In Vitro Photodynamic Studies of a BODIPY-Based Photosensitizer

Article abstract of DOI:10.1002/ejoc.201601054

A new halogenated 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) derivative was designed as a singlet-oxygen photosensitizer (PS) for use as a photodynamic therapy (PDT) agent, and its photophysical properties were characterized. The PS, having two i

Full text of DOI:10.1002/ejoc.201601054

A Journal of
Accepted Article
Title: In Vitro Photodynamic Studies of a Novel BODIPY-based
Photosensitizer
Authors: Bosung Kim, Binglin Sui, Xiling Yue, Simon Tang, Michael G.
Tichy, and Kevin D. Belfield
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To be cited as: Eur. J. Org. Chem. 10.1002/ejoc.201601054
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Supported by

European Journal of Organic Chemistry
10.1002/ejoc.201601054
COMMUNICATION
In Vitro Photodynamic Studies of a Novel BODIPY-based
Photosensitizer
[
b]
[a]
[b]
[b]
[b]
[a]
Bosung Kim, Binglin Sui, Xiling Yue, Simon Tang, Michael G. Tichy, and Kevin D. Belfield*
Abstract: A new halogenated BODIPY derivative was designed as a
singlet oxygen photosensitizer (PS) for use as a photodynamic
therapy (PDT) agent and its photophysical properties were
characterized. The PS, having two iodine atom substituents,
exhibited low fluorescence quantum yield (ΦFL) of 0.02 and high
considerable research efforts in developing BODIPY dyes for
PDT. By introducing heavy atoms, such as halogens, to the
BODIPY core, BODIPY derivatives showed improved SOG,
[
8]
which renders them to be potential photosensitizers for PDT.
This is most likely due to the internal heavy-atom effect that
enhances spin-orbit coupling and consequently, the efficiency of
singlet oxygen generation quantum yield (Φ
in vitro photodynamic activities were evaluated to assess the
potential of using the PS for PDT. propidium iodide (PI)
∆
) of 0.93. Furthermore,
ISC from an excited singlet state (S
n
) to an excited triplet state
[
2a]
A
1
(T ).
fluorescence assay and live cell imaging in LLC cells demonstrated
that photosensitization of the agent induced cellular damage,
subsequently leading to cell death by necrosis.
In this study, we report not only the synthesis of a novel
halogenated BODIPY dye (3), but the analysis of its
photophysical properties, including SOG. In order to
demonstrate preliminary potential PDT efficacies of the agent, in
vitro photodynamic activities were conducted.
Photodynamic therapy (PDT) has emerged as a potential
modality for the treatment of a variety of cancers. By combining
PDT agents with light and oxygen, the light-activated
The synthesis of 3 began with 4-hydroxybenzaldehyde,
which was used to prepare compound 1 (4,4-difluoro-8-(4-
hydroxyphenyl)-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-
indacene), a BODIPY derivative (Scheme 1). Full experimental
details are provided in the Supporting Information. According to
photosensitizer transfers energy to surrounding ground state
3
oxygen molecules ( O
2
), resulting in the formation of singlet
1
oxygen ( O
2
)
and eventual production of reactive oxygen
[9]
a literature procedure, 1 was prepared via a condensation
species (ROS). The generated ROS can induce cellular damage,
reaction of 4-hydroxybenzaldehyde with 2,4-dimethylpyrrole in
[
1]
leading to cell death by apoptotic and/or necrotic process. As
CH
acid at room temperature, followed by an oxidation reaction with
DDQ (2,3-dichloro-5,6-dicyano-1,4-benzoquinone) and
subsequent chelation reaction with BF ·OEt in the presence of
2 2
Cl in the presence of a catalytic amount of trifluoroacetic
1
2
O plays key roles in PDT therapeutic efficacy, it is essential
that the optimized photosensitizers possess high efficiency of
singlet oxygen generation (SOG) together with minimal dark
toxicity and high photostability. In an effort to develop efficient
PDT agents, heavy-atom effects have been considered as an
effective approach to enhance singlet-triplet intersystem
a
3
2
triethylamine at room temperature. Hydroxyphenyl BODIPY 1
was then reacted with triethyleneglycol monotosylate in the
presence of potassium carbonate in acetonitrile to produce
compound 2. Iodination of 2 with N-iodosuccinimide in the dark
[
2]
crossing (ISC) in several chromophores.
The 4,4-difluoro-4-bora-3a,4a,-diaza-s-indacene (BODIPY)
at room temperature afforded
3 (4,4-difluoro-8-(4-{2-[2-(2-
[
3]
chromophore, since its first discovery, has gained substantial
attention. BODIPY dyes have extraordinary photophysical
properties, including generally high fluorescence quantum yields,
excellent photostability, and high extinction coefficients (molar
hydroxyethoxy)ethoxy]ethoxy}phenyl)-1,3,5,7-tetramethyl-2,6-
diiodo-4-bora-3a,4a-diaza-s-indacene) as red solid.
[
4]
absorptivity).
Having readily tunable characteristics, these
compounds have been widely studied for various applications,
such as fluorescent probes for one-photon and two-photon
[
5]
[6]
fluorescence bioimaging, sensors for various analytes, and
[
7]
for dye-sensitized solar cells (DSSC). Recently, there has been
[
a]
b]
Dr. B. Sui, Dr. K. D. Belfield
Department of Chemistry and Environmental Science, College of
Science and Liberal Arts
New Jersey Institute of Technology
University Heights, Newark, NJ 07102, (USA)
E-mail: belfield@njit.edu, http://csla.njit.edu/belfield/
Dr. B. Kim, Dr. X. Yue, Dr. S. Tang, M. G. Tichy
Department of Chemistry
[
University of Central Florida
P.O. Box 162366, Orlando, FL 32816-2366, (USA)
Supporting information for this article is given via a link at the end of
the document.
Scheme 1. Synthetic route for photosensitizer 3.
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European Journal of Organic Chemistry
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COMMUNICATION
Photophysical properties of 3 were characterized to assess
its potential as a PDT agent and are summarized in Table 1
along with those of acridine, a commonly used photosensitizer
via a (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-
2-(4-sulfophenyl)-2H-tetrazolium) (MTS) assay. As shown in
Figure 2(A), cell viability remained over 70% until 15 µM,
providing good cellular biocompatibility of the agent. At a dye
[
10]
for oxidative stress studies,
for comparison purposes. As
2
shown in Figure 1(A), 3 in acetonitrile (ACN) exhibited sharp
steady-state absorption and fluorescence emission spectra with
a fluorescence quantum yield (ΦFL) of 0.02 (see Supporting
Information for details of photophysical characterization). The
low fluorescence quantum efficiency of the dye can be attributed
to the introduction of two iodine atoms at the 2- and 6- positions,
which enhances the ISC efficiency from an excited singlet state
concentration of 10 µM with a light dose of 3.5 mW/cm (band
pass filter HQ522/40, Chroma Technology Corp.), ~50% of the
cells appeared to be killed indicating, efficient light-induced
toxicity of the agent (Figure 2(B)).
A
B
100
120
PDT
Only Irradiation
100
(
S
n
) to an excited triplet state (T
1
). Additionally, the ability of the
was assessed by a direct measurement of
near-infrared luminescence at 1270 nm. As presented in Figure
80
1
80
agent to generate O
2
60
60
40
20
0
1
40
20
0
1
(B), 3 exhibited characteristic luminescence of O
2
at 1270 nm
) of 0.93,
with a singlet oxygen generation quantum yield (Φ
∆
which is higher than that of acridine (0.82). This result is a clear
1
µΜ
5 µΜ
10 µΜ
15 µΜ
20 µΜ
0 min
1 min
5 min
10 min
indication that 3 is a potentially promising PDT agent.
Concentration (µΜ)
Irradiation time (min)
Figure 2. (A) Dark cytotoxicity toward LLC cells after 24 h incubation with
increasing concentrations of 3. (B) Cell viability of LLC cells in the absence
Table 1. Photophysical Properties for 3 and Acridine.
Dye
3
Acridine
(
blue) or presence (red) of 10 µM of 3 under differing periods of irradiation
2
time; 3.5 mW/cm from a filtered Hg light source (522 ± 40 nm).
Solvent
ACN
528 ± 1
ACN
356 ± 1
403 ± 1
47 ± 2
λ
Abs (nm)
Furthermore, PDT-induced cell necrosis was demonstrated
by a propidium iodide (PI) fluorescence assay. PI, a membrane
impermeant marker, can stain only dead cells by penetrating
λ
Em (nm)
546 ± 1
[
11]
dead cells mainly due to the loss of membrane integrity.
Control cells without exhibited healthy morphology and
∆
λ (nm)
18 ± 2
3
negative staining with PI (Figure 3(A) to (C)), suggesting
irradiation alone in this wavelength range (502-542 nm) is
nontoxic to the cells. In contrast, cells treated with the
combination of 3 and irradiation exhibited red fluorescence from
PI (Figure 3(D) to (F)). These results suggest that
photosensitization of the agent can induce cellular damage,
subsequently leading to cell death consistent with a necrosis.
max
-3
-1
-1
ε
× 10 (M cm )
75.5 ± 8
0.02 ± 0.002
9.3 ± 1
ND
Φ
FL
Φ
∆
0.
ꢀ
93 ± 0.09
0.82 ± 0.08
Maxima of Absorption λAbs and Fluorescence λEm, Stokes shift ∆λ, Maximum
max
molar absorptivity ε , Fluorescence quantum yield ΦFL, and singlet oxygen
generation quantum yield Φ
∆
. ND, not determined.
A
B
C
A
B 200000
Abs.
Em.
Exc.
Acridine
BDP-3
1
0
0
0
0
0
.0
.8
.6
.4
.2
.0
1
50000
100000
D
E
F
5
0000
0
3
00
400
500
600
700
800
1200
1240
1280
1320
1360
Wavelength (nm)
Wavelength (nm)
Figure 1. (A) Normalized absorption (black), emission (red), and excitation
green) spectra of 3 in ACN. (B) Near-infrared singlet oxygen luminescence
spectra of 3 (red) and acridine (black) in ACN.
(
ꢀ
Figure 3. Differential interference contrast, DIC, (A, D) and confocal
fluorescence (B, E) images of LLC cells stained with propidium iodide after
2
In vitro photodynamic activities of 3 were evaluated in LLC
cells, a Lewis lung carcinoma cell line. First, dark cytotoxicity
with the dye concentration ranging from 1 to 20 µM and
photoinduced cytotoxicity of the photosensitizer were assessed
irradiation (0.6 mW/cm from a filtered Hg light source; 522 ± 40 nm) in the
absence (A-C) or presence (D-F) of 3. Scale bars are 50 µm.
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European Journal of Organic Chemistry
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Finally, we examined time-lapsed live cell imaging to
provide insight on cell morphological changes. Cells incubated
with 3 showed cell swelling-like activities, a characteristic of
necrosis, immediately after irradiation (Figure 4(C) and (D)).
Eight minutes after irradiation, all the cells in the observation
window appeared to undergo necrosis, supporting that cell death
is not caused via an apoptotic mechanism but rather necrosis.
As shown in Figure 4(A) and (B), control cells without the dye
exhibited no detectable cellular damage even 30 min after
irradiation, confirming nontoxicity of the light alone.
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Figure 4. Differential interference contrast, DIC images of LLC cells after
2
irradiation (0.2 mW/cm from a filtered Hg light source; 522 ± 40 nm) in the
absence (A, B) or presence (C, D) of 3. Scale bars are 100 µm.
Experimental Section
1
Full experimental details for synthesis of intermediate 2 and PS 3,
H
1
3
NMR and C NMR spectra of new compounds (2 and 3), photophysical
characterization, cell viability, and PDT experimental details are reported
in the Supporting Information. This material is available free of charge via
the Internet.
Acknowledgements
This work was supported by the National Science Foundation
(
CBET-1517273).
Keywords: BODIPY • photodynamic therapy • singlet oxygen
generation • photosensitizer
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Entry for the Table of Contents
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Bosung Kim, Binglin Sui, Xiling Yue,
Simon Tang, Michael G. Tichy, and
Kevin D. Belfield*
Page No. – Page No.
In Vitro Photodynamic Studies of a
Novel BODIPY-based Photosensitizer
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