Design of novel photosensitizers and controlled singlet oxygen generation for photodynamic therapy

Article abstract of DOI:10.1039/d1nj02656a

Photodynamic therapy (PDT) is a promising strategy in cancer treatment with its relatively lower side effect profile. Undoubtedly, the key component of PDT is the photosensitizers with a high ability to produce singlet oxygen. In this work, iodinated or brominated boron dipyrromethene-bearing trimers (5and6) were prepared as photosensitizers and their molecular structures were characterized by mass spectrometry and NMR (¹H and¹³C) spectroscopy. Their photophysical and photochemical abilities were also evaluated in detail. The molar absorption coefficient (ε) of compound5was found to be 2.5 times higher than that of compound6. The photosensitizer (5), bearing iodinated BODIPY units, exhibited almost a non-fluorescent profile with a fluorescence lifetime of 0.12 ns, whereas the other photosensitizer (6) exhibited a more moderate fluorescence character with a fluorescence lifetime of 1.22 ns. The singlet oxygen quantum yields of the photosensitizers were determined to be 0.88 and 0.76, respectively, by the chemical trapping method. Moreover, the biological assessment of the novel compounds showed that these PSs were internalized into the cells and triggered cell death in a light source-dependent manner, underlying the success of the compounds in PDTin vitro.

Full text of DOI:10.1039/d1nj02656a

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Design of Novel Photosensitizers and Controlled Singlet Oxygen
Generation for Photodynamic Therapy
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Esra $#%"&'(")* Eçik*a, Onur Bulutb,c, Hasan Huseyin Kazand, Elif ;(%,0730a and Bünyemin
>?@03e
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aDepartment of Chemistry, Atatürk University, Erzurum, Turkey
bDepartment of Bioengineering, Konya Food and Agriculture University, Konya, Turkey
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cResearch and Development Center for Diagnostic Kits (KIT-ARGEM), Konya Food and Agriculture University, Konya,
Turkey
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dDepartment of Biological Sciences, Middle East Technical University, Ankara, Turkey
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eDepartment of Chemistry, Gebze Technical University, Kocaeli, Turkey
E-mail: esra.ecik@atauni.edu.tr;
Tel: +90 442 231 4496
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KEYWORDS: Triazine, Photosensitizer, Singlet Oxygen, In vitro Photodynamic Therapy
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ABSTRACT
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Photodynamic therapy (PDT) is a promising strategy in cancer treatment with its relatively
lower side effect profile. Undoubtedly, the key component of PDT is the photosensitizers with
a high ability to produce singlet oxygen. In this work, iodinated or brominated boron-
dipyrromethene-bearing trimers (5 and 6) were prepared as photosensitizers and their molecular
structures were characterized by mass spectrometry and NMR (1H and 13C) spectroscopy. Their
photophysical and photochemical abilities were also evaluated in detail. The molar absorption
coefficient I_N of compound 5 was found to be 2.5 times higher than that of compound 6. The
photosensitizer (5), bearing iodinated-BODIPY units, exhibited almost non-fluorescent profile
with 0.12 ns lifetime whereas the other photosensitizer (6) exhibited more moderate
fluorescence character with 1.22 ns fluorescence lifetime. The singlet oxygen quantum yields
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of photosensitizers were determined to be 0.88 and 0.76, respectively by chemical trapping
method. Moreover, the biological assessment of the novel compounds showed that these PSs
internalized into the cells and triggered cell death in a light source-dependent manner,
underlying the success of the compounds in PDT in vitro.
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1. Introduction
Cancer is a global health problem and one of the most fatal diseases. Although conventional
methods such as surgical intervention, chemotherapy and radiation therapy are frequently
preferred in cancer treatment, low success rates and negative effects of these methods on the
quality of the patient life are widely reported disadvantages.1 Photodynamic therapy (PDT),
which has a lower side effect than the traditional methods, has emerged as a promising
technique in cancer treatment.2-5
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The success of PDT depends on the ability of the photosensitizer (PS), which is ineffective in
the dark, to destroy the cancerous cells through controlled production of singlet oxygen (1O2)
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under light.
Photoactivatable PSs are generally designed from heteroatom-containing
macrocycles, modified organic dyes, organic/inorganic nanostructures or metal-organic
frameworks.11-16
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Even though many PSs have been developed, the disadvantages such as low singlet oxygen
production yield, high dark toxicity and low photostability are frequently reported to be
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overcome.
To improve photosensitizing ability of PS, one way is to increase its
concentration, usually resulting in the aggregation effect.17 Another way is to enhance the
photosensitizing abilities of PS molecule by the suitable structural modification.10-12,15
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Boron dipyrromethene (BODIPY) unit is considered as a remarkable candidate for PDT
applications since it can easily be modified and structure-activity relationship can be
improved.6,11, 12, 15, 18 The heavy atoms such as iodine or bromine covalently linked to the
BODIPY unit strongly promote the intersystem crossing (ISC) and increase the production of
singlet oxygen.19 Some researchers have focused on multiple BODIPY derivatives, such as
orthogonal dimers and oligomers to improve the photosensitizing ability.20-22 Cyanuric chloride
(triazine), possessing three chloro atoms, is an ideal structure to construct complexes baring
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2. Experimental section
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2.1. General methods
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All chemicals and solvents were procured from commercial suppliers. Analytical thin-layer
chromatography (TLC) was performed on silica gel plates (Merck, Kieselgel 60 Å, 0.25 mm
thickness) with the F254 indicator. Column chromatography was performed on silica gel
(Merck, Kieselgel 60 Å, 230-400 mesh). Mass spectra were recorded on a Bruker Daltonics
Microflex mass spectrometer using dithranol as the MALDI matrix. All NMR spectra (1H and
13C NMR) were recorded on a Varian INOVA 500 MHz spectrometer. Electronic absorption
spectra were analyzed with a Shimadzu 2101 UV spectrophotometer. Fluorescence excitation
and emission spectra were obtained on a Varian Eclipse spectrofluorometer using 1.0 cm
pathlength cuvettes at room temperature. The fluorescence lifetimes were recorded using
Horiba- Jobin-Yvon-SPEX Fluorolog 3-2iHR instrument with Fluoro Hub-B Single Photon
Counting Controller at an excitation wavelength of 390 nm for compounds. Signal acquisition
was performed using a TCSPC module.
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The meso-azido-substituted BODIPY derivative (1), di-iodinated BODIPY monomer (2), di-
brominated BODIPY monomer (3), and tris-alkyne-carrying triazine core (5) were synthesized
and purified as in the literature.22, 27, 28
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2.2.1. Synthesis of compound 5
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Compound 2 (100.0 mg, 0.145 mmol) and compound 4 (11.36 mg, 0.046 mmol) were dissolved
in the solvent mixture (4.0 mL dichloromethane: 1.0 mL methanol: 1.0 mL water). Sodium
ascorbate (7.5 mg; 0.03 mmol), CuSO4.5H2O (6.0 mg, 0.03 mmol) and one drop of
triethylamine were added to this reaction medium and the reaction mixture was stirred at room
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temperature for two days. The crude product was extracted with CH2Cl2: H2O (50.0 mL : 50.0
mL) and the organic phase was dried over Na2SO4. The solvent was removed on the rotary
evaporator. The reaction mixture was subjected to column chromatography on silica gel using
MeOH-CH2Cl2 (4: 96) as the mobile phase. The pure product (5, 36.0 mg, 33 %) was obtained
as red solid. MALDI TOF (m/z) Calc. 2310.51, Found: 2309.97 [M]+, 2288.91 [M+H-F]+ (Fig
S1). 1H NMR (500 MHz, CDCl3) pH 7.8 (s, 3H), 7.1 (d, J = 8.1 Hz, 6H), 7.0 (d, J = 8.1 Hz, 6H),
5.6 (s, 6H), 4.5 (t, J = 6.4 Hz, 6H), 4.1 (m, 6H), 2.6 (s, 18H), 2.2-2.1 (m, 6H), 1.9-1.8 (m, 6H),
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1.4 (s, 18H) ppm (Fig. S2). C NMR (126 MHz, CDCl3) pC 172.78, 159.66, 156.58, 145.29,
142.25, 141.42, 131.68, 129.16, 126.87, 123.84, 115.28, 85.59, 67.07, 61.51, 50.11, 27.26,
26.21, 17.23, 16.02 ppm (Fig. S3).
2.2.2. Synthesis of compound 6
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Compound 3 (100.0 mg, 0.17 mmol) and compound 4 (13.2 mg, 0.054 mmol) were dissolved
in the solvent mixture (4.0 mL dichloromethane: 1.0 mL methanol: 1.0 mL water). Sodium
ascorbate (7.5 mg; 0.03 mmol), CuSO4.5H2O (6.0 mg, 0.03 mmol) and one drop of
triethylamine were added to this reaction medium and the reaction mixture was stirred at room
temperature for two days. The crude product was extracted with CH2Cl2: H2O (50.0 mL : 50.0
mL) and the organic phase was dried over Na2SO4. The solvent was removed on the rotary
evaporator. The reaction mixture was subjected to column chromatography on silica gel using
MeOH-CH2Cl2 (4: 96) as the mobile phase. The pure product (6, 41.0 mg, 37 %) was obtained
as red solid. MALDI TOF (m/z) Calc. 2028.50; found: 2028.76 [M]+, 2007.82 [M+H-F]+ (Fig.
S4). 1H NMR (500 MHz, CDCl3) pH 7.8 (s, 3H), 7.2 (d, J = 8.3 Hz, 6H), 7.0 (d, J = 8.3 Hz, 6H),
5.6 (s, 6H), 4.5 (t, J = 6.9 Hz, 6H), 4.1 (t, J = 5.7 Hz, 6H), 2.6 (s, 18H), 2.2-2.1 (m, 6H), 1.9-
1.8 (m, 6H), 1.4 (s, 18H) ppm (Fig. S5). 13C NMR (126 MHz, CDCl3) pC 172.78, 159.66,
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153.76, 142.14, 140.56, 130.76, 129.17, 126.53, 123.81, 115.24, 111.66, 67.05, 61.52, 50.11,
27.27, 26.21, 13.95, 13.80 ppm (Fig. S6).
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2.3. Photophysical parameters
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The fluorescence quantum yield IrF) was calculated in ethanol using the Rhodamine 6G IrF =
0.90 in water) as a reference molecule.29
F. AStd. n2
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r (Std)
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(1)
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In the above equation, F is the area under the fluorescence emission curve of sample. A is the
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respective absorbance of the sample.
is the refractive index of solvent.
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2.4. Singlet Oxygen Sensitization
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The singlet oxygen generation capacities Itu) of the trimers were evaluated by the comparative
method. Rose Bengal Itu=0.68 in ethanol)30 was employed as a reference compound and 1,3-
diphenylisobenzofuran (DPBF) was used as a singlet oxygen scavenger. The trimers and Rose
Bengal were irradiated with the light source Iv = 516 nm, 2.1 mWcm-2) from a 6.0 cm distance.
The following equation (2) was used to calculate the singlet oxygen quantum yield.15
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tu (samp) = tu (ref) × [m (samp)/m(ref)]×[F(ref)/F(samp)]
(2)
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In the above equation, m is the slope of difference in change in absorbance of DPBF (414 nm)
with the irradiation time and F is the absorption correction factor, which is given by F = 1 – 10-
OD (OD at the irradiation wavelength).
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As the second method, singlet oxygen formation Iru) were determined via the phosphorescence
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of O2, using Horiba Jobin-Yvon Fluorimeter with Hamamatsu NIR PMT 5509.31 The ability
of singlet oxygen formation was obtained via according to the equation,
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! (&*+&) ,#$%&'
! = !($%&)( )
(3)
)! ($%&) ,#&*+&'
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where, ru (std) is the singlet oxygen quantum yield of the Rose Bengal Iu is the area under the
curve of peak of singlet oxygen at 1270 nm; and A is the absorbance which was set to 0.391,
0.362 and 0.366 for 5, 6 and RB, respectively.
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2.5. In vitro photodynamic therapy
Photodynamic therapy efficacy of the compounds were in vitro evaluated using a human
cervical cancer cell line (HeLa). Briefly, 1!104 cells/well were seeded into the wells of 96-well
plate and incubated for 24 h in RPMI 1640 medium (Biochrom AG, Germany) containing 10
% PBS (v/v; Biochrom AG, Germany) and 1 % gentamycin (v/v; Biological Industries, Israel)
at 37°C and 5 % CO2. Next, cells were washed with phosphate-buffered saline (PBS) twice and
the medium was renewed. Cells were treated with compound 5 and 6 dissolved in dimethyl
sulfoxide (DMSO) to minimize the solvent toxicity at a concentration of 0.0125-0.1 mg/mL as
less than 1 % DMSO volume. The compounds 2-4 (each 0.1 mg/mL) which were also dissolved
in DMSO were used to evaluate the PDT efficacy of the precursors and all compounds and
DMSO were used as cell-free medium control to eliminate the background signal. These
concentration range was the optimal one to see the stepwise cell death under these conditions.
Cells were incubated at 37°C and 5 % CO2 whether in dark or under green light source ("= 516
nm, 2.1 mWcm-2) for 5 h. Next, 10.0 {f of the 3-(4,5-dimethylthiazol-2-yl)-2,5
diphenyltetrazolium bromide (MTT) solution (5.0 mg mL|Y; dissolved in PBS) was added onto
cells and incubated for 4 h. Cells were disrupted with an SDS–HCl solution overnight and the
microplates were read using a microplate spectrophotometer (Synergy H1, BioTek Instruments,
USA) at 570 nm. Optical densities were converted to % viability by considering untreated cells
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to have 100 % viability and all the treatments were correlated with this group. All biological
experiments were performed as three replica each of which including four technical replica.
Data were analyzed by two-way ANOVA by GraphPad Prism 8.0 software (GraphPadInc,
USA). The results were considered significant at the level of 0.05.
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2.6. Live cell imaging
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To further characterize the novel compounds and their precursors biologically, we evaluated
the cellular imaging properties and internalization patterns of the compounds. 1!105 cells/well
were seeded into the wells of 12-well plate and the cells were incubated overnight. Next, the
cells were washed by PBS twice and incubated with 0.1 mg/mL of the compounds 2-6 for 5
min at room temperature in PBS. Then, the cells were washed by PBS twice and treated with
Trypan Blue (0.4 %) for 5 min at room temperature to block the extracellular fluorescent signal,
and the cells were washed by PBS twice. Images were obtained by Zeiss Axio Observer (Carl
Zeiss Microscopy GmbH, Germany) using the default settings and filters.
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3. Results and Discussion
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3.1. Synthesis and structural characterization of triazine-based BODIPY trimers
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Molecular scaffolds that allow fine tuning for desired pharmacological properties are extremely
important in drug or biomolecule discovery. The triazine, a heterocyclic ring, has frequently
been preferred as molecular platforms in novel biomolecule designs due to its predictable
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pharmacological properties, biocompatibility and chemically stable structure.
In this
study, we focused on preparing novel triazine-based BODIPY trimers and determining their
PDT activities, inspired by excellent pharmacological properties of triazine core and
photochemical character of BODIPY. Synthesis pathway for the triazine-based BODIPY
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trimers (5 and 6) are depicted in Scheme 1. The meso-azido-substituted BODIPY (1) was
synthesized from the two-step reaction of 2,4-dimethyl pyrrole with 4-azidobenzaldehyde in
dichloromethane (DCM) in the presence of trifluoro acetic acid, p-chloranil, Et3N and boron
trifluoride diethyl etherate. To promote the intersystem crossing, 2 and 6 positions of the
compound 1 was iodinated with iodine and iodic acid in ethanol and di-iodinated monomer (2)
was obtained.27 Then, the compound 1 was brominated in the presence of N-boromosuccinimide
(NBS) in DCM to get di-brominated monomer (3).28 The nucleophilic substitution reaction of
cyanuric chloride (triazine) with propargyl alcohol in the presence of NaH in THF was carried
out and tris-alkyne-carrying triazine (4) formed.23 The click reactions of triazine core (4) with
BODIPY monomers (2 and 3) in the presence of copper sulfate pentahydrate and sodium
ascorbate yielded triazine-based BODIPY trimers (5 and 6). All compounds were purified by
column chromatography and then novel compounds (5 and 6) were characterized by mass
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spectrometry and H, and C NMR spectroscopy. The molecular ion peaks of the trimers (5
and 6) were marked as 2309.97 and 2028.76 Da by MALDI-TOF mass spectrometer (Fig. S1
and S4). Also, [M+H-F]+ ion peaks were observed as 2288.91 and 2007.82 for compound 5 and
6, respectively. The aromatic carbons of trimers were marked between 173.0 and 111.0 ppm in
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the C NMR spectra (Fig. S3 and S6).The aliphatic carbon signals were seen between 85.0-
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13.0 ppm regions. The H NMR spectra of the trimers (5 and 6; Table 1) were evaluated in
comparison to that of the monomers (2 and 3).27,28 The 1H NMR spectra of the trimers exhibited
1
doublet signals for meso aromatic protons around 7.0 ppm. When the H NMR spectra of
monomers and trimers were compared, it was seen that a new peak (-NCH2-) was formed at
around 4.0 ppm instead of CH2N3 proton peak. It was also observed that two new peaks
belonging to the triazole ring protons at 7.8 ppm and -COCH2 protons at 5.6 ppm for the trimers
formed. While the -OCH2 protons were marked as triplet at 4.5 ppm, the -CH3 protons on the
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Table 1. H NMR parameters for monomers (2, 3)27,28 and trimers (5, 6).
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5
Compounds OPH (ppm)]a
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7
8
9
2
3
5
6
7.1 (d, J =7.6 Hz)
7.0 (d, J =7.6 Hz)
7.1 (d, J =8.4 Hz) 7.2(d, J =8.3 Hz)
7.0 (d, J =8.4 Hz) 7.0 (d, J =8.3 Hz)
7.1 (d, J=8.1 Hz)
7.0 (d, J =8.1 Hz)
Aromatic-CH-
-CH-N
-
-
-
-
7.8 (s)
5.6 (s)
7.8 (s)
5.6 (s)
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-COCH2-
-OCH2-
N3CH2-
-NCH2-
4.1 (t, J = 6.0 Hz)
3.4 (t, J = 6.3 Hz)
-
4.1 (t, J = 6.0 Hz) 4.5 (t, J = 6.9 Hz)
3.4 (t, J = 6.6 Hz)
4.5 (t, J = 6.4 Hz)
-
-
-
4.1 (t, J = 5.7 Hz)
2.2-2.1 (m)
1.9-1.8 (m)
2.6 (s)
4.1 (m)
2.0-1.9 (m)
2.0-1.8 (m)
2.2-2.1 (m)
1.9-1.4 (m)
2.6 (s)
Aliphatic -CH-
2.6 (s)
1.4 (s)
2.6 (s)
1.4 (s)
Ar-CH3
1.4 (s)
1.4 (s)
a
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500 MHz 1H NMR chemical shifts (ppm) in CDCl3 solution. 3J(HH) [Hz], m: multiplet, s: singlet, d: doublet, t:triplet.
3.2. Photophysical properties of triazine-based BODIPY trimers
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The optical properties of the triazine-based BODIPY trimers (5 and 6) were investigated in
ethanol at room temperature (Fig.2). The basic photophysical data of trimers were given at
Table 2. The absorption spectra of trimers exhibited major bands at 635 and 525 nm, which can
be assigned to the S0-S1 transitions, for compound 5 and 6, respectively. The absorption
maximum of compound 5 was slightly red-shifted (10 nm) compared to compound 6, which
can be attributed to the minor contribution of halogen atom size to the molecular ~K!7!3(=P The
molar absorption coefficient I_N of the trimer 5 (# = 22.30 !104 cm-1 M-1) was determined to be
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2.5 times higher than that of the trimer 6 (# = 6.94 !104 cm-1 M-1; Fig. S7 and S8). As shown
in Fig. 2, the excitation of compound 5 at 520 nm gave a very weak emission band at 551 nm
(rF=0.05). This high emission quenching can be explained by the effect of the heavy atom.34
The trimer 6 showed emission peak at 543 nm when excited at 500 nm and florescence quantum
yield was calculated as 0.11. Fluorescence lifetimes IÄF) of the trimers were measured with
mono-exponential calculation (Fig. S9). The lifetime values were found to be 0.12 and 1.22 ns
for trimers 5 and 6, respectively. The difference in fluorescence lifetime values is compatible
with the fluorescence quantum yields. The trimer, bearing iodinated- BODIPY units (5)
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exhibited almost non-fluorescent profile whereas trimer 6 showed more moderate fluorescence
characteristics. The lower fluorescence profile of trimer 5 compared to 6 can be attributed to
promotion of more intersystem crossing (ISC) in the molecule by iodine atoms.19,34,35
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6 Ext.
6 Ems.
6 Abs.
5 Ext.
5 Ems.
5 Abs.
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Wavelength (nm)
Wavelength (nm)
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Figure 2. Absorbance, excitation and emission spectra of 5 Ivex= 520 nm) and 6 Ivex= 500
nm; 2.0 µM) in ethanol.
Table 2. Photophysical and photochemical properties of triazine-based BODIPY trimers (5 and
6)a
d
rF
1ab,
nm
1em,
nm
4b , 104
ÇStokes,
(nm)
e
Compound
ÄF (ns)c
tÇ
M-1 cm-1
0.12
5
6
535
525
551
543
22.30
8.94
16
18
0.05
0.11
0.88
0.76
(CHISQ=0.89)
1.22
(CHISQ=0.86)
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a Ethanol. b Molar extinction coefficients. c Lifetime, d Fluorescence quantum yield, e Singlet oxygen quantum
yield.
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3.3. Photosensitizing properties of triazine-based BODIPY trimers
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The controlled singlet oxygen generation from the photosensitizer is essential for PDT
applications.16 In this study, the photosensitizing abilities of triazine-based BODIPY trimers (5
and 6) under controlled light were determined by two different techniques: chemical
comparative method and singlet-oxygen phosphorescence peaks at 1270 nm. In the chemical
method, 1,3- diphenylisobenzofuran (DPBF), a common singlet oxygen scavenger agent, was
used to monitor the singlet oxygen production. Before starting the measurements, the solutions
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containing the trimer (5 and 6) and DPBF were kept in the dark for 30 min and it was determined
that the trimers was inactive in the dark. Upon irradiation of solutions with a LED light ("= 516
nm, 2.1 mWcm-2), gradual decrease in the absorbance of DPBF at 414 nm was detected at time
interval between 0 and 35 s (Fig. 3). The same measurement was repeated under the same
conditions for Rose Bengal (Fig. S10). The singlet oxygen quantum yields Iru) were
determined as 0.88 and 0.76 for trimers bearing iodinated BODIPY units (5) and brominated
BODIPY units (6), respectively. The iodine-substituted trimer exhibited the highest singlet-
oxygen generation capacity, followed by the bromine-substituted trimer and finally RB
Iru=0.68). As the second method, singlet oxygen generation capacities of the trimers were
determined via monitoring the phosphorescence signal of singlet oxygen at 1270 nm. The
photosensitizers (5 and 6) were triggered by using a xenon lamp at their regarding absorption
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1
maxima and monitored with a near-IR detector (Fig.4). Based on the O2 phosphorescence
emission signal, the singlet oxygen quantum yields Iru) were calculated as 0.85 and 0.82 for
compound 5 and 6, respectively. In this study, the stability of trimers under light was also tested.
The solution of compound 5 and 6 without DPBF were stimulated with LED light ("= 516 nm,
2.1 mWcm-2) for 30 min and no significant mobility was observed in the absorbance intensities.
The photophysical and photochemical results showed that compound 5 had the highest ability
to generate singlet oxygen and had a rather low fluorescence profile. The differences in
1
fluorescence characteristics and O2 productions of the trimers can be explained by halogen
atom size, known as the most popular method to facilitate the ISC of the molecule. To
demonstrate the advantage of trimer structures on the singlet oxygen generation capacity
compared to their monomer precursors, the ability of monomers (2 and 3) to produce singlet
oxygen was also determined by the chemical method. Based on the oxidation data of the trap
1
molecule in the presence of O2, the values of ru were calculated to be 0.83 and 0.65 for
monomer 2 and 3, respectively (Fig. S13 and S14). Both trimers but drastically brominated one
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(2-4) with maximum concentration alone (0.1 mg/mL) in dark or green light source for 5 h and
MTT cell viability assay was performed. Our results showed that the compounds were not toxic
in dark but green light source was needed to induce the cell death. The minimum concentration
to induce cell death was 0.025 mg/mL and 0.05 mg/mL was the optimal concentration to trigger
the death of about all cells for both trimers (5 and 6; Fig. 5). Importantly, with the maximum
concentration (0.1 mg/mL) used in the study, BODIPY precursors (2 and 3) was also effective
to induce cell death as the novel compounds, 5 and 6. In the previous studies, iodine- and
bromide-containing BODIPY were proposed as well photosensitizers.5 Thus, the results
obtained in the present study correlated with the literature. As expected, triazine core alone (4)
was not able to induce cell death even under green light. However, the ability of triazine
complexes to be able to target some biological entities specifically36 makes the triazine-based
complexes instead of BODIPY alone units favorable in the PDT studies and those studies are
under investigation by our research group.
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Dark
Green Light
100
****
*
****
****
****
*
****
****
50
0
)
)
)
l
)
l
)
)
l
)
l
)
l
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T
l
l
l
O
U
S
m
m
m
m
m
m
m
m
/
/
/
/
/
/
/
/
M
g
g
g
g
g
g
g
g
D
m
m
m
5
m
m
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m
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m
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1
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0
.
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.
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(
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(
(
0
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6
(
(
5
6
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6
Treatments
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Figure 5. Effects of compounds on viabilities of HeLa cells in dark or under green
light.*p<0.05; ****p<0.0001.
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To further characterize the compounds biologically, we determined the internalization pattern
of the compounds. We treated the cells with 0.1 mg/mL of the compounds 2, 3, 5 and 6 and
applied live cell imaging in the presence of Trypan Blue which blocks the extracellular signal
using a fluorescence microscope with a maximal excitation wavelength of 475 nm and a
maximal emission wavelength of 547 nm. Our results pointed that all compounds internalized
into cells and stained the all detectable compartments of the cells (Fig. 6). Intracellular and/or
extracellular singlet oxygen has been shown to trigger cell death.37 In the present study, we
propose that the cell death was performed by internalization of the compounds and intracellular
production of the singlet oxygen. Moreover, according to the results, the novel compounds can
also be evaluated as live cell imaging agents. Finally, the results of in vitro photodynamic
therapy efficacies of the novel compounds make them well candidates for in vivo photodynamic
therapy in further studies.
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Figure 6. Cellular imaging properties of the compounds. Ex: 475 nm and Em: 547nm.
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4. Conclusions
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In this study, we described the molecular design, synthesis and characterization of novel
triazine-based BODIPY trimers (5 and 6) comprising the triazine platform and iodinated/
brominated boron-dipyrromethene branch. Also, the photophysical, and photochemical
characteristics and PDT activities of the photosensitizers were evaluated. The photosensitizers
that were chemically stable under light excitation exhibited strong absorption bands at about
530 nm. These compounds, which were inactive in the dark, were able to produce highly
efficient singlet oxygen when excited by light source. The singlet oxygen quantum yields of the
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compounds determined by chemical and phosphorescence methods have shown that these
materials are very suitable designs and convenient for PDT applications. Especially, compound
5 exhibited a fluorescence quantum yield of 0.05 with very high chemical and phosphorescence
singlet oxygen quantum yields of 0.88 and 0.85, respectively. Undoubtedly, our results
provided critical candidate agents for in vitro and in vivo, as further studies, PDT applications.
Moreover, triazine-based cellular targeting strategies may also combined to PDT studies to
effectively and locally destroy the cancer cells, which is now under investigation by our
laboratory. Still, the novel compounds are proved to be in vitro PDT agents with desirable
photochemical and photophysical properties.
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Electronic supplementary information
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The mass and NMR spectra of the compounds and details of the photophysical studies were
given in SI file.
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Conflicts of interest
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The authors declare no competing financial interest.
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