficial for
their applications.
We here report the aqueous coꢀselfꢀassembly of two
1
chemistry and bioimaging. By using building blocks such as
structurally comparable BODIPY amphiphiles
1 and 2 with
2
3
4
functional dyes,
πꢀconjugated oligomers, amphiphilic polymers,
distinct emission colour (Scheme 1). Of particularly interesting,
the fluorescence of both dyes is fully quenched in the coꢀ
assembled vesicular nanoparticles, but dequenchable upon the
disassembly driven by solvent variation or cell uptake.
Accordingly, the potential application of the nanoaggregates of
5
6
7
graphenes, metal complexes, peptides, etc., a variety of
supramolecular imaging nanoꢀprobes have been conveniently
8
prepared. Moreover, the supramolecular strategy provides
effective pathways to improve the sensitivity of the fluorescent
probes. In particular, the on/off switching of the fluorescence of
imaging probes can be modulated by the control of aggregation
and disaggregation of fluorophores to enhance the targetꢀtoꢀ
coꢀassembled dyes
was examined.
1 and 2 for dual colour imaging of live cells
9
background ratio of the signals. Nevertheless, imaging
inaccuracy can also stem from the intensity fluctuation of
excitation light, the probe concentration, inner filter effect, and
10
other issues. To minimize these perturbations, efforts have
been devoted to the development of dual colour imaging probe
systems containing different fluorophores which are able to
11
generate insusceptible, selfꢀcalibrating emission signals. To this
end, the coꢀselfꢀassembly of two or more functional dyes with
distinct and dequenchable fluorescence signals could be a
promising approach, but there is still a lack of exploration in this
aspect.
12
Boronꢀdipyrromethene (BODIPY) dyes have been widely
used for bioimaging and sensing owing to their intense
fluorescence, narrow emitting band, and high stability in
physiological conditions. For biological applications, a large
Scheme 1 Schematic illustration for the formation of fluorescence-quenched
nanovesicular co-aggregates of and and the dequenching of fluorescence of
both dyes upon the disassembly driven by cell uptake or solvent variation.
1
2
13
number of waterꢀsoluble BODIPY dyes have been synthesized
by attaching ionic groups or neutral oligoꢀethyleneglycol chains
at the dipyrromethene core. On the other hand, BODIPYꢀbased
The BODIPY dyes
1 and 2, both bearing a hydrophobic 3,4,5ꢀ
tridodecyloxyphenyl substituent at the mesoꢀposition and two
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hydrophilic quaternary ammonium groups attached to the boron chromophores and the formation of Jꢀtype coꢀaggregates of the
atom (dye ) or the 2,6ꢀpositions (dye ), were synthesized in BODIPY mixtures.
1
2
several steps starting from 3,4,5ꢀtridodecyloxybenzaldehyde and
fully characterized (see Electronic Supplementary Information).
The UV/Vis absorption spectrum of BODIPY
displayed absorption bands between 450 and 525 nm for the S
transition with λmax = 501 nm (Fig. 1). For dye
was bathochromically shifted 37 nm as compared with that of
1 in DMSO
0
2
.5
DMSO content
a)
→S
1
2, the λmax
1
2
0.5%
15%
26%
1
,
2.0
1.5
indicating the electronic interactions between the 2,6ꢀalkynyl
substituents and the BODIPY core. For both compounds, the
34%
50%
54%
emission bands are approximately the mirror image of their S
absorption bands. The emission maxima are located at 510
nm for dye and 558 nm for dye in DMSO. The fluorescence
quantum yields of
in DMSO, respectively.
0
→
A
1.0
0.5
0.0
S
1
57%
0%
6
1
2
63%
1
and
2
were determined to be 0.17 and 0.23
4
50
500
550
/ nm
600
650
1
.0
.8
.6
.4
.2
.0
1.0
λ
o
1
b)
10 C
0
2
0.8
0.6
2.5
2.0
o
1
5 C
o
2
0 C
0
-1
o
ε
/
1
2
25 C
5
-1
Ifl. / a.u.
o
1
0 M cm
30 C
1
1
0
0
.5
.0
.5
.0
o
0
0
0
0.4
0.2
0.0
3
5 C
o
A
4
0 C
o
45 C
o
5
0 C
o
5
5 C
o
450
500
550
/ nm
Fig. 1 UV/Vis absorption (solid lines) and fluorescence spectra (dashed lines) of
dyes and in DMSO (λex = 470 nm for and λex = 510 nm for ).
600
650
60 C
λ
4
50
500
550
600
650
λ
/ nm
1
2
1
2
2
.0 c)
DMSO content
The UV/Vis absorption spectra of
1
and
for nonꢀaggregated BODIPYs. However, upon addition of the
poor solvent water into the DMSO solutions of or , distinct
absorption spectral changes were observed (Fig. S9ꢀ10 ), which
2
in DMSO are typical
1
7
7
5%
1%
1
6
1.6
1.2
0.8
0.4
0.0
1
2
68%
66%
†
63%
are highly indicative for the formation of aggregates of both dyes.
On the basis of these observations, the coꢀselfꢀassembly
Ifl / a.u.
50%
30%
2
0.5%
properties of dye
1 and 2 were further investigated DMSOꢀ
containing water. When the content of DMSO in solvent is
greater than or equal to 63% (Fig. 2a), the UV/Vis absorption
−4
spectrum of
interpreted as a simple sum of the spectra of molecularly
dissolved BODIPY and and the S
transition bands of the Fig. 2 (a) UV/Vis absorption spectra of
1
/2
mixture ([
1
] = [
2
] = 2.5 × 10 M) can be
510
540
570
600
630
660
690
λ
/ nm
−
] = [ ] = 2.5 × 10 M) in
4
1
2
0
→S
1
1
/
2
mixture ([
1
2
DMSO-containing water with decreasing DMSO content (63% to 0.5%, v/v). (b)
Temperature-dependent UV/Vis absorption spectra of and ([ ] = [ ] = 2.5 ×
M) in DMSO-containing water (0.5% DMSO, v/v) and the arrows indicate the
two components can be easily distinguished. With decreasing
DMSO content in the system, the two monomer bands gradually
1
2
1
2
−
0
4
1
decrease and a new bathochromically shifted band with λmax spectra changes with increasing temperature. (c) Fluorescence spectra of
1/2
mixture ([1] = [2] = 2.5 × 10 M, λex = 460 nm) in DMSO-containing water with
−
4
around 564 nm appears, indicating a wellꢀdefined transition from
monomeric species to coꢀaggregates. The same transition was
also observed in temperatureꢀdependent spectroscopic studies
increasing DMSO content (0.5% to 75%, v/v).
More interestingly, the fluorescence of both dyes was fully
quenched when they coꢀaggregate in DMSOꢀcontaining water.
The recovery of fluorescence of both dyes was observed when
(
Fig. 2b) in water containing DMSO. With stepwisely decreasing
o
o
the temperature from 60 C to 5 C, gradually increase of the
aggregation band with λmax = 564 nm was observed. In contrast,
the coꢀassemblies of
1 and 2 were disaggregated by increasing
this aggregation band was not observed for pure
1 or 2 under
the content of DMSO in solution (Fig. 2c). The dual colour
fluorescence dequenching upon dissembling of the coꢀ
same solvent or temperature variation. All these pronounced
spectral changes indicate strong electronic interactions between
aggregates based on amphiphilic BODIPY
1 and 2 can be
2
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DOI: 10.1039/C5CC047 A
considered as “turn on” fluorescence that could be used for cell of coꢀaggregated dyes remained quenched in aboveꢀmentioned
imaging to increase the signalꢀtoꢀnoise ratio. medium. To use the nanoaggregates for dualꢀcolour imaging of
For the BODIPY amphiphiles and , their Israelachvili’s live cells, the suspension of nanovesicles ([
1
9
1
2
1]/[2] = 1/5) in
17
packing parameters (or shape factors) were estimated to be 0.8 DMSOꢀcontaining water (0.5%, v/v) was injected into culture
and 0.9 respectively (see ESI). This result implies the formation medium containing human cervical cancer (HeLa) cells and
of vesicular aggregates in the aqueous solution of coꢀassembled studied by confocal laser scanning microscopy (Fig. 4). After
dyes
1
and
2
with various molar ratios. To verify this prediction, incubation of two hours, a series of Zꢀstack images indicated
and were prepared by solventꢀswitch good internalization of both dyes (Fig. S16 ). Then the
the coꢀaggregates of
1
2
†
18
method in DMSOꢀcontaining water (0.5% DMSO, v/v) and were intracellular localization of BODIPYs was investigated by
visualized by transmission electron microscopy (TEM). For the studying their coꢀlocalization with known organelle probes. The
coꢀaggregates with a molar ratio of [
1
]/[
2
] = 1:5, spherical hollow results showed that BODIPYs
1
and
2
partially coꢀlocalized with
) while they mainly coꢀ
vesicles with a mean diameter of 110 nm were observed (Fig. 3a) lysosome and mitochondria (Fig. S17ꢀ18
†
in TEM study. The wall thickness of the vesicles is c.a. 10 nm, localized with Endoplasmic Reticulum (ER) Tracker, suggesting
suggesting that the vesicle wall has a multipleꢀlayered structure that the disaggregated dye molecules accumulated generally on
(length of c.a. 2.5 nm for 1 and c.a. 2.3 nm for 2 with fully ER in living cells (Fig. 4aꢀg).
extended alkyl chains from molecular modelling). Dynamic light
scattering (DLS) studies for the selfꢀassembled sample (Fig. 3b)
indicated that the average hydrodynamic size of particles was
1
20 nm with a polydispersity index of 0.24. The nanomorphology
of coꢀaggregates of and was also confirmed by tappingꢀmode
atom force microscopy and scanning electron microscopy (Fig.
c, S11,12 ). Furthermore, the vesicular morphology was also
observed for the coꢀaggregates of and with molar ratios of
]/[ ] = 1:3 and [ ]/[ ] = 1:1 in TEM studies (Fig. S12 ). For the
1
2
3
†
1
2
[1
2
1
2
†
coꢀaggregates with 1:1 ratio of the dyes, a wellꢀresolved lamellar
structure was observed for the vesicle wall with a interlayer
spacing of c.a. 5 nm, corresponding the thickness of a bilayer of
the dyes.
Fig. 4 Confocal microscope images of HeLa cells using Hoechst 33342, BODIPY
vesicles ([ ]/[ ]=1/5), and ER Tracker Red as imaging probes. (a) Bright-field
image. (b) Image for Hoechst. (c) Image for dye vesicles (channel 1, λex = 488 nm,
em = 525 ± 25 nm). (d) Image for dye vesicles (channel 2, λex = 543 nm, λem = 575
25 nm). (e) Image for ER Tracker Red (channel 3, λex = 594 nm, λem = 625 ± 25
1
2
λ
±
nm). (f) Merged images. (g) Correlation plot of BODIPYs and ER Tracker Red
emission intensities (Dual channel). (h) Merged image of channels 1 and 2. (i)
Normalized intensity profile of ROIs across the red arrow in image (h).
From the intensity correlation plots (Fig. S19
†), a high
Fig. 3 (a) TEM images of co-aggregates of dye
mg/mL). (b) Size distribution of the nanoaggregates by DLS measurements in
DMSO-containing water (0.5% DMSO, v/v, [ ]/[ ]= 1:5, [ ] = 0.2 mg/mL). (c) AFM
amplitude image of vesicles ([ ]/[ ]= 1:5 ) on mica.
1 and 2 ([1]/[2]= 1:5, [2
] = 0.2 Pearson’s coefficient of 0.66 and an overlap coefficient of 0.83 (0:
no coꢀlocalization, 1: all pixels coꢀlocalized) were obtained
respectively, confirming that the BODIPY coꢀaggregates can
stain ER specifically. Typically a Pearson’s coefficient above 0.4
1
2
2
1
2
20
is good. In contrast, when using molecularly dissolved BODIPY
alone as imaging probe, the resultant Pearson’s coefficient
decreased to 0.18 (Fig. S20, Table S1 ), implying the coꢀselfꢀ
The defined vesicular morphology and the dequenchable
fluorescence of the coꢀassembled nanoaggregates of and
1
1
2
†
are beneficial for their application as probes in monitoring
complex physiological processes. For such applications, the coꢀ
assembly of the two dyes plays a key role for the specificity of
the imaging probes. When the dualꢀcolour signal readout was
evaluated by measuring the intensity ratio of green and red
signals in region of interests (ROIs) (Fig. 4h), almost constant
ratiometric intensity of the fluorescence was obtained, ensuring
aggregation of dyes
1 and 2 was further examined in serumꢀfree
as well as serumꢀcontaining medium by UV/Vis spectroscopy
and DLS. The results indicate that the dye coꢀaggregates are
invulnerable in these medium (Fig. S13, S14†). The fluorescence
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the accuracy of imaging. In addition, the realꢀtime confocal
microscopic imaging studies indicated that the BODIPY coꢀ
aggregates remain fluorescenceꢀ quenched in the extracelluar
space while it becomes highly fluorescent only inside the cells,
thereby providing high targetꢀtoꢀbackground ratio of fluorescence
D Zhang, Y.ꢀX. Zhao, Z.ꢀY. Qiao, U. Mayerhöffer, P. Spenst, X.ꢀJ. Li,
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signals (Fig. S21
dyes and was assessed with the cell counting kitꢀ8 (CCKꢀ8)
assay. The viability of Hela cells incubated with increasing
amounts (5 ꢀM to 40 ꢀM) of a PBS dispersion of dyes and 4 (
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†
). Furthermore, the cytotoxicity of the BODIPY 3 (
a
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1
2
and L. Brunsveld, Chem. Eur. J., 2013, 19, 16646; (b) C. Zhu, Q. Yang,
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) M. C. L. Yeung, K. M. C. Wong, Y. K. T. Tsang and V. W.ꢀW.
) Y. Ding, Y. Yi, H. Xu, Z.
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,
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a
b
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†
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5
6
Conclusions
(a
) M. C. L. Yeung, K. M. C. Wong, Y. K. T. Tsang and V. W.ꢀW Yam,
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amphiphiles and was demonstrated by spectroscopic and
Chem. Commun., 2010, 46, 7709; ( ) R. Liu, J. Zhai, L. Liu, Y. Wang,
b
1
2
Y. Wei, X. Jiang, L. Gao, H. Zhu, Y. Zhao, Z. Chai and X. Gao, Chem.
Commun., 2014, 50, 3560
microscopic methods and nanovesicular coꢀaggregates of two
dyes with various mixing ratio were obtained in aqueous solution.
These fluorescenceꢀquenched nanoaggregates can disassemble
into fluorescent dye molecules with distinct emission bands upon
solvent variation or cell uptake. Dual color and ratiometric
fluorescence imaging of liveꢀcells indicated that the
disaggregated dyes are localized on the endoplasmic reticulum.
With their unique hollow vesicular morphology, the coꢀassembled
BODIPY nanoaggregates have the potential to be further
modified for the intracellular delivery and realꢀtime imaging.
7
8
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China (No. 21176184), National Basic Research Program of
China (973 Program, 2013CB932701), Program for New Century
Excellent Talents by the Ministry of Education (NCETꢀ09ꢀ0584),
the 100ꢀTalent Program of the Chinese Academy of Sciences,
and Beijing Natural Science Foundation (2132053) for financial
support for this work.
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