Study on Liposomal Encapsulation of New Bodipy Sensitizers for Photodynamic Therapy

Article abstract of DOI:10.1021/acsmedchemlett.7b00490

We report a series of four efficient photosensitizers (PSs) based on a Bodipy core for photodynamic therapy (PDT). In the absence of hydrophilic functional groups, these PSs have been encapsulated in liposomes and examined for photocytotoxicity against hu

Full text of DOI:10.1021/acsmedchemlett.7b00490

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Letter
A Study on Liposomal-Encapsulation of New
Bodipy Sensitizers for Photodynamic Therapy
Thumuganti Gayathri, A. Vijayalakshmi, Sreejith Mangalath, Joshy
Joseph, Nalam Madhusudhana Rao, and Surya Prakash Singh
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.7b00490 • Publication Date (Web): 04 Feb 2018
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A Study on LiposomalꢀEncapsulation of New Bodipy Sensitizers for
Photodynamic Therapy
Thumuganti Gayathri,†,‡ A. Vijayalakshmi,§ Sreejith Mangalath,# Joshy Joseph,#* N. Madhusudhana Rao, §* and Surya Pra-
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kash Singh*†,‡
†Inorganic and Physical Chemistry Division, CSIR-Indian Institute of Chemical Technology, Uppal road, Tarnaka, Hyderabad-
500007, India.
§Centre for Cellular & Molecular Biology, Hyderabad-500007, India.
#CSIR-National Institute for Interdisciplinary Science and Technology, Thiruvananthapuram, Kerala 695019, India.
‡Academy of Scientific and Innovative Research
KEYWORDS: Bodipy, photodynamic therapy, liposomes, photosensitizer, photocytotoxicity, cancers
ABSTRACT: We report a series of four efficient photosensitizers (PS) based on Bodipy core for photodynamic therapy (PDT). In the
absence of hydrophilic functional groups, these PS’s have been encapsulated in liposomes and examined for photocytotoxicity
against human ovarian carcinoma cell line (SK-OV-3). The IC50 values obtained are as low as 0.350 µM, which compete with the
classical photosensitizer Chlorine E6 (IC50= 0.39 µM) under similar experimental conditions.
Photodynamic therapy (PDT) represents a minimally inva-
sive therapeutic approach and emerged as a better clinical
modality for treatment of various neoplastic and non-
neoplastic diseases.1-3 PDT requires a photosensitizer (PS),
light source of a specific wavelength suitable for the absorp-
tion characteristics of the PS and molecular oxygen. This
combination leads to the formation of highly cytotoxic singlet
oxygen accompanied by the generation of reactive oxygen
species (ROS), which cause tumor cell death. The most stud-
ied photosensitizers for photodynamic therapy include
tetrapyrrole-based compounds (porphyrins, chlorins, and
bacteriochlorins) such as porfimer sodium (Photofrin), proto-
porphyrin IX, and temoporfin etc.4-6 In general, majority of
PS’s possess limited solubility in water; hence require various
formulations that enhance their bioavailability. These formu-
lations generally include micelles, liposomes, dendrimers and
nanoparticles etc.7-10 Among various drug delivery formula-
tions tested, liposomes were studied widely and several clini-
cal trials using drug-loaded liposomes are in progress.11,12
tractive photophysical properties along with the ability of
generating singlet oxygen with efficient triplet excited life
times.16 Bodipy based photosensitizers for PDT have been
reported earlier.17-23 However, majority of them were linked
with polar functional groups or (and) oligoethylene glycol
chains to enhance the hydrophilicity and cellular uptake.17-28
In brief, dominant sector of these Bodipy photosensitizers
were designed on the perception of hydrophilicity. On the
other hand, photosensitizers with hydrophilic functional
groups such as carboxylic acid, sulfonic acid, or sodium sul-
fonate result in significant reduction of the photocytotoxic
activity.29 Until the present, potent hydrophobic Bodipy based
photosensitizers were not explored for PDT. This manuscript
describes the liposomal encapsulation of Bodipy analogues
and assessing their efficacy in PDT mediated cell death. To the
best of our knowledge, this is the first study of Bodipy sensi-
tizers for PDT via liposomal encapsulation.
Sensitizers for PDT are required to be designed in such a
way that they acquire moderate to high triplet quantum
yields in order to obtain long-lived triplet states. High fluores-
cence quantum yields are actually unwanted for PDT sensitiz-
ers as the fluorescence occurs via relaxation from singlet ex-
cited state, which means the energy absorbed on excitation
does not cross to triplet states.30 Nagano’s group investigated
that a 2,6-diiodo-analog of Bodipy resulted in enhanced sin-
glet oxygen generation in comparison to the unsubstituted
one. The halogen atoms (preferably I or Br) improve singlet-
to-triplet intersystem crossing by heavy atom effect.31 They
also remarked that high oxidation potential would be advan-
tageous for the PS to avoid self-oxidation.
Liposomes are artificially constructed membrane vesicles,
composed of phospholipids and other amphiphilic compo-
nents. They are widely used as vehicles for drug delivery as
they can encapsulate hydrophilic drugs within the aqueous
regions, and also lipophilic molecules within their lipid bi-
layers.13,14 Though, porphyrin based PS’s are well known for
PDT, there are few drawbacks, which include low molar ex-
tinction coefficients (especially in the range of 650–900 nm),
involve difficult synthetic routes and their scale up is tedious.
Therefore, design and developing PS’s with non-porphyrin
scaffolds has become important.15 In this regard, 4,4-difluoro-
4-bora-3a,4a-diaza-s-indacene (Bodipy) derivatives have at-
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Figure 2. Normalized absorbance and emission spectra of Bodipy
Bodipy PS’s in DMSO
Considering all these factors, we investigated four 2,6-
diiodo 3,5-symmetric distyryl based Bodipy PS, synthesized
from meso-p-nitrophenyl Bodipy (1) and meso-phenyl Bodipy
(1a) dyes (Supporting information). These photosensitizers
were named as NBDP-SAL, NBDP-NVER, NBDP-VER and PBDP-
VER (Figure 1). meso-p-nitrophenyl Bodipy (1) and meso-
phenyl Bodipy (1a) dyes were prepared using the classic one
pot procedure with 2,4-dimethylpyrrole followed by the oxi-
dation with DDQ to form dipyrrin, which in turn complexed
with BF3.Et2O in presence of base (Scheme S1, Supporting
information).32 To facilitate the intersystem crossing, 2 and 6
positions of Bodipy core were iodinated using N-
Iodosuccinimide. A 2-fold Knoevenagel reaction of 3 and 5
methyl substituents of the Bodipy core33 with aryl carboxal-
dehyde using catalytic amount of piperidine and AcOH in re-
fluxing dry benzene resulted corresponding symmetrical de-
rivatives in 35-50% yield (Scheme S2 and S3, Supporting in-
formation). All the compounds were characterized by NMR
(1H, 13C) analysis and MALDI-TOF. The complete synthetic
procedures along with characterization spectra are included
in supporting information.
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Since the core iodinated Bodipy sensitizers are expected to
present good triplet quantum yields owing to heavy atom
effect, nanosecond laser flash photolysis studies were carried
out to understand the transient intermediates involved under
laser irradiation. All the samples showed good triplet absorp-
tion, under 355 nm upon laser excitation (Figure S2, Support-
ing information). Figure 3 shows the transient absorption
spectrum of NBDP-NVER in DMSO, which shows maxima at
380, 480 and 760 nm with bleach around 660 nm correspond-
ing to ground state absorption. The transient intermediate
from the sample, NBDP-NVER decayed by a first-order pro-
cess with a lifetime of 0.76 μs, shown in inset of Figure 3. The
triplet excited state quantum yields (ФT) of the compounds
were determined by an earlier reported procedure of energy
transfer to β-carotene, using Ru(bpy)32+ as the reference mol-
ecule.34 The data are summarized in Table 1 (A detailed de-
scription is given in supporting information). Excellent triplet
state quantum yield of 0.90±0.03 for NBDP-NVER and moder-
ate yields for other Bodipy PS’s were obtained.
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Figure 3. Transient absorption spectra of NBDP-NVER, excited at
355 nm LASER. Triplet decay at 480 nm is shown in inset.
0.12
Figure 1. Molecular structures of Bodipy sensitizers for PDT
Methylene Blue
PBDP-VER
NBDP-VER
NBDP-SAL
Electronic absorbance and fluorescence emission spectra of
the Bodipy PS’s were recorded in dimethyl sulfoxide (DMSO)
(Figure 2). All of them exhibit moderate Stokes shifts ranging
from 31-55 nm and possess higher extinction coefficients
(Table 1). The photostability of Bodipy PS’s was tested by
continuous irradiation of light (200W xenon lamp) for 2
hours. The absorbance spectra measured before and after
irradiation confirm that these PS are highly photostable (Fig-
ure S1, Supporting information).
NBDP-NVER
0.08
0.04
0.00
0
5
10
15
20
Time, S
NBDP-NVER
1.0
0.8
0.6
0.4
0.2
0.0
1.0
0.8
0.6
0.4
0.2
0.0
NBDP-NVER
NBDP-SAL
NBDP-VER
PBDP-VER
NBDP-SAL
NBDP-VER
PBDP-VER
Figure 4a. Plot of ꢀA of DPBF at 410 nm vs. irradiation time with
Bodipy PS’s along with methylene blue as the standard.
0.60
0
NBDP-NVER + DPBF
0.45
20 s
350 400 450 500 550 600 650 700 750 800 850
Wavelength, nm
650
700
750
800
0.30
Wavelength, nm
0.15
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NBDP-VER
NBDP-NVER
PBDP-VER
NBDP-SAL
60
40
Figure 4b. Decrease in absorption of DPBF in presence of NBDP-
NVER by photo-irradiation
20
0
The ability of these compounds in generating singlet oxygen
can be determined by the rate of decay of 1,3-
diphenylisobenzofuran (DPBF), which is a singlet-oxygen
quencher. The singlet oxygen generation quantum yields (Ф
1O2) were measured in DMSO using methylene blue as stand-
ard (Figure 4a). All the Bodipy PS’s demonstrated good singlet
oxygen generation efficiencies, with the highest of 0.80 ob-
tained for NBDP-NVER (Figure 4b & Table 1). The generation
of singlet oxygen is further confirmed from the emission of
samples at 1273 nm, which is characteristic luminescence of
singlet oxygen35 (Figure S3, Supporting information). All sam-
ples exhibited considerable singlet oxygen luminescence by
exciting at 600 nm.
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0.1
1
PS, ꢁM
Figure 5. Cell viability of SK-OV-3 cells in the presence of free PS
and light treatment. Cell viability was estimated by MTT assay
using the dark plate as control plate. Cell death in the presence
of PS and absence of light was negligible.
Initially, we have conducted an experiment with variation of
light dosages and their effect in the presence of PS. Based on
these experiments 20 Jcm-2 was selected as the optimal dose
for PDT treatment. Light-mediated cell death was investigat-
ed with these Bodipy PS’s on SK-OV-3 cells. In the PDT stud-
ies, each of the light treatments was performed in triplicate
for all the four Bodipy photosensitizers in three sets. First set
was subjected to light exposure which was treated with free
PS (without using liposomal encapsulation) (Figure 5), where
as second set was treated with liposome encapsulated PS
under light exposure (Figure 6) and the third set was with
liposomal encapsulated PS protected even from stray light
and was treated as a dark control (Figure 7).
Table 1. The photophysical properties of Bodipy PS’s in DMSO
PS
Λmax
Λmax Stokes
ԑd
Singlet
Oxygen
Q.Y e
Triplet
Quantum
Yield f
Triplet
Lifetime
µs g
Absa Ems b shift c (M−1⋅cm−1
)
nm
nm
PBDP-VER
675
730
55
119197.21
0.46
0.490
1.07
NBDP-VER
684
657
725
700
41
43
120917.41
92822.28
0.37
0.80
0.39
0.80
0.76
NBDP-NVER
0.903
NBDP-SAL
664
695
31
82364.86
0.40
0.411
1.20
Light mediated cell death in the presence of sensitizers was
insignificant for the first set i.e., with free PS (Figure 5). The
IC50 value (IC50 is concentration of PS required for killing 50%
of cancerous cells) could not be determined for free PS in the
tested concentration range. Whereas the cell death was very
pronounced for the second set, in which cells were treated
with liposome-encapsulated PS (Figure 6). It can also be seen
that all of them are essentially non-cytotoxic in the absence
of light (Figure 7). The IC50 of these photosensitizers in lipo-
somal formulations (shown in inset of Figure 6) were compa-
rable to that of the classical PS Chlorine E6 (IC50= 0.39 µM)
under the same experimental conditions. Of all the Bodipy
PS’s in the series, NBDP-VER exhibits the lowest IC50 of 0.35
µM.
a) Absorbance maxima b) Emission maxima c) Difference be-
tween absorbance and emission maxima d) Molar extinction
coefficients at the absorbance maxima e) Singlet oxygen quan-
tum yields were measured using methylene blue standard f) Tri-
plet excited state yields were determined by energy transfer to
2+
β-carotene using Ru(bpy)3 as the reference molecule g) Triplet
2+
Lifetime of the photosensitizers calculated using Ru(bpy)3
standard (0.61 µs)
Liposomes were prepared by reconstituting chloroform
stocks of egg L-α-phosphatidylcholine (EPC), 1,2-dioleoyl-sn-
glycero-3-phosphoethanolamine (DOPE) and with PS at a
mole ratio of 70:25:5 (Detailed procedure included in Sup-
porting information). To estimate the liposome associated PS,
the PS loaded liposomes were dissolved in DMSO. The ob-
served absorbance, determined for each PS, was used to cal-
culate the PS concentration from the standard graphs con-
structed with each pure PS in DMSO.
120
IC , ꢁM
50
NBDP-VER 0.35
PBDP-VER 0.88
NBDP-SAL 0.65
NBDP-NVER 0.86
100
80
60
40
20
0
0.0
0.5
1.0 1.5
2.0
PS, ꢁM
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NBDP-VER
PBDP-VER
NBDP-SAL
NBDP-NVER
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Figure 7. Cell viability of SK-OV-3 cells in the presence of various
PS in liposomal formulations and without light treatment.
In conclusion, we have developed a new series of Bodipy
based PS and evaluated their in vitro photodynamic activity
using liposomal encapsulation. PDT efficacy and photophysi-
cal characteristics of these compounds noticeably depend on
substituent present on them. Nitro group substitution on the
3,5 styryl moieties resulted in higher singlet oxygen and tri-
plet quantum yields. The photosensitizers reported herein
obtained competing IC50 values to those of classic PS, endors-
ing a scope to access advancements in Bodipy modification,
without the limitations of hydrophilicity as much as it was
focused earlier. This research paves the way for designing and
developing potent formulations of Bodipy sensitizers, circum-
venting the need to incorporate hydrophilic functional
groups.
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ASSOCIATED CONTENT
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17 Chen, Y.; Zhao, J.; Xie, L.; Guo, H.; Li, Q. Thienyl-substituted
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19 Lim, S. H.; Thivierge, C.; Sliwinska, P. N.; Han, J.; Bergh, H. V.
D.; Wagnieres, G.; Burgess, K.; Lee, H. B. In vitro and in vivo
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20 Wu, W.; Guo, H.; Wu, W.; Ji, S.; Zhao, J. Organic triplet sensi-
tizer library derived from a single chromophore (bodipy) with
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based upconversion. J. Org. Chem. 2011, 76, 7056-7064.
21 Umezawa, K.; Matsui, A.; Nakamura, Y.; Citterio, D.; Suzuki,
K. Bright, Color-tunable fluorescent dyes in the Vis/NIR re-
gion: Establishment of new "tailor-made" multicolor fluoro-
The Supporting Information is available free of charge on the ACS
Publications website.
Experimental procedures, characterization for the synthesis of com-
pounds mentioned in Figure 1, liposomal preparation, photobleach-
ing experiments, determination of triplet and singlet oxygen quan-
tum yields and Figures S1−S3 (PDF)
AUTHOR INFORMATION
Corresponding Authors
*
*
*
Tel: +91-40-27191384, E-mail: spsingh@iict.res.in
Tel: +91-40-27192504, E-mail: madhu@ccmb.res.in
Tel: 0471-2515476, E-mail: joshyja@gmail.com
Acknowledgment
TG and SM thank UGC and CSIR respectively for senior research
fellowship.
phores based on borondipyrromethene. Chem. Eur. J. 2009
15, 1096-1106.
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Insert Table of Contents artwork here
4
5
6
PDT effect
7
8
100
80
9
NBDPꢀVER
NBDPꢀNVER
60
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
PBDPꢀVER
NBDPꢀSAL
40
Light
20
Tumor
0
0.1
1
PS, ꢁM
120
100
80
60
40
20
0
IC
, ꢁM
50
NBDPꢀVER 0.35
PBDPꢀVER 0.88
NBDPꢀSAL 0.65
NBDPꢀNVER 0.86
Light
Tumor
0.0
0.5
1.0 1.5
2.0
PS, ꢁM
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