Photostable BODIPY-based molecule with simultaneous type i and type II photosensitization for selective photodynamic cancer therapy

Article abstract of DOI:10.1039/c3tb21547d

We introduce a new class of photostable, efficient photosensitizers based on boron-dipyrromethene (BODIPY) derivatives that can generate singlet oxygen and superoxide simultaneously under irradiation. First, we report the synthesis and design of how to control the generation of a specifically substituted position of BODIPY. Second, after biologically evaluating the uptake, localization and phototoxicity in cell lines, we conclude that 2,6-di-anisole substituted BODIPY is a potentially selective photodynamic therapy candidate because its photodamage is more efficient in cancer cells than in normal cells without significant dark toxicity.

Full text of DOI:10.1039/c3tb21547d

Journal of
Materials Chemistry B
View Article Online
View Journal | View Issue
PAPER
Photostable BODIPY-based molecule with
simultaneous type I and type II photosensitization
Cite this: J. Mater. Chem. B, 2014, 2,
for selective photodynamic cancer therapy†
1576
*
Yen-Chih Lai and Cheng-Chung Chang
We introduce a new class of photostable, efficient photosensitizers based on boron-dipyrromethene
(BODIPY) derivatives that can generate singlet oxygen and superoxide simultaneously under irradiation.
First, we report the synthesis and design of how to control the generation of a specifically substituted
position of BODIPY. Second, after biologically evaluating the uptake, localization and phototoxicity in cell
lines, we conclude that 2,6-di-anisole substituted BODIPY is a potentially selective photodynamic
therapy candidate because its photodamage is more efficient in cancer cells than in normal cells without
significant dark toxicity.
Received 1st November 2013
Accepted 19th December 2013
DOI: 10.1039/c3tb21547d
www.rsc.org/MaterialsB
proteins,9 oligonucleotides,10 monoclonal antibodies,11
epidermal growth factors,12 carbohydrates,13 and hydrophilic
polymers.14 However, these high molecular weight branched-
peptide conjugates are difficult to synthesize, purify, and
characterize. Although enhanced cellular uptake and selec-
tivity for tumour tissues have been achieved with some
porphyrin sensitizers, most of these are usually found in
cytoplasmic membranes rather than in sensitive intracellular
sites, such as the mitochondria, the endoplasmic reticulum
(ER), and the nuclei.
BODIPY (boron-dipyrromethene) derivatives offer large
absorption extinction coefficients, sharp uorescence emis-
sions with high uorescence quantum yields, photostability
and low sensitivity to environmental variation.15 Thus, BODIPY
derivatives have attracted much research interest and have been
used for many applications.16 The new application of BODIPY
derivatives for photosensitizers was achieved by attaching a
functional group which can offer electrons to stabilize that
photoexcited BODIPY core and to quench the emission from the
singlet state, thereby promoting a triplet state lifetime.17
Specically, heavy atoms are incorporated into the structure as a
strategy to facilitate intersystem crossing and to promote the
singlet oxygen yield to become a new generation of photosen-
sitizers for photodynamic therapy.18,19 This appears to satisfy
the required criteria for a good photosensitizer (high singlet
oxygen yield, photostability and long wavelength absorption);
however, this approach disobeys the principle of dark-non-
toxicity for a photosensitizer to be a PDT candidate.20 More
importantly, based on the above considerations, it is rare to nd
BODIPY-based photosensitizers which present selective photo-
damage to cancer but not normal cells or tissues. Hence, it is
possible to design and produce, with appropriate chemical
modication, a new generation of BODIPY-based photosensi-
tizers that can overcome the issues of dark-toxicity and
1. Introduction
Photodynamic therapy (PDT) involves the uptake of a photo-
sensitizer by cancerous tissues or other sites of the therapeutic
target, followed by selective irradiation with visible or near IR
light of an appropriate wavelength that is absorbed by the
photosensitizer. There are three fundamental requirements for
PDT that are oxygen, a light source, and a photosensitizer. Each
factor is harmless by itself, but their combination can produce
cytotoxic agents.1–3 There are many reported or commercially
available photosensitizers. By comparison, cyclic tetrapyrroles
(porphyrins, chlorins, and bacteriochlorins) are difficult to
synthesize and modify; while phenothiazinium-based struc-
tures might be made more easily but present low light-to-dark
toxicity ratios.4–6 Most photosensitizers present application
limitations, such as a low photostability, structural instability,
or usable range of solvent conditions.7 Thus, it is important to
develop a new generation of photosensitizers with high effi-
ciency and photostability that are widely applicable in various
conditions.
Selective photodamage is the most important criterion for
a superior PDT reagent. The efficient delivery of a photo-
sensitizer to cancer cells and its subcellular distribution not
only depend on cellular processes but also on the physico-
chemical properties and structural characteristics of the
molecule itself.8 Therefore, several strategies have been
developed to improve the selective delivery of sensitizers to
tumour tissues, including their conjugation to carrier
Graduate Institute of Biomedical Engineering, National Chung Hsing University, 250
Kuo Kuang Road, Taichung, 402, Taiwan, ROC. E-mail: ccchang555@dragon.nchu.
edu.tw; Fax: +886-4-22852422
† Electronic supplementary information (ESI) available: Synthesis and
characterization, Fig. S1 and S2. See DOI: 10.1039/c3tb21547d
1576 | J. Mater. Chem. B, 2014, 2, 1576–1583
This journal is © The Royal Society of Chemistry 2014

View Article Online
Paper
Journal of Materials Chemistry B
nonselective photodamage without losing their unique optical
characteristics.
2.4 Light-induced cytotoxicity assay
Three different cell types were examined in the assay: the
human lung MRC-5 normal cells, lung A549 cancer cells and
human cervical HeLa cancer cells. Varying concentrations of
compounds were incubated with the cells in the dark for 12 h.
Subsequently, the culture medium was removed, and fresh
culture medium was added to each well. The plates were irra-
diated using a light source that is described in the next section
and then incubated for a further 24 h at 37 ꢁC. All assay
experiments were carried out in triplicate, and an average of the
three individual runs are presented. Methylene blue and Foscan
were used as comparative standard controls and were assayed
according to previously documented procedures.21
In this study, we report a novel photosensitizer that is highly
efficient, photostable, and more importantly, showed selective
PDT behaviour. This strategy can be easily achieved by attaching
an aryl group directly onto the pyrrole moiety of BODIPY, but
this is not more expensive than dimethyl pyrrole. By controlling
the bromination sequence, 3,5- or 2,6-diaryl substituted BOD-
IPY derivatives can be collected with good yield. Preliminary
biological studies on one of these dyes indicate that this type of
compound is highly membrane permeable, and it can selec-
tively localize within specic subcellular organelles, suggesting
promising PDT applications for these molecules.
2.5 Extraction and quantication of compounds in cells
2. Experimental
2.1 Materials
HeLa cells (2 ꢂ 106) were seeded into 60 mm ꢂ 15 mm cell
culture dishes. Before the cells reached ꢃ100% conuence, a
compound was added to one dish and incubated for 12 h. For
the dishes that were nearly 100% conuent, the conjugate
solution was added and incubated from 15 min to 4 h. Aer
incubation with the conjugates, all dishes were washed with
PBS. The cells were removed from the substrate with trypsin and
then pelleted. The cell suspension was sonicated for 1 h, and
1.8 mL of ethyl acetate was added to each tube to extract the
compound. Both absorbance and uorescence in the organic
phase were recorded.
The general chemicals employed in this study were the highest
grade available and were obtained from Acros Organic Co.,
Merck Ltd., or Aldrich Chemical Co. and used without further
purication. All of the solvents were of spectrometric grade.
2.2 Apparatus
The absorption spectra were generated using a Thermo Genesys
6 UV-visible spectrophotometer, and the uorescence spectra
were recorded using a HORIBA JOBIN-YVON Fluoromas-4
spectrouorometer with a 1 nm band-pass and a 1 cm cell
length at room temperature. The cellular uorescence images
and dual staining uorescence images were obtained using a
Leica AF6000 uorescence microscope with a DFC310 FX Digital
colour camera and a Leica TCS SP5 confocal uorescence
microscope. Fluorescence photographs were taken through the
related wavelength ranges using photomultiplier tubes (PMT).
The light source used to measure the singlet oxygen yield and
PDT cell death was a Xenon Light Source LAX-Cute (Asahi
Spectra).
2.6 Measurement
The light source for the measurement of singlet oxygen yield
and PDT cell death was a white light (a 20 W xenon lamp that
passes through a 400–700 nm mirror module). Before the light
was output from the optional light guide and collimator lens,
the light passed through a 510 nm long pass (lp) lter (the light
power was 6 mW cmꢀ2 on the dish surface). The singlet oxygen
quantum yields from compounds that were in an organic
solvent were determined by a photo-steady-state method using
1,3-diphenylisobenzofuran (DPBF) as the scavenger. Finally, the
following equation was utilized to calculate the singlet oxygen
quantum yields (FD):
2.3 Cell culture conditions and compound incubation
FD ¼ [(n/nMB) ꢂ (AMB/A)] ꢂ FD,MB
Human embryonal lung MRC-5 normal broblast cells and
HeLa human cervical cancer cells were maintained in Modied where n is the DPBF oxidation rate, A, the area of absorbance
Eagle's Medium (MEM) containing non-essential amino acids, maxima of the compound and MB means methylene blue. The
Earle's salts, L-glutamine, 1 mM sodium bicarbonate, 1 mM value of singlet oxygen quantum yield (FD) for MB is 0.52 in
sodium pyruvate, 1% (penicillin + streptomycin) and 10% fetal several media and measured with different techniques.22,23 The
bovine serum (FBS); the human lung adenocarcinoma cell line superoxide generation from compounds that were in an
A549 was maintained in RPMI-1640 medium containing 1% aqueous solution was determined by a photo-steady-state
(penicillin + streptomycin), sodium bicarbonate (2.0 g Lꢀ1) and method using 4-((9-acridinecarbonyl)amino)-2,2,6,6-tetramethyl-
10% FBS. The cells were cultured at 37 ꢁC in a humid atmo- piperidin-1-oxyl (TEMPO-9-ac, Invitrogen, Carlsbad, CA) as the
sphere with 95% air and 5% CO2. Before the observation of radical detector. Dual staining of tracker and compound: the cells
cellular localization, cells were seeded onto coverslips and were rst incubated with a compound for 12 h, which was fol-
incubated for 24 h. The next day, cells were incubated with lowed by incubation with 5 mM organelle probes (Mito Tracker
different concentrations of compounds for 12 h, for which the Green FM for 20 min at 37 ꢁC, ER Tracker™ Green for 30 min at
ꢁ
DMSO stock solutions were diluted in serum-free medium 37 C), and then washed with PBS before observation. The exci-
before use (1/100 v/v).
tation source was a green light cube, in which the light passed
This journal is © The Royal Society of Chemistry 2014
J. Mater. Chem. B, 2014, 2, 1576–1583 | 1577

View Article Online
Journal of Materials Chemistry B
Paper
through a 535 ꢄ 25 nm band pass (bp) lter and the emission was complexation of compound 1. Then, the 2,6-dibromo
collected through a 590 nm long pass (lp) lter (red emission of substituted BODIPY can be prepared by treating one equivalent
compound) and a blue light cube, in which light passed through of 8-(4-methoxyphenyl) boron-dipyrromethane with 2.2 equiva-
a 470 ꢄ 20 nm bp lter and the emission was collected through a lents of N-bromosuccinimide (NBS). Alternatively, when
510 nm lp lter to collect green emission of probes. Once the compound 1 was brominated with NBS rst, the main product
denite yellow color image was presented, we said that the was 3,5-dibromo substituted dipyrromethane, which is unstable
compound was co-localized with the probes. PDT-induced because it is easily protonated, especially in the column ush-
phototoxicity was determined using Hoechst 33342 (H-1399, ing purication process. That is why we prepared compound
Invitrogen, USA) and propidium iodide (PI, P3566, Invitrogen, 3,5-A in a sequence of steps in a one pot reaction directly from
USA) under a uorescence microscope and conrmed by a trypan compound 1 to 3,5-A. Notably, the use of 2.2 equivalents of NBS
blue exclusion and staining assay. Determination of quantum provided dibromo-BODIPY in good yield along with a minor
yields is relative to Rhodamine B.24
amount of mono-substituted bromine. Here we claim that even
with an excess amount of the brominating agent, no regioiso-
meric products, such as 3,5-dibromo-substituted product in
2,6-A or 2,6-dibromo-substituted product in 3,5-A, were detected
in the reactions. Additionally, in the last step, compounds 2,6-A
and 3,5-A were both coupled with compound 2 with similar
yield in the same reaction conditions. Recently, most literature
presented the molecular constructions of unitary 3,5- or
2,6-disubstituted BODIPY were based on dimethylpyrrole
coupling with the Wittig reaction. Here we want to emphasize
that we can collect the 3,5-disubstituted and 2,6-disubstituted
BODIPY products, by using pyrrole as the starting material
instead of more expensive dimethylpyrrole. This breakthrough
result can be widely applied to organic synthesis.
3. Results and discussion
3.1 Molecular preparation and basic spectroscopic
properties
BODIPY derivatives 2,6-DAB and 3,5-DAB were synthesized by
following different routes as outlined in Scheme 1. Bromination
is the key reaction of molecule preparation in this study. The
8-(4-methoxyphenyl) boron-dipyrromethane, an intermediate
precursor of compound 2,6-A, was obtained upon BF3$Et2O
In this investigation, the BODIPY moiety was selected as the
electron acceptor and anisole was chosen as the electron-
donating unit. The basic spectral properties in Fig. 1 show that
no signicant solvent polarity-dependent spectral shis in the
absorption and emission spectra were observed for the two
compounds 2,6-DAB and 3,5-DAB. However, when comparing
the spectral properties of these chromophores, 2,6-disubsti-
tuted product 2,6-DAB showed lower transition energy in all
investigated solvents with broader peak patterns in absorption
or emission spectra. Meanwhile, higher extinction coefficient
values but lower quantum yields were obtained. This nding
indicates that, in the ground states, these chromophores are
signicantly stabilized by solvation and the auxiliary electron-
donating ability of anisole units offered better effective
Scheme 1 Synthetic route to BODIPY-based photosensitizers 2,6-
DAB and 3,5-DAB: (i) pyrrole, TFA, rt. (ii) NBS, THF, ice bath. (iii) DDQ, Fig. 1 Absorption (left) and emission (right) spectra of compound
THF, rt. then Et3N–BF3$OEt2, toluene, reflux. (iv) K2CO3, (o-tol)3P, 2,6-DAB (left) and 3,5-DAB (right) in different solvents; characterized
Pd(OAc)2, DME/H2O ¼ 5/1, compound 2, reflux. (v) Pd(PPh3)2Cl2, by extinction coefficient and quantum yield, respectively. Excited
pinacolborane, dioxane/Et3N, reflux.
wavelengths are depicted as absorption maxima.
1578 | J. Mater. Chem. B, 2014, 2, 1576–1583
This journal is © The Royal Society of Chemistry 2014

View Article Online
Paper
Journal of Materials Chemistry B
conjugation to the BODIPY core. However, 3,5-disubstituted
product compound 3,5-DAB exhibited more stable absorption
and luminescence properties in most organic solvents, espe-
cially with quantum yields ranging from 0.4 to 0.6. Based on our
knowledge, uorophores that can be successfully applied were
based on a single type of uorophore structure, common to all
integrated sensor molecules. The uorescent optical property of
3,5-DAB makes this proposal possible once we design and
synthesize derivatives with suitable spacers or functional
groups, so that it becomes 3,5-dialkoxyphenyl substituted
BODIPY, which will be applied in optical sensors, non-doped
OLEDS, or laser dyes. Furthermore, we expect that this type of
uorophore will be useful as a cellular probe because of its
unique optical property.
3.2 Singlet oxygen generation and photostability
The singlet-oxygen production study was undertaken by moni-
toring the reaction of the known singlet oxygen acceptor 1,3-
diphenylisobenzofuran (DPBF) with photosensitizer-generated
singlet oxygen.25 This production was achieved experimentally
by following the disappearance of the 410 nm absorbance band
of DPBF at an initial concentration of 20 mM (2 fold the
concentration of the compound). The rates of DPBF oxidation
by these two compounds in DMSO solvent were investigated by
exciting the absorptions of the compounds (Fig. 2). When
placed under the white light source (400–700 nm, no lter) or
visible light source (400–700 nm with a 510 nm long pass lter)
for the irradiation period, compound 2,6-DAB presented a
slightly better DPBF oxidation rate than compound 3,5-DAB.
Based on the result of Fig. 2, the estimated singlet oxygen yields
were about equal to the level of methylene blue (MB). Here, as
described in the Experimental, the singlet oxygen quantum
yields (FD) of compound 2,6-DAB and 3,5-DAB were obtained as
0.48 and 0.53 by the relative method using methylene blue as
the reference (FD ¼ 0.52). However, the singlet oxygen genera-
tions apparently increase when the system was irradiated under
a white light source. This nding indicates that the S0–S2
transition absorption bands of the compound at approximately
400 nm contribute some singlet oxygen yield. Additionally, the
inset in Fig. 2b shows that under this irradiation condition, the
photostabilities of compounds are better than MB. It is known
that good photostability upon repetitive excitation is highly
desirable in PDT application because photobleaching of
photosensitizers usually produces photodegradation side
products that may lower the efficiency of reactive oxygen species
(ROS) generation. Moreover, when a photosensitizer is used as a
tumour-visualizing tool in PDT, high photostability is required
to allow monitoring for a sufficiently long period.26
Fig. 2 Absorption spectra variations of 20 mM DPBF consumption in
the presence of 10 mM (a) 2,6-DAB and (b) 3,5-DAB in DMSO after
irradiation with an average 6 mW cmꢀ2 for the 510 nm lp through the
filter. Relative DPBF oxidation rates by singlet oxygen, which were
generated from the compounds, are plotted in (c); the data without the
510 nm lp filter are also shown. Each data point represents the average
from three separate experiments. (Inset: (a) control DPBF without
adding a compound, (b) identical experimental condition but with
methylene blue (MB).)
system measured uorescence using a 535 ꢄ 25 nm band pass
lter as an exciting light source and collected through a red
lter (590 nm long pass). It is clear that plasma membrane bleb
formation and acute cell death were caused by illumination
with green light for cells that were incubated with a compound.
Here, irradiated time dependent uorescent images show
nearly constant emission intensities, which further conrms
the photostability behaviour in Fig. 2; these two compounds
both cause apparent photodamage to cancer cells, but only
compound 2,6-DAB presents little or no collateral damage to
normal cells. Here, the important message is that in our studied
system, 2,6-disubstituted BODIPY (2,6-DAB) possibly shows
selective PDT to cancer cells rather than 3,5-disubstituted
BODIPY (3,5-DAB), even in unclear uorescent images because
3.3 Real-time photodamage and photostability in cells
Once these two compounds presented the characteristic
behaviours of singlet oxygen generation and photostability, we
examined whether the compounds can be used as a tool for cell
photosensitization. Fig. 3 illustrates the photodamage process
from a real-time video recorded under a uorescence micro-
scope with a colour CCD. This distinction was revealed by the
This journal is © The Royal Society of Chemistry 2014
J. Mater. Chem. B, 2014, 2, 1576–1583 | 1579

View Article Online
Journal of Materials Chemistry B
Paper
kinetics of compound uptake was determined by the extrac-
tion of the compound from cells.27 By monitoring the absor-
bance and uorescence intensities of compounds in the ethyl
acetate layer aer extracting from the cells, as in the Experi-
mental section description, the increase in the compound
concentration in the cells was quantied (Fig. 4). It is clearly
conrmed that the cellular uptake of 3,5-DAB appears no
different between cancer and normal cells, whereas 2,6-DAB
accumulated in the cancer cells more than in the normal cells.
This notable observation supports us to propose that the
uptake difference between cancer and normal cells is the
dominant factor for selective photodamage.
3.5 Sub-cellular localization
It is known that the intracellular localization of photosensi-
tizers strongly affects the mechanism of cell death. The bio-
logical efficacy for most photodynamic therapy (PDT)
sensitizers of tumors depends on their efficient translocation
across cellular membranes and their delivery into specic
organelles within cancer cells.28 To determine the localization of
each compound, as detailed in the Experimental section, the
cells incubated with compound 2,6-DAB were double stained
with cellular tracker green, which merged with the red light
emission region of the compound. Fig. 5 reveals that 2,6-DAB is
Fig. 3 Several selected fluorescent and bright-field photodamage
images from a real-time video of (a and c) HeLa cancer cells, and (b
and d) MRC-5 normal cells incubated with 10 mM 2,6-DAB (a and b) or
3,5-DAB (c and d) for 12 h after 535 ꢄ 25 nm irradiation from a mercury
lamp for fluorescent microscopy.
of low quantum yield. This result suggests that 2,6-DAB is a
potentially useful reagent for cell photosensitization, studies on
oxidative stress, or PDT.
3.4 Intracellular accumulation
Following the experimental conditions of Fig. 3, we tried to
determine the quantitative accumulation of compounds in the
cells using red (590 nm long pass) lters, as in Fig. S1.† The
compound 3,5-DAB presented clearer concentration-depen-
dent intracellular uorescent signals because of the com-
pound's higher emission quantum yield, and these
intracellular accumulation images appear no different
between cancer cells and normal cells. The intracellular
accumulation of compound 2,6-DAB is difficult to observe
Fig. 4 Intracellular accumulations of 2,6-DAB (a) and 3,5-DAB (b) in
normal (MRC-5) and cancer (HeLa) cells. The cells were incubated with
variable concentrations of compounds for 12 h, and the intracellular
uptake was determined from a fluorescence calibration curve of the
ethyl acetate layer after extracting from the cells. Each data point
because of its low uorescent quantum yield. Thus, the represents the average from three separate experiments.
1580 | J. Mater. Chem. B, 2014, 2, 1576–1583
This journal is © The Royal Society of Chemistry 2014

View Article Online
Paper
Journal of Materials Chemistry B
Fig. 5 Sub-cellular localization of 2,6-DAB in HeLa cancer cells: the
images of tracker green were excited by a blue light cube that passed
light through a 470 ꢄ 20 nm bp filter, and emission was collected
through a 510 nm lp filter. The red images of the compound were
excited by a green light cube that passed light through a 535 ꢄ 25 nm
bp filter, and emission was collected through a 590 nm lp filter.
Fig. 6 Phototoxicity of cells treated with variable concentrations of
2,6-DAB for 12 h before irradiation and irradiated with a 510 nm lp light
source (average 8 J cmꢀ2); cell death was tested overnight. Each data
point represents the average from three separate experiments and the
irradiation condition details are described in the Experimental section.
localized predominantly in the endoplasmic reticulum (ER) and
less in the mitochondria of cancer cells.
Generally, mitochondrial photodamage has been reported to
be the primary cause of apoptosis during PDT because the
mitochondria play an integral role in various cell biological
processes, such as energy production, apoptotic cell death,
molecular metabolism, calcium signaling, and cell redox
status.29–31 However, ER is also a very attractive site for the
localization of the photosensitizers to improve the efficiency of
PDT. It is known that oxidation of ER proteins due to PDT may
cause changes in ER Ca2+ homeostasis and/or aggregation of
unfolded and misfolded proteins.32 For example, the ER-local-
izing photosensitizer Foscan was recently shown to cause Bcl-2
photodamage, possibly specically affecting the ER pool of Bcl-
2 in cancer cells.33,34 Based on the literature, PDT with photo-
sensitizers that target mitochondria and ER will very possibly
cause photodamage to Bcl-2 and Bcl-Xl,35 and this type of pho-
todamage to these antiapoptotic proteins is observed immedi-
ately upon light exposure,36 as in Fig. 3. It is doubtless that the
ability of PDT to affect Bcl-2 function plays a crucial role in
tumour treatment.
blue emission enhancement in the nucleus. This result is
because of the interaction between Hoechst and condensed
DNA. This occurred when the red uorescence from PI became
dominant in the nucleus when the irradiated cell line was
cultured overnight. It is likely that the nuclear membrane is
damaged during a later period aer irradiation. This cell death
pathway is likely because of an anti-apoptosis protein of mito-
chondria or ER, as discussed above. However, we provided an
additional PDT reagent as another choice, and this is the rst
report for a BODIPY derivative to show selective PDT to cancer
cells but not normal cells.
3.7 New reactive oxygen species generation
It is notable that the PDT effects of 2,6-DAB are better than we
expected. Hence, we examined the possibility for this com-
pound's PDT progress by other possible mechanisms, such as
type I PDT. The type I mechanism involves hydrogen-atom
abstraction or electron-transfer between the excited sensitizer
and a substrate, yielding free radicals. These radicals can
react with oxygen to form an active oxygen species, such as the
3.6 Phototoxicity
The combination of compound 2,6-DAB and light illumination superoxide radical anion.39,40 Most studies generally infer
resulted in quantitative cellular toxicity. Fig. 6 reveals that three that singlet oxygen formation of type II PDT is primarily
different cell lines displayed no determinable dark toxicity with responsible for the biological PDT effect; however, several
2,6-DAB up to a concentration of 15 mM. By contrast, irradiation recent studies indicate that radical species from the type I
with 8 J cmꢀ2 of 510 nm long pass light dose showed a signif- mechanism may lead to an amplied PDT response, particularly
icant light-induced toxicity with EC50 values determined for under low oxygen conditions.41,42 It is reported that these two
HeLa, A549, and MRC-5 as 5.7, 8.2 and 18.5 mM, respectively. A competing mechanisms can occur simultaneously.28,43 The free
more apparent phototoxicity result was observed when the radical probe 4-((9-acridinecarbonyl)amino)-2,2,6,6-tetramethyl-
system was irradiated under white light (400–700 nm) as the piperidin-1-oxyl (TEMPO-9-ac, Invitrogen, Carlsbad, CA) captures
PDT light source. On the other hand, an interesting result was radicals, resulting in uorescence turn-on (lex/em
¼
358/
observed when the cell death was determined with double 440 nm).44 Irradiation of 2,6-DAB also generated a more apparent
staining Hoechst 33342 as well as PI.37,38 Fig. S2† shows that in increase in signal from TEMPO-9-ac compared to unchanged
the early stage of cell death (two probes stained once immedi- levels of the control (Fig. 7). This nding indicates that
ately aer illuminating), uorescent images result in apparent compound 2,6-DAB may undergo type
I and II PDT
This journal is © The Royal Society of Chemistry 2014
J. Mater. Chem. B, 2014, 2, 1576–1583 | 1581

View Article Online
Journal of Materials Chemistry B
Paper
3 M. Ochsner, J. Photochem. Photobiol., B, 1997, 39, 1–18.
4 E. S. Nyman and P. H. Hynninen, J. Photochem. Photobiol., B,
2004, 73, 1–28.
5 M. Wainwright, Photodiagn. Photodyn. Ther., 2005, 2, 263–272.
6 M. Wainwright, D. A. Phoenix, L. Rice, S. M. Burrow and
J. Waring, J. Photochem. Photobiol., B, 1997, 40, 233–239.
7 E. A. Lissi, M. V. Encinas, E. Lemp and M. A. Rubio, Chem.
Rev., 1993, 93, 699–723.
8 D. Kessel, J. Porphyrins Phthalocyanines, 2004, 8, 1009–1014.
9 M. R. Hamblin and E. L. Newman, J. Photochem. Photobiol., B,
1994, 26, 147–157.
10 B. Mestre, M. Pitie, C. Loup, C. Claparols, G. Pratviel and
B. Meunier, Nucleic Acids Res., 1997, 25, 1022–1027.
11 R. Hudson, M. Carcenac, K. Smith, L. Madden, O. J. Clarke,
A. Pelegrin, J. Greenman and R. W. Boyle, Br. J. Cancer, 2005,
92, 1442–1449.
12 A. Gijsens, L. Missiaen, W. Merlevede and P. de Witte, Cancer
Res., 2000, 60, 2197–2202.
13 G. Li, S. K. Pandey, A. Graham, M. P. Dobhal, R. Mehta,
Y. Chen, A. Gryshuk, K. Rittenhouse-Olson, A. Oseroff and
R. K. Pandey, J. Org. Chem., 2004, 69, 158–172.
14 M. Tijerina, P. Kopeckova and J. Kopecek, Pharm. Res., 2003,
20, 728–737.
15 A. Loudet and K. Burgess, Chem. Rev., 2007, 107, 4891–4932.
16 H. Kabayashi, M. Ogawa, R. Alford, P. L. Choylle and
Y. Urano, Chem. Rev., 2010, 110, 2620–2640.
Fig. 7 (a) Absorption spectra of TEMPO-9ac upon irradiation with
compound 2,6-DAB. Experimental conditions follow Fig. 2. Emission
(lex ¼ 360 nm) spectra of TEMPO-9ac upon irradiation without
compound (b) and with compound 2,6-DAB (c).
17 A. Kamkaew, S. H. Lim, H. B. Lee, L. V. Kiew, L. Y. Chung and
K. Burgess, Chem. Soc. Rev., 2013, 42, 77–88.
18 N. Adarsh, R. R. Avirah and D. Ramaiah, Org. Lett., 2010, 12,
5720–5723.
19 T. Yogo, Y. Urano, Y. Ishitsuka, F. Maniwa and T. Nagano, J.
Am. Chem. Soc., 2005, 127, 12162–12163.
simultaneously when it stays in the cells. We proposed that is why
the PDT effects of 2,6-DAB are better than we expected.
4. Conclusions
20 S. H. Lim, C. Thivierge, P. Nowak-Sliwinska, J. Han, H. van
den Bergh, G. Wagnieres, K. Burgess and H. B. Lee, J. Med.
Chem., 2010, 53, 2865–2874.
BODIPY offers many superior optical properties. In this study,
we have successfully applied BODIPY derivatives for cancer
targeting and demonstrated that the 2,6-di-anisole substituted
product 2,6-DAB may be a potent tumor-specic photosensitizer
for PDT. First, we presented a cheap and convenient synthesis
procedure that can be widely applied to prepare diversied
BODIPY derivatives. Second, ROS generation as well as biolog-
ical evolution of cellular uptake, localization and phototoxicity
was determined. Therefore, the PDT characteristics of the
2,6-DAB compound include (1) target selectivity for the PDT of
cancer cells; (2) higher photostability than methylene blue; (3)
supported extra phototoxicity with superoxide generation.
`
21 M. H. Teiten, L. Bezdetnaya, P. Morliere, R. Santus and
F. Guillemin, Br. J. Cancer, 2003, 88, 146–152.
22 F. Wilkinson, W. P. Helman and A. B. Ross, J. Phys. Chem.
Ref. Data, 1993, 22, 113–262.
23 R. W. Redmond and J. N. Gamlin, Photochem. Photobiol.,
1999, 70, 391–475.
24 R. A. Velapoldi and H. H. Tonnesen, J. Fluoresc., 2004, 14,
465–471.
25 K. Gollnick and A. Griesbeck, Tetrahedron, 1985, 41, 2057–
2068.
26 J. Moan, Cancer Lett., 1986, 33, 45–53.
27 Y. Cheng, A. C. Samia, J. Li, M. E. Kenney, A. Resnick and
C. Burda, Langmuir, 2010, 26, 2248–2255.
28 T. J. Dougherty, C. J. Gomer, B. W. Henderson, G. Jori,
D. Kessel, M. Korbelik, J. Moan and Q. Peng, J. Natl.
Cancer Inst., 1998, 90, 889–905.
Acknowledgements
This work was supported nancially by the National Science
Council (NSC 101-2113-M-005-016-MY3) of Taiwan.
29 A. P. Castano, T. N. Demidova and M. R. Hamblin,
Photodiagn. Photodyn. Ther., 2005, 2, 1–23.
30 N. Dias and C. Bailly, Biochem. Pharmacol., 2005, 70, 1–12.
Notes and references
1 J. C. Kennedy, R. H. Pottier and D. C. Pross, J. Photochem. 31 M. E. Rodriguez, K. Azizuddin, P. Zhang, S. M. Chiu, M. Lam,
Photobiol., B, 1990, 6, 143–148.
2 J. D. Spikes, Ann. N. Y. Acad. Sci., 1975, 244, 496–508.
M. E. Kenney, C. Burda and N. L. Oleinick, Mitochondrion,
2008, 8, 237–246.
1582 | J. Mater. Chem. B, 2014, 2, 1576–1583
This journal is © The Royal Society of Chemistry 2014

View Article Online
Paper
Journal of Materials Chemistry B
32 R. Michael and P. M. Hamblin, Advances in Photodynamic
Therapy: Basic, Translational, and Clinical, Artech House,
Incorporated, 2008, 28.36.37.55.71-74.
33 H. R. Kim, Y. Luo, G. Li and D. Kessel, Cancer Res., 1999, 59,
3429–3432.
34 L. Y. Xue, S. M. Chiu and N. L. Oleinick, Oncogene, 2001, 20,
3420–3427.
35 D. Kessel and A. S. Arroyo, Photochem. Photobiol. Sci., 2007, 6,
1290–1295.
36 F. Belloc, P. Dumain, M. R. Boisseau, C. Jalloustre, J. Reiffers,
P. Bernard and F. Lacombe, Cytometry, 1994, 17, 59–65.
37 M. C. DeRosa and J. Robert, Coord. Chem. Rev., 2002, 233,
351–357.
38 A. P. Castano, P. Mroz and M. R. Hamblin, Nat. Rev. Cancer,
2006, 6, 535–545.
M. M. Pereira and L. G. Arnaut, Chem.–Eur. J., 2010, 16,
9273–9286.
40 Y. Vakrat-Haglili, L. Weiner, V. Brumfeld, A. Brandis,
Y. Salomon, B. McIl-roy, B. C. Wilson, A. Pawlak,
M. Rozanowska, T. Sarna and A. Scherz, J. Am. Chem. Soc.,
2005, 127, 6487–6497.
41 M. E. Bulina, D. M. Chudakov, O. V. Britanova,
Y. G. Yanushevich, D. B. Staroverov, T. V. Chepurnykh,
E. M. Merzlyak, M. A. Shkrob, S. Lukyanov and
K. A. Lukyanov, Nat. Biotechnol., 2006, 24, 95–99.
42 A. Roy, P. Carpentier, D. Bourgeois and M. Field, Photochem.
Photobiol. Sci., 2010, 9, 1342–1350.
43 H. Ding, H. Yu, Y. Dong, R. Tian, G. Huang, D. A. Boothman,
B. D. Sumer, J. Gao, B. D. Sumerb and J. Gao, J. Controlled
Release, 2011, 156, 276–280.
39 E. F. F. Silva, C. Serpa, J. M. Dabrowski, C. J. P. Monteiro, 44 M. E. Milanesio, M. G. Alvarez, V. Rivarola, J. J. Silber and
S. J. Formosinho, G. Stochel, K. Urbanska, S. Simoes,
E. N. Durantini, Photochem. Photobiol., 2005, 81, 891–897.
This journal is © The Royal Society of Chemistry 2014
J. Mater. Chem. B, 2014, 2, 1576–1583 | 1583