Synthesis and photophysical characterization of highly water-soluble PEGylated BODIPY derivatives for cellular imaging

Article abstract of DOI:10.1016/j.jphotochem.2019.03.050

A series of poly(ethylene glycol) (PEG)-modified water-soluble boron dipyrromethene (BODIPY) dyes (EtBOD-PEG and BOD-PEG) were synthesized and their photophysical properties in various solutions were investigated. The presence of ethyl groups at the 2,6-p

Full text of DOI:10.1016/j.jphotochem.2019.03.050

Journal of Photochemistry & Photobiology A: Chemistry 377 (2019) 214–219
Contents lists available at ScienceDirect
Journal of Photochemistry & Photobiology A: Chemistry
journal homepage: www.elsevier.com/locate/jphotochem
Synthesis and photophysical characterization of highly water-soluble
PEGylated BODIPY derivatives for cellular imaging
Isabel Wen Badona,1, Joomin Leeb,1, Temmy Pegarro Vales , Byoung Ki Chod,⁎, Ho-Joong Kima,⁎
a,c
a
Department of Chemistry, Chosun University, Gwangju, 61452, Republic of Korea
Department of Food and Nutrition, Chosun University, Gwangju, 61452, Republic of Korea
Department of Natural Sciences, Caraga State University, Butuan City, 8600, Philippines
Department of Chemistry, Dankook University, Chungnam, 31116, Republic of Korea
b
c
d
A R T I C L E I N F O
A B S T R A C T
Keywords:
BODIPY
Fluorescent probes
Poly(ethylene glycol)
Water-soluble
Bioimaging agents
A series of poly(ethylene glycol) (PEG)-modified water-soluble boron dipyrromethene (BODIPY) dyes (EtBOD-
PEG and BOD-PEG) were synthesized and their photophysical properties in various solutions were investigated.
The presence of ethyl groups at the 2,6-positions of the BODIPY core in EtBOD-PEG resulted in bathochromic
shifts in both the absorption and emission spectra relative to those of the unsubstituted BOD-PEG. Importantly,
bulky di-branched PEG chains were introduced at the meso position of the BODIPY core, leading to enhanced
solubilities and high fluorescence quantum yields in aqueous solutions. The water-soluble BODIPY dyes were
explored for their applicability in fluorescent bio-imaging using MCF-7 human breast cancer cells. The dyes were
cell-permeable and were concentrated in cellular cytoplasm area, suggesting that they have potential applica-
tions as bio-imaging agents.
1. Introduction
quantum yields (QYs; generally > 0.5), high molar extinction coeffi-
−
1
cients (typically > 80,000 L (mol·cm) ), modifiability, neutral total
charges, and excellent photostability [7–10]. Although BODIPY and
derivates have been employed for fluorescence imaging studies, a vast
majority of the research concerning the photophysical and electro-
chemical properties of BODIPY has been carried out in organic en-
vironments, whereas counterpart studies conducted in aqueous media
are scarce [11]. Various bio-imaging BODIPY dyes have been reported,
however they would accumulate in endoplasmic reticulum, mitochon-
dria and subcellular membranes because of their lipophilicity, rigidity
and total charge [7,12–14]. Hence, it is of paramount importance to
develop BODIPY dyes that are relatively water-soluble under physio-
logical conditions and resist the formation of non-fluorescent ag-
gregates, thereby being suitable for biological and medical use [15].
Appending hydrophilic moieties, such as poly(ethylene glycol) (PEG),
N,N-bis(2-hydroxyethyl) amine, carbohydrates, nucleotides, or ionic
groups like carboxylic acids, sulfonic acids, or ammonium groups, to
the BODIPY core are some of the approaches recently used to synthesize
water-soluble BODIPY dyes [15–20].
Over the last decade, fluorescence imaging techniques have proved
to be powerful tools for visualizing cell biology at many levels and for
revealing spatiotemporal details about cellular dynamics [1–4]. They
have paved the way for the development of various fluorescent probes,
including fluorescent proteins, nanocrystals (quantum dots), and small
organic fluorescent dyes, to provide highly sensitive, minimally in-
vasive, and safe detection of cells and tissues [2,5]. However, such
fluorescent systems often suffer from several shortcomings which im-
pede their potential application as biological probes. In particular, or-
ganic dyes commonly suffer from aggregation-caused fluorescence
quenching (ACQ) originating from the formation of non-emissive ex-
cimers or energy transfer to quenching sites [6]. To avoid the un-
desirable quenching effects, bulky protective groups have frequently
been introduced to the periphery of the fluorescent core. By this ap-
proach, high fluorescence efficiencies can be retained in concentrated
solutions, because the sterically bulky protective substituents can pre-
vent intermolecular interactions and unfavorable aggregation.
Among the various organic fluorophores available, 4,4′-difluoro-4-
bora-3a,4a-diaza-s-indacene (BODIPY) and its derivatives are widely
used due to their sharp fluorescence emissions, large fluorescence
Neutral water-soluble BODIPY dyes, such as PEGylated BODIPY,
have an advantage over ionic dyes in that they avoid potential elec-
trostatic interactions between the dyes and biomolecules in biological
⁎
Corresponding authors at: Department of Chemistry, Chosun University, Gwangju, 501-759, Republic of Korea.
E-mail addresses: chobk@dankook.ac.kr (B.K. Cho), hjkim@chosun.ac.kr (H.-J. Kim).
These authors equally contributed to this work.
1
https://doi.org/10.1016/j.jphotochem.2019.03.050
Received 14 February 2019; Received in revised form 20 March 2019; Accepted 30 March 2019
Available online 04 April 2019
1010-6030/ © 2019 Elsevier B.V. All rights reserved.

I.W. Badon, et al.
Journal of Photochemistry & Photobiology A: Chemistry 377 (2019) 214–219
and medical applications [21]. Thus, the use of PEG to increase the
water solubility of BODIPY dyes is a technique still widely used among
researchers. It is also well known that PEG possesses several biological
and medical advantages such as long circulation time, satisfactory
biocompatibility, and a tendency to accumulate in tumor sites via the
enhanced permeability and retention (EPR) effect of leaky tumor neo-
vasculature [22]. Previous methods for introducing PEG chains as hy-
drophilic groups to the BODIPY core often involved arylation at the
meso position of the dye core. This modification would result in an
appreciable decrease in the fluorescence QY due to the free rotation of
the meso substituent, causing energy losses via nonradiative deactiva-
tion of excited states [23,24]. In light of the above discussion, we de-
signed a facile synthetic route to prepare highly water-soluble PEGy-
lated BODIPY dyes. Bulky di-branched PEG chains were prepared and
introduced at the meso position of the BODIPY core, without the in-
corporation of a rotationally free phenyl ring. We investigated the op-
tical properties and fluorescence quantum yields of the resultant dyes in
both organic and aqueous solutions. The BODIPY dyes were well-dis-
tributed in the cellular cytoplasm area, wherein they exhibited high
fluorescence intensity and low cytotoxicity, and are thus quite pro-
mising as bio-imaging agents.
Fig. 1. Synthesis of PEG-functionalized BODIPY fluorescent dyes BOD-PEG and
EtBOD-PEG.
Table 1
Photophysical properties of BOD-PEG and EtBOD-PEG dyes in different solvents
a
at room temperature (concentration = 17.2 μM). For φ measurements, λ
fl
ex
b
c
= 500 nm. For φ measurements, λ = 520 nm. QY = Absolute fluores-
fl
ex
cence quantum yield.
QYc
Dye
Solvent
THF
λ
abs /nm
λem /nm
BOD-PEGa
498
500
498
496
522
524
522
520
516
517
515
511
541
541
539
535
0.681
0.663
0.668
0.512
0.659
0.658
0.624
0.254
CH
2
Cl
2
2. Experimental
EtOH
H O
2
All reagents were obtained from commercial sources and were used
without further purification unless specifically mentioned. 2,4-di-
EtBOD-PEGb
THF
CH Cl
EtOH
2
2
methyl-3-ethylpyrrole, 2,4-dimethylpyrrole, polyethylene glycol
−
1
2
H O
monomethyl ether (MPEG, M
n
= 350 g mol
), 3,5-dihydroxybenzoic
acid and boron trifluoride diethyl etherate were purchased from Sigma-
Aldrich Chemical Company (St. Louis, MO, USA). 5-Bromovaleryl
chloride was obtained from Tokyo Chemical Industry (Tokyo, Japan).
Tetrabutylammonium bromide was obtained from Junsei Chemical Co.,
Ltd. (Tokyo, Japan). Triethylamine and tosyl chloride were bought from
Daejung Chemicals & Metals Co., Ltd. (Siheung, South Korea). Solvents
used were of analytical grade and purified using standard techniques.
Thin layer chromatography was performed on precoated TLC plates
from Merck (Silica Gel 60 F254). NMR Spectroscopic data were ob-
1
tained from the Korea Basic Science Institute, Gwangju branch. H and
1
3
C NMR spectra were referenced to the residual proton resonance of
CDCl
3
(δ 7.26) and recorded on a JEOL JNM-AL300 spectrometer
11
(
(
Japan) with tetramethylsilane (TMS) as the internal standard.
400 MHz) NMR spectra were referenced to BF
B
1
9
3
Et
2
O. F (500 MHz)
NMR chemical shifts were calibrated using CF
3
COOH as external
standard. Absorption spectra were measured with Hitachi 5300 spec-
trophotometer from 200 to 900 nm using 10 mm quartz cuvette.
Fluorescence emission intensities were measured with Hitachi F-7000
fluorescence spectrophotometer. Absolute fluorescence quantum yields
were measured with Hamamatsu Photonics Quantarius – QY Absolute
PL quantum yield spectrometer (Hamamatsu Photonics K.K.,
Hamamatsu, Japan). Cell images were captured using a Zeiss confocal
laser scanning microscope. Bruker Ultraflextreme MALDI-TOF/TOF
mass spectrometer (Berlin, Germany) at the Ochang Branch of the
Korean Basic Science Institute was used to measure the mass of the final
compounds.
Fig. 2. Photographs of BOD-PEG and EtBOD-PEG under UV illumination
365 nm) in different solvents: (A) THF; (B) CH Cl ; (C) EtOH; (D) H O (con-
centration = 17.2 μM).
(
2
2
2
added dropwise. The reaction mixture was refluxed for 1.5 h at 50 °C
2
2
.1. Synthesis of halogenated dyes
and the solvent was vacuum evaporated. The crude product was pur-
ified by silica gel column chromatography eluting with CH
2
Cl
, 400 MHz): δ = 6.07 (s, 2 H), 3.46 (t, 2 H),
2.99 (t, 2 H), 2.52 (s, 6 H), 2.43 (s, 6 H), 2.06 (t, 2 H), 1.82 (t, 2 H) ppm.
2
-hexane.
1
.1.1. BODIPY-(CH
2
)
4
-Br
Yield 49%. H NMR (CDCl
3
The halogenated dye was prepared according to a previously pub-
lished method [25]. 5-Bromovaleryl chloride (0.352 ml, 1.00 eq) was
added dropwise to a stirred solution of 2,4-dimethylpyrrole (0.500 g,
2.1.2. Ethyl-BODIPY-(CH
2 4
) -Br
2.00 eq) in anhydrous dichloromethane at 50 °C for 2 h. After the sol-
This compound was prepared in the same way as BODIPY-(CH ) -
2 4
vent was evaporated, the residual solid was dissolved in toluene (35 ml)
and dichloromethane (15 ml). Triethylamine (1.54 ml, 4.20 eq) was
added, and the mixture was stirred under argon atmosphere for 30 min,
and then boron trifluoride diethyl etherate (1.62 ml, 5.00 eq) was
Br, using 2,4-dimethyl-3-ethylpyrrole (0.500 g, 2.00 eq). All other re-
agents were scaled accordingly. The crude product was purified by si-
lica gel column chromatography eluting with CH
2 2
Cl -hexane. Yield
56%. 1H NMR (CDCl
, 400 MHz): δ = 3.46 (t, 2 H), 3.03 (t, 2 H), 2.50
3
215

I.W. Badon, et al.
Journal of Photochemistry & Photobiology A: Chemistry 377 (2019) 214–219
Fig. 3. Absorption (left) and emission (right) spectra (λex = 496–500 nm) of BOD-PEG dye in different solvents (concentration = 17.2 μM).
Fig. 4. Absorption (left) and emission (right) spectra (λex = 520–524 nm) of EtBOD-PEG dye in different solvents (concentration = 17.2 μM).
Fig. 5. Emission spectra of BOD-PEG (left, λex = 506 nm) and EtBOD-PEG (right, λex = 530 nm) dyes in water at different concentrations.
Table 2
glycol (5.00 g, 1.00 eq) was dissolved in 5.00 mL of anhydrous pyridine.
Absolute fluorescence quantum yields of BOD-PEG and EtBOD-PEG dyes in
Tosyl chloride (3.27 g, 1.20 eq) was dissolved in anhydrous methylene
chloride and slowly injected into the solution. The solution was con-
tinuously stirred under Ar gas for 8 h at RT. Then, 6 N HCl solution (5
times the total volume of pyridine used) was added into the solution.
The mixture was extracted with dichloromethane three (3) times and
the organic layer was washed with 2 N HCl solution. After drying with
water at different concentrations. a
λ
ex = 500 nm. b
λex = 520 nm.
Dye
Conc. (μM)
φ
fl
BOD-PEGa
1.0
0.514
0.561
0.361
0.471
0.292
0.138
1
3
0.0
0.0
EtBOD-PEGb
MgSO
colorless oil. Yield 76%. H NMR (CDCl
7.36 (d, 2 H), 4.16 (t, 2 H), 3.68-3.61 (m, 32 H), 3.58 (t, 2 H), 3.38 (s,
4
, the solvent was evaporated to afford the tosylated PEG as a
1.0
1
1
3
0.0
0.0
3
, 400 MHz): δ = 7.81 (d, 2 H),
3
H), 2.45 (s, 3 H) ppm.
(
s, 6 H), 2.41 (m, 4 H), 2.35 (s, 6 H), 2.07 (t, 2 H), 1.83 (t, 2 H), 1.05 (t,
6
H) ppm.
2.3. Synthesis of Ethyl-3,5-dialkoxy benzoate
2.2. Synthesis of Poly(ethylene glycol) methyl ether tosylate
Ethyl-3,5-dihydroxy benzoate (0.636 g, 1.00 eq), PEG methyl ether
2 3
tosylate (4.00 g, 2.2 eq) and anhydrous K CO (2.12 g, 4.4 eq) were
The PEG moiety was synthesized according to the previously re-
ported procedure [26]. Briefly, 350 MW monomethyl polyethylene
dissolved in anhydrous DMF and the solution was refluxed for 12 h at
80 °C under Ar atmosphere [26]. Then, the solution was poured into ice-
216

I.W. Badon, et al.
Journal of Photochemistry & Photobiology A: Chemistry 377 (2019) 214–219
1
1
5
5
61.47, 160.01, 156.01, 135.43, 132.00, 130.11, 121.96, 108.13,
06.93, 101.70, 92.41, 72.02, 71.45, 70.92, 70.75, 70.66, 69.67, 67.78,
19
3
9.13, 42.78, 32.20, 29.74, 27.90, 16.39, 14.49. F NMR (CDCl ,
00 MHz)= -147.10 (m) ppm. 11B NMR (CDC+l
, 400 MHz): δ = 0.410
m/z: calculated for
3
(
t) ppm. MALDI-TOF/TOF MS: [M + Na]
C
58
H
95BF
EtBOD-PEG was synthesized using the same procedure described
above but with ethyl-BODIPY-(CH -Br (40.0 mg, 1.00 eq). All the
other reagents were scaled accordingly, and the crude product was
purified by silica gel column chromatography eluting with CH Cl
MeOH to afford the PEGylated BODIPY as a dark magenta oil. Yield
2 2
N NaO20, 1211.64; found, 1213.713.
2 4
)
2
2
:
1
25%. H NMR (CDCl
3
, 400 MHz): δ = 7.17 (d-s, 2 H), 6.70 (s, 1 H), 4.37
(
t, 2 H), 4.12 (t, 4 H), 3.71-3.63 (m, 52 H), 3.56 (t, 4 H), 3.38 (s, 6 H),
3
1
1
1
6
.07 (m, 2 H), 2.49 (s, 6 H), 2.40 (m, 2 H), 2.33 (s, 6 H), 2.05 (m, 2 H),
.99 (t, 2 H), 1.03 (t, 6 H) ppm. 13C NMR (CDCl
66.45, 160.02, 152.55, 144.06, 138.63, 135.75, 132.91, 132.06,
31.05, 108.18, 106.82, 100.06, 72.03, 71.46, 70.92, 70.76, 69.67,
3
, 300 MHz): δ 187.42,
7.78, 59.14, 42.79, 29.75, 29.18, 17.19, 16.09, 14.86, 13.37, 12.43.
1
9
11
3
F NMR (CDCl3, 500 MHz): δ = -146.43 (m) ppm. B NMR (CDCl ,
+
400 MHz): δ = 0.458 (t) ppm. MALDI-TOF/TOF MS: [M + Na] m/z:
2 2
calculated for C62H103BF N
NaO20, 1267.71; found, 1267.786.
2.6. Cell proliferation assay
The human breast MCF-7 cells (3 × 103 cells/well) were seeded in
6-well plates. After the cells were maintained for 24 h, cells were
9
treated with either BOD-PEG or EtBOD-PEG dye for 24 h. Cell pro-
liferation assay was measured using CellTiter 96 ® AQueous One
Solution Cell Proliferation Assay (Promega, Madison, WI, USA) ac-
cording to the manufacturer's instruction. This assay contains MTS [3-
Fig. 6. The relative number of viable cells after incubation with different
concentrations of BOD-PEG (top) and EtBOD-PEG (bottom) dyes for 24 h.
(
4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfo-
cold water and extracted with dichloromethane three (3) times. The
organic layer was washed with water and brine, and dried over MgSO ,
4
following which the solvent was removed by rotary evaporation. The
phenyl)-2H-tetrazolium] which is reduced to formazan by the action of
NADH or NADPH in metabolically active cells [27]. The absorbance
value of the wells containing solutions of MTS (background) was sub-
tracted from those of the wells containing the treated and control cells.
crude product was purified by silica gel column chromatography using
1
CH
2
Cl
2
and MeOH as eluent. Yield 53%. H NMR (CDCl
3
, 400 MHz):
δ = 7.20 (s, 2 H), 6.69 (s, 1 H), 4.37 (m, 2 H), 4.16 (t, 4 H), 3.76-3.60
2.7. Confocal microscopy
(
m, 52 H), 3.54 (t, 4 H), 3.38 (s, 6 H), 1.38 (t, 3 H) ppm.
MCF-7 cells were grown on sterilized coverslips in 12-well overnight
and then treated with either BOD-PEG or EtBOD-PEG dye for 24 h.
After washing with phosphate-buffered saline (PBS), cells were fixed
with 4% paraformaldehyde for 60 min and permeabilized with 0.5%
Triton X-100 for 10 min. at room temperature. The slides were mounted
with Vectashield mounting medium with 4′,6-Diamidino-2-pheny-
lindole (DAPI) (Vector Laboratories, Burlingame, CA, USA) following
the manufacturer's instructions. Images were captured with a confocal
microscope at 20× magnification (LSM-700; Carl Zeiss Microimaging
Inc., Stockholm, Sweden).
2
.4. Synthesis of 3,5-dialkoxy benzoic acid
In a THF-H O (1:1) mixed solvent system, ethyl-3,5-dialkoxy
2
benzoate (3.47 g, 1.00 eq) and sodium hydroxide (0.473 g, 3.00 eq)
were dissolved and refluxed for 24 h at 80 °C. After the solution cools to
room temperature, the pH was adjusted to 3–4 using 10% v/v HCl.
Then it was extracted using CH
via rotary evaporation to afford a brown oil. Yield 13%. H NMR
CDCl
2 2 4
Cl , dried over MgSO and concentrated
1
(
3
3
, 400 MHz): δ = 7.25 (s, 2 H), 6.70 (s, 2 H), 4.16 (t, 4 H), 3.71-
.65 (m, 52 H), 3.57 (m, 4 H), 3.38 (s, 6 H) ppm.
3. Results and discussion
2.5. Synthesis of PEGylated BODIPYs
3.1. Synthesis of PEGylated BODIPY dyes
To KOH (5.00 mg, 1.00 eq) dissolved in MeOH, 3,5-dialkoxy benzoic
acid (150 mg, 2.00 eq) was added and stirred. Then the solvent was
completely removed to obtain potassium 3,5-dialkoxy benzoate.
Fig. 1 outlines the synthesis of the fluorescent BODIPY dyes con-
taining di-branched PEG chains. The di-branched PEG chains were
prepared according to previously reported methods. [26] First, Wil-
liamson etherification between ethyl 3,5-dihydroxybenzoate and tosy-
lated PEG produced di-PEGylated benzoates. Their subsequent hydro-
lysis yielded di-PEGylated benzoic acid. Meso-1-bromo-butyl-
substituted BODIPY dyes were prepared via the condensation of 5-
bromovaleryl chloride with either 2,4-dimethyl-3-ethylpyrrole or 2,4-
BODIPY-(CH
2 4
) -Br (43.0 mg, 20 eq), potassium 3,5-dialkoxy benzoate
(
200 mg, 40 eq) and tetrabutylammonium bromide (1.81 mg, 1 eq)
were dissolved in anhydrous DMF and refluxed at 40 °C for 48 h. After
cooling to room temperature, the solvent was vacuum distilled, and the
crude product was purified by silica gel column chromatography
eluting with CH
2
Cl
2
: MeOH to afford BOD-PEG dye as a dark orange
oil. Yield 17%. H NMR (CDCl , 400 MHz): δ = 7.16 (d-s, 2 H), 6.69 (s,
H), 6.06 (s, 2 H), 4.36 (t, 2 H), 4.12 (t, 4 H), 3.69-3.64 (m, 52 H), 3.54
t, 4 H), 3.38 (s, 6 H), 3.03(m, 2 H), 2.51 (s, 6 H), 2.41 (s, 6 H), 1.96 (m,
3 2
dimethylpyrrole and subsequent complexation with BF ·OEt in the
1
3
presence of triethylamine. Finally, the esterification between the bro-
mine-containing BODIPYs and the carboxyl ends of the di-PEGylated
benzoic acid afforded the water-soluble BODIPY dyes in moderate
yields; 16.6% and 25.2% for BOD-PEG and EtBOD-PEG, respectively.
1
(
1
3
2
3
H), 1.80 (m, 2 H) ppm. C NMR (CDCl , 300 MHz): δ 186.30, 177.96,
217

I.W. Badon, et al.
Journal of Photochemistry & Photobiology A: Chemistry 377 (2019) 214–219
Fig. 7. CLSM images of MCF-7 human breast cancer cells with either BOD-PEG or EtBOD-PEG dye at various concentrations, with the nucleus labeled with 4′,6-
diamidino-2-phenylindole (DAPI). Top: DAPI images; middle: BOD-PEG/ EtBOD-PEG dye images; bottom: merged images of both (concentration = 10.0 μM).
The formation of an ester bond is evidenced by the appearance of triplet
peaks at 4.36/4.37 ppm in the 1H NMR spectra (Figs. S1 & S6). All
(520–524 nm), to 539–541 nm in organic solvents and 535 nm in water.
Theoretical studies have indicated that electron-withdrawing sub-
stituents stabilize the lowest unoccupied molecular orbital (LUMO) of
BODIPY units, while electron-donating substituents destabilize the
LUMO [29,30]. Therefore, electron-donating ethyl units resulted in the
red-shifted emission of EtBOD-PEG relative to that of BOD-PEG.
In the case of EtBOD-PEG, broadening of the peak at 480–500 nm in
the absorption profile in water seemed to suggest dye aggregation. To
understand this behavior further, we studied the fluorescence spectra
and fluorescence QY of both dyes in aqueous solution as a function of
concentration, as depicted in Fig. 5. The fluorescence spectra of BODIPY
dyes were red-shifted by about 30 nm and the emission intensities de-
creased exponentially as the dye concentrations were increased from
1
intermediate compounds were characterized by H NMR, and final
1
3
19
11
products were also characterized by C NMR, F NMR and B NMR
SI). High-resolution mass spectral analysis showed the PEG chains to
be approximately 6–8 units in length (SI Figs. S5 & S10).
(
3.2. Photophysical properties
The photophysical properties of BOD-PEG and EtBOD-PEG were
investigated in different solvents, and are summarized in Table 1. Upon
UV light excitation, organic and aqueous solutions of BOD-PEG emit
green light while those of EtBOD-PEG emit yellow-green light (Fig. 2).
As depicted in Figs. 3 and 4, narrow absorption spectra mirroring the
respective emission spectra were recorded for both fluorescent dyes.
The absorption and emission profiles correspond to those of typical
BODIPY derivatives [28], indicating that the appended PEG did not
exert a significant effect on the photophysical properties of the dye in
any of the solvents used in this study. The absorption spectra of BOD-
PEG exhibited maximum intensity (λab) at 498–500 nm in organic sol-
vents and at 496 nm in water, due to the π-π* transition of the BODIPY
unit. The λab generally blue-shifts with an increase in the polarity of the
solvent, but only by a few nanometers. The emission peaks of BOD-PEG
were observed at 515–517 nm in organic solvents and at 511 nm in
water. The presence of ethyl groups at the 2,6 positions of the BODIPY
core in the dye EtBOD-PEG resulted in red-shifts in the absorption
spectra of ˜20 nm relative to its unsubstituted counterpart
1.0 μM to 10.0 μM. This phenomenon may be ascribed to inter-
molecular interactions or aggregation in aqueous solution induced by
the increasing concentration of the dyes [31]. The QYs of the dyes in
water at 1.0 μM reached 0.514 for BOD-PEG and 0.471 for EtBOD-PEG.
At concentrations of 30.0 μM, the fluorescence QYs decreased to 0.361
for BOD-PEG and 0.138 for EtBOD-PEG (Table 2). Notably, both dyes
exhibited relatively high fluorescence QYs even at concentrations of
30.0 μM, because the aggregation of the dyes in water was suppressed
by the hydrophilic and sterically hindering groups. Further, relative to
the unsubstituted BOD-PEG, the ethyl groups at the 2- and 6-positions
in EtBOD-PEG increased the hydrophobic character of the dye core. In a
study conducted by Nepomnyashchii et al., 2,6-ethyl-substituted
BODIPY dyes displayed lower fluorescence than their unsubstituted
counterparts [11], suggesting that hydrophobic interactions between
218

I.W. Badon, et al.
Journal of Photochemistry & Photobiology A: Chemistry 377 (2019) 214–219
the ethyl-substituted BODIPY dyes may play a role in this phenomenon.
References
3
.3. Cell viability and imaging
[1] C.J. Weijer, Visualizing signals moving in cells, Science 300 (5616) (2003) 96–100.
[
2] B.N. Giepmans, S.R. Adams, M.H. Ellisman, R.Y. Tsien, The fluorescent toolbox for
assessing protein location and function, Science 312 (5771) (2006) 217–224.
3] M.J. Pittet, R. Weissleder, Intravital imaging, Cell 147 (5) (2011) 983–991.
One of the critical requirements for fluorescent probes is good
[
biocompatibility [8]. The CellTiter 96 ® AQueous One Solution Cell
Proliferation Assay was used to assess the cytotoxicity of the BODIPY
dyes. This method is based on the bioreduction of the tetrazolium
compound MTS (Owen’s reagent) to a colored formazan product by the
cells. The quantity of formazan produced, as measured by absorbance at
[4] R.N. Germain, E.A. Robey, M.D. Cahalan, A decade of imaging cellular motility and
interaction dynamics in the immune system, Science 336 (6089) (2012)
1
676–1681.
[
5] S.H. Lee, H.T. Bui, T.P. Vales, S. Cho, H.J. Kim, Multi-color fluorescence of pNIPAM-
Based nanogels modulated by dual stimuli-responsive FRET processes, Dye.
Pigment. 145 (2017) 216–221.
[
6] D. Khuong Mai, J. Lee, I. Min, T. Vales, K.H. Choi, B. Park, et al., Aggregation-
induced emission of tetraphenylethene-conjugated phenanthrene derivatives and
their bio-imaging applications, Nanomaterials 8 (9) (2018) 728.
490 nm, is directly proportional to the number of viable cells [27]. Cell
viability experiments for the BODIPY dyes synthesized in this study
were carried out employing MCF-7 human breast cancer cells. The test
cells were incubated with either BOD-PEG or EtBOD-PEG dye at con-
centrations ranging from 2.5 to 10 μM. As shown in Fig. 6, there was no
significant reduction in the viability of the cells after 24 h incubation
with the fluorescent dyes. The BODIPY probes did not exhibit signs of
toxicity towards the tested cells at low concentrations. Moreover, it is
noteworthy that the number of living cells was maintained at ap-
proximately 90% and 80% of the original count for BOD-PEG and
EtBOD-PEG, respectively, even after treatment with 10 μM of the dye.
These results indicate that neither dye is significantly cytotoxic under
the tested conditions over 24 h.
[7] T. Kowada, H. Maeda, K. Kikuchi, BODIPY-based probes for the fluorescence ima-
ging of biomolecules in living cells, Chem. Soc. Rev. 44 (14) (2015) 4953–4972.
[
8] T. Gao, H. He, R. Huang, M. Zheng, F.F. Wang, Y.J. Hu, et al., BODIPY-based
fluorescent probes for mitochondria-targeted cell imaging with superior brightness,
low cytotoxicity, and high photostability, Dye. Pigment. 141 (2017) 530–535.
9] L. Quan, S. Liu, T. Sun, X. Guan, W. Lin, Z. Xie, et al., Near-infrared emitting
fluorescent BODIPY nanovesicles for in vivo molecular imaging and drug delivery,
ACS Appl. Mater. Interfaces 6 (18) (2014) 16166–16173.
[
[10] H. Xiao, J. Li, K. Wu, G. Yin, Y. Quan, R. Wang, A turn-on BODIPY-based fluorescent
probe for Hg (II) and its biological applications, Sens. Actuators B Chem. 213 (2015)
3
43–350.
[
[
11] A.B. Nepomnyashchii, A.J. Pistner, A.J. Bard, J. Rosenthal, Synthesis, photophysics,
electrochemistry and electrogenerated chemiluminescence of PEG-modified
BODIPY dyes in organic and aqueous solutions, J. Phys. Chem. C 117 (11) (2013)
5
599–5609.
To evaluate the potential application of the fluorescent dyes for
bioimaging, MCF-7 cells were treated with the dyes for 24 h and imaged
12] L. Jiao, C. Yu, T. Uppal, M. Liu, Y. Li, Y. Zhou, et al., Long wavelength red fluor-
escent dyes from 3, 5-diiodo-BODIPYs, Org. Biomol. Chem. 8 (11) (2010)
2517–2519.
13] X.D. Jiang, R. Gao, Y. Yue, G.T. Sun, W. Zhao, A NIR BODIPY dye bearing 3, 4, 4 a-
trihydroxanthene moieties, Org. Biomol. Chem. 10 (34) (2012) 6861–6865.
14] T. Uppal, X. Hu, F.R. Fronczek, S. Maschek, P. Bobadova‐Parvanova,
M.G.H. Vicente, Synthesis, computational modeling, and properties of benzo‐ap-
pended BODIPYs, Chem. Eur. J. 18 (13) (2012) 3893–3905.
by confocal microscopy. 4′,6-diamidino-2-phenylindole (DAPI),
a
[
fluorescent stain commonly used to visualize the nucleus, is employed
to confirm the localization of the fluorescent dyes within the cells. [32]
As shown in Fig. 7, there was no fluorescence emission from the control
untreated cells, while the test cells incubated with the dyes at con-
centrations of 10 μM emitted bright green fluorescence of high in-
tensity. Additionally, the fluorescence microscopy images of the MCF-7
cells with the fluorescent probes showed that BODIPY dyes were suc-
cessfully taken in by the cells, efficiently localized, and then fluoresced
upon excitation.
[
[
15] L. Li, J. Han, B. Nguyen, K. Burgess, Syntheses and spectral properties of functio-
nalized, water-soluble BODIPY derivatives, J. Org. Chem. 73 (5) (2008) 1963–1970.
[16] Y. Vo‐Hoang, L. Micouin, C. Ronet, G. Gachelin, M. Bonin, Total Enantioselective
Synthesis and In Vivo Biological Evaluation of a Novel Fluorescent BODIPY
α‐Galactosylceramide, ChemBioChem 4 (1) (2003) 27–33.
[
[
[
[
17] S.C. Dodani, Q. He, C.J. Chang, A turn-on fluorescent sensor for detecting nickel in
living cells, J. Am. Chem. Soc. 131 (50) (2009) 18020–18021.
18] S.L. Niu, G. Ulrich, R. Ziessel, A. Kiss, P.Y. Renard, A. Romieu, Water-soluble
BODIPY derivatives, Org. Lett. 11 (10) (2009) 2049–2052.
19] L. Jiao, J. Li, S. Zhang, C. Wei, E. Hao, M.G.H. Vicente, A selective fluorescent
sensor for imaging Cu 2+ in living cells, New J. Chem. 33 (9) (2009) 1888–1893.
20] A. Romieu, C. Massif, S. Rihn, G. Ulrich, R. Ziessel, P.Y. Renard, The first com-
parative study of the ability of different hydrophilic groups to water-solubilise
fluorescent BODIPY dyes, New J. Chem. 37 (4) (2013) 1016–1027.
4. Conclusion
In this study, a series of water-soluble PEGylated BODIPY dyes
(
BOD-PEG and EtBOD-PEG) were prepared, and their photophysical
properties were investigated. Bulky di-branched PEG chains were in-
troduced at the meso position of the BODIPY core to reduce the ag-
gregation tendencies of the dyes. The dye BOD-PEG, which has no
substitutions at positions 2 and 6 of the BODIPY core, exhibited ab-
sorption and emission maxima at shorter wavelengths relative to those
of EtBOD-PEG, which has electron-donating ethyl groups at the 2 and 6
positions of the core. Notably, the fluorescence QYs of the dyes at 1 μM
in water were 0.514 for BOD-PEG and 0.471 for EtBOD-PEG, which are
higher than those of other BODIPY-based water-soluble probes reported
previously. The PEGylated BODIPY dyes were able to permeate MCF-7
cells and localized in the cellular cytoplasm, exhibiting good water
solubility and biocompatibility. This work may provide new strategies
for the design and fabrication of highly efficient fluorescent probes in
aqueous environments.
[21] M.S.T. Gonçalves, Fluorescent labeling of biomolecules with organic probes, Chem.
Rev. 109 (1) (2008) 190–212 22.
[
22] X. Liu, B. Chen, X. Li, L. Zhang, Y. Xu, Z. Liu, et al., Self-assembly of BODIPY based
pH-sensitive near-infrared polymeric micelles for drug controlled delivery and
fluorescence imaging applications, Nanoscale 7 (39) (2015) 16399–16416.
[23] A. Loudet, K. Burgess, BODIPY dyes and their derivatives: syntheses and spectro-
scopic properties, Chem. Rev. 107 (11) (2007) 4891–4932.
[
24] R. Hu, E. Lager, A. Aguilar-Aguilar, J. Liu, J.W. Lam, H.H. Sung, et al., Twisted
intramolecular charge transfer and aggregation-induced emission of BODIPY deri-
vatives, J. Phys. Chem. C 113 (36) (2009) 15845–15853.
[
25] Y. Kajiwara, A. Nagai, Y. Chujo, Microwave-assisted preparation of intense lumi-
nescent BODIPY-containing hybrids with high photostability and low leachability,
J. Mater. Chem. 20 (15) (2010) 2985–2992.
[26] R. Nguyen, N. Jouault, S. Zanirati, M. Rawiso, L. Allouche, G. Fuks, et al., Core–shell
inversion by pH modulation in dynamic covalent micelles, Soft Matter 10 (22)
(2014) 3926–3937.
[
[
27] A.G. Protocol, CellTiter 96® AQueous One Solution Cell Proliferation Assay.
Promega, USA, (2001).
28] S. Hoogendoorn, A.E. Blom, L.I. Willems, G.A. van der Marel, H.S. Overkleeft,
Synthesis of pH-activatable red fluorescent BODIPY dyes with distinct functional-
ities, Org. Lett. 13 (20) (2011) 5656–5659.
Acknowledgment
[
[
29] Y. Mao, M. Head-Gordon, Y. Shao, Unraveling substituent effects on frontier orbitals
of conjugated molecules using an absolutely localized molecular orbital based
analysis, Chem. Sci. 9 (45) (2018) 8598–8607.
30] I.K. Petrushenko, K.B. Petrushenko, Effect of meso-substituents on the electronic
transitions of BODIPY dyes: DFT and RI-CC2 study, Spectrochim. Acta A. Mol.
Biomol. Spectrosc. 138 (2015) 623–627.
31] Y. Li, H. Lin, C. Luo, Y. Wang, C. Jiang, R. Qi, et al., Aggregation induced red shift
emission of phosphorus doped carbon dots, RSC Adv. 7 (51) (2017) 32225–32228.
32] A.K. Estandarte, S. Botchway, C. Lynch, M. Yusuf, I. Robinson, The use of DAPI
fluorescence lifetime imaging for investigating chromatin condensation in human
chromosomes, Sci. Rep. 6 (2016) 31417.
This study was supported by research funds provided by Chosun
University in 2017.
Appendix A. Supplementary data
[
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.jphotochem.2019.03.
[
050.
219