In Vitro and in Vivo Demonstration of Human-Ovarian-Cancer Necrosis through a Water-Soluble and Near-Infrared-Absorbing Chlorin

Article abstract of DOI:10.1021/acs.jmedchem.8b00460

With the objective of developing efficient sensitizers for therapeutic applications, we synthesized a water-soluble 5,10,15,20-tetrakis(3,4-dihydroxyphenyl)chlorin (TDC) and investigated its in vitro and in vivo biological efficacy, comparing it with the

Full text of DOI:10.1021/acs.jmedchem.8b00460

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Article
In Vitro and In Vivo Demonstration of Human Ovarian Cancer Necrosis
through a Water Soluble and Near Infrared Absorbing Chlorin
Betsy Marydasan, Bollapalli Madhuri, Shirisha Cherukommu, Jedy Jose,
Mambattakkara Viji, Suneesh C Karunakaran, Tavarekere K. Chandrashekar,
Kunchala Sridhar Rao, Ch. Mohan Rao, and Danaboyina Ramaiah
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00460 • Publication Date (Web): 16 May 2018
Downloaded from http://pubs.acs.org on May 16, 2018
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In Vitro and In Vivo Demonstration of Human Ovarian Cancer
Necrosis through a Water Soluble and Near Infrared Absorbing
Chlorin
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Betsy Marydasan,† Bollapalli Madhuri,‡ Shirisha Cherukommu,‡ Jedy Jose,‡ Mambattakkara
Viji,† Suneesh C. Karunakaran,† Tavarekere K. Chandrashekar,§ Kunchala Sridhar Rao,*,#
Ch. Mohan Rao,*,‡ and Danaboyina Ramaiah*,∥
†Chemical Sciences and Technology Division, CSIR-National Institute for Interdisciplinary
Science and Technology (CSIR-NIIST), Trivandrum - 695 019, India
‡CSIR-Centre for Cellular and Molecular Biology (CSIR-CCMB), Hyderabad - 500 007, India
§National Institute of Science Education and Research (NISER), Bhubaneswar - 751 005, India
∥CSIR-North East Institute of Science and Technology (CSIR-NEIST), Jorhat - 785 006, India
# Indo-American Cancer Research Foundation (IACRF), Basavatarakam Indo-American Cancer
Hospital and Research Institute, Hyderabad 500034
* Corresponding authors:
E-mail: rama@neist.res.in., Tel: +91 3762370012; Fax: +91 3762370011
E-mail: research.director@induscancer.com., Telefax: 91-40-23540348
E-mail: mohan@ccmb.res.in., Tel: 91-40-27195630; Fax: 91-40-27195633
Keywords: Dihydroxychlorin; Singlet oxygen; Photodynamic therapy; Ovarian cancer; Necrosis
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ABSTRACT
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With an objective to develop efficient sensitizers for therapeutical applications, we
synthesized a water soluble, 5,10,15,20-tetrakis(3,4-dihydroxyphenyl)chlorin (TDC), and
investigated its in vitro and in vivo biological efficacy and compared with the commercially
available sensitizers. TDC showed high water solubility (6-fold), when compared to Foscan and
exhibited excellent triplet excited state () and singlet oxygen () yields. In vitro
photobiological investigations in human ovarian cancer cell lines SKOV-3 showed high
photocytotoxicity, negligible dark toxicity, rapid cellular uptake and specific localization of
TDC in neoplastic cells as assessed by flow cytometric cell-cycle and propidium iodide staining
analysis. The photodynamic effects of TDC have confirmed the reactive oxygen species induced
mitochondrial damage leading to necrosis in SKOV-3 cell lines. The in vivo photodynamic
activity in nude mice models demonstrated abrogation of tumor growth without any detectable
pathology in skin, liver, spleen, kidney, thereby TDC application as an efficient and safe
photosensitizer.
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INTRODUCTION
Photodynamic therapy (PDT) is a new approach for a safe and effective eradication of
different types of cancerous tissues without affecting the normal cells. This emerging technique
is based on the light activation of the sensitizer and the production of reactive oxygen species
(ROS) in presence of molecular oxygen.1-5 PDT induces oxidative damage and can trigger either
necrosis or apoptosis mediated cell death depending on the intensity of the insult.6-9 The reactive
oxygen species can also prevent tumor growth indirectly through vascular shutdown and
activation of immune response.4,5,10-17 In this modality, the sensitizer plays a key role in
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determining the efficacy of the photodynamic therapy. A series of sensitizers such as porphyrins,
porphycenes, phthalocyanines, chlorins, bacteriochlorins, squaraines, rose bengal, methylene
blue, and their derivatives, which exhibit good singlet oxygen generation efficiency have been
evaluated for the photodynamic treatment.18-24 Among these, the first generation sensitizer,
hematoporphyrin derivative (HpD), which is commercially known as Photofrin@ is being in
clinical use for the treatment of several types of cancers.25 However, HpD has disadvantages like
it is a mixture of nine components, has relatively weak absorption at 630 nm and is known to
cause skin photosensitivity.26,27
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In the last few decades, the major challenge in the field of photodynamic therapy has
been to overcome the drawbacks of HpD and to discover second generation photosensitizers by
red shifting the absorption of Q bands together with high extinction coefficients in the red region.
In this effort, several compounds have been investigated for anti-cancer PDT activity.28 Among
these, 5,10,15,20-tetra(m-hydroxyphenyl)chlorin, commercially known as Foscan, is one of the
promising sensitizer, which is a reduced form of the porphyrin.29 But the main disadvantages
associated with Foscan are its poor solubility in aqueous media and its high photosensitivity to
the patients even up to twenty days after PDT treatment.30 The porphyrin based photosensitizers
developed so far are either heterogeneous in their chemical composition, water insolubility, and
have less photodynamic efficiencies or associated with extended cutaneous sensitivity.31-33 The
second generation chlorin derived photosensitizers are chemically pure, but are minimally
soluble in aqueous medium.33,34 Several attempts are being made to overcome these drawbacks
by functionalizing the photosensitizers by attaching folic acid, lipid encapsulation etc.35
However, these additional methodologies compromise on the targeting, toxicity and shelf life.
Both Photofrin and Foscan that are approved for clinical use have extended photosensitivity or
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required prolonged treatment of light exposure for periods ranging for up to one week. Herein,
we report a novel water soluble sensitizer, 5,10,15,20-tetrakis(3,4-dihydroxyphenyl)chlorin
(TDC) and investigation of its photophysical and in vitro and in vivo photobiological aspects as
compared to Photofrin and Foscan. Uniquely, TDC exhibited high absorption and photodynamic
activity in human ovarian cancer cells-SKOV-3 and acted as anti-tumor agent in vivo, using nude
mice models, thereby demonstrating its use as an effective sensitizer in PDT applications.
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RESULTS AND DISCUSSION
Synthesis and Photophysical Properties. The synthesis of the 3,4-dimethoxyporphyrin
was achieved through the modified procedure of Lindsey method,36 while the chlorin derivative,
TDC was accomplished by the reduction of the corresponding porphyrin as shown in Scheme 1.
The starting materials and chlorin derivatives were purified through column chromatography as
well as through recrystallization and were characterized on the basis of various analytical and
spectral evidences. TDC showed the characteristic chlorin absorption having a Soret band at 421
nm, and four Q-bands at 521, 550, 596 and 650 nm. When compared to the porphyrins, the Q-
band at 650 nm in TDC was found to be relatively intense and exhibited an extinction coefficient
value of 1.5 x 104 M-1cm-1. The emission spectrum of dihydroxychlorin TDC exhibited two
maxima at 654 and 722 nm with a fluorescence quantum yield (ΦF) of 0.01 ± 0.002 in methanol
(Figure 1A).
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Scheme 1. Synthesis of the chlorin derivative TDC.
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TDC
To understand the transient intermediates involved in the case of the chlorin derivative
TDC, we have carried out the nanosecond laser flash photolysis experiments. As the first step of
this investigation, we have monitored the transient absorption spectrum of TDC in DMSO
immediately after 355 nm laser pulse excitation. The transient absorption of TDC showed an
absorption maximum at 360 nm with bleach at 420 nm and the transient intermediate formed
from TDC decayed by a first-order process with a lifetime of 60 µs. Further, we quantified the
triplet quantum yield of (ΦT) TDC employing triplet-triplet energy transfer method and using β-
carotene as the energy acceptor.37 We obtained a triplet quantum yield value of ca. ΦT = 0.84 ±
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0.02 (Figure 1B) for TDC, which is better than the values reported for Photofrin and Foscan
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(Table 1) under similar conditions.
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A)
B)
Figure 1. A) UV-Vis absorption and fluorescence (inset) spectra of TDC (6 µM) in methanol;
ex, 430 nm and B) transient absorption spectra of TDC (30 μM) following 355 nm laser pulse
excitation; time-resolved absorption spectra recorded at 10 µs. The inset shows the transient
decay of TDC at 440 nm.
To have a better understanding of the PDT efficacy of TDC, we have determined the
singlet oxygen generation efficiency using chemical trapping method using 1,3-
diphenylisobenzofuran (DPBF) as the singlet oxygen scavenger38 as well as by quantification of
its emission intensity at 1270 nm.39 To determine the singlet oxygen quantum yields [Φ (1O2)],
irradiation of a solution containing the chlorin system TDC along with DPBF was carried out
using a 200 W mercury lamp as light source with a 515 nm long pass filter at 0-60 s time
intervals (Figure S1, Supporting Information). The singlet oxygen generation efficiency [Φ
(1O2)] of TDC was found to be ca. 0.80 ± 0.03. Interestingly, a comparative study showed higher
singlet oxygen generation efficiency for TDC in comparison with commercially available
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sensitizers, Photofrin and Foscan (Table 1). Moreover, the hydrophilicity of TDC due to the
presence of hydroxyl groups imparts better solubility for the compound in biological media
(Table S1, Supporting Information). For example, the compound TDC exhibited ca. 6-fold
enhancement (3.64 mM) in solubility in 25% aqueous ethanolic medium, when compared to the
commercially available sensitizer, Foscan (0.6 mM). In addition to that, the increased solubility
decreases the aggregation in aqueous medium, enhances its cellular uptake and bioavailability,
aiding in faster plasma clearance and thereby contributing to the higher photodynamic efficacy
of TDC, when compared to Photofrin and Foscan under similar conditions.40
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Table 1. Quantum yields of triplet (T) and singlet oxygen [ (1O2)] yields of sensitizers.a
ab, nm (, M-1cm-1)
628 (3.0 x 103)
651 (1.3 x 104)
650 (1.5 x 104)
F
T
(1O2)
0.30
Sensitizer
Photofrin
Foscan
TDC
0.10
0.08
0.01
0.60
0.80
0.84
0.59
0.80
a
Average of more than three independent experiments. The quantum yields of fluorescence, triplet
excited states and singlet oxygen was determined under similar experimental conditions. The values
obtained for Photofrin and Foscan are similar to those reported in the literature.25,33
Investigation of In Vitro Photocytotoxicity. To investigate the photobiological effects
of TDC, we selected human ovarian cancer cell lines SKOV-3 and a tunable light source-
Waldman PDT 1200L (Germany), with an emission spectrum in the range of 600-720 nm. We
have used a dose of 200 J/cm2 at 50 mW/cm2 for irradiation of cells in culture and 100 J/cm2
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(100 mW/cm2) for irradiation in animal model experiments. To understand the cellular uptake
and localization of TDC, we performed fluorescence and confocal microscopic images of TDC
with SKOV-3 cell lines using blue stain Hoechst 3342 at different conditions. It is interesting to
observe that when SKOV-3 cells were incubated with 20 M TDC for 1 h, the cells readily
uptake the sensitizer. Moreover, intracellularly, TDC showed a high and punctuate fluorescence
indicating its probable vesicular/mitochondrial association and also a diffused fluorescence along
the membrane periphery as shown in Figure 2. The dye localization to F-actin and to.
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Figure 2. Uptake of TDC by SKOV-3 cells. A) Red fluorescence in SKOV-3 cells after
treatment with 20 M TDC and B) merged image showing SKOV-3 cell nuclei with a vital
nuclear dye Hoechst 33342 (blue) and TDC (red).
mitochondria has been subsequently confirmed by co-localization experiments using specific
probes (Figures 6 and 7). It has been reported earlier, that even low levels of singlet oxygen
produced in close proximity to the target organelle such as mitochondria is far more efficient in
its phototoxic effects, than a large quantities generated at a distant cellular location.41 The
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proximity of localization of TDC to vital intracellular organelles, could therefore yield more
efficient phototoxicity.
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Cell viability as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) assay showed a clear dose dependent photo-toxicity (Figure 3A). We observed a
significant loss of viability of SKOV-3 cells at concentrations beyond 10 µM and followed
typical biological protocols. Under these experimental conditions, the IC50 value of TDC was
Figure 3. Cytotoxic and phototoxic effects of TDC: A) Dark control: SKOV-3 cells treated with
TDC but not exposed to light and B) cells after photo-irradiation (100X). C) Viability of SKOV-
3 cells that were exposed to varied concentrations of TDC left unexposed to light (dark control,
blue line) and after light exposure (red line); cell viabilities were measured at 24 h after their
respective treatments (the results are an average of 3 independent experiments).
calculated to be 11.16±2.08 M for SKOV-3 cells. TDC exhibited excellent photo-toxic effects
with the IC50 values varying from 4-20 M depending on the cancer cell line used ((IMR32
(3.5±0.84 μM), MCF7 (4.4±1.62 μM) and MDA MB 231 (6.1±1.16 μM)). Phase contrast
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microscopic images of TDC treated SKOV-3 cells, in the absence of light exposure, displayed
minimal alterations in the cell morphology. However, dye treated cells when exposed to light,
displayed extensive changes in cell morphology, indicating cell death).
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Further, to understand the dark cytotoxicity and cellular mechanism behind the
photodynamic efficacy of TDC on SKOV-3 cells, we have analyzed SKOV-3 cells by staining
with propidium iodide (PI) and flow cytometric cell cycle analysis (Figure 4).42 In a typical
distribution, sub G1 population represents the hypo-diploid, dead cells. The experimental
controls displayed ca. 1-7% cells in sub-G1 stage, whereas the cells treated with TDC and
exposed to light showed a sub-G1 population of ca. 60% after 12 h and ca. 77% after 24 h. A
gradual shift of cells to sub-G1 population indicates the persistence of photo-induced effects of
TDC over a period of time. This gradual shift of cells to sub-G1 phase represents the immediate
and late events connected to the photodynamic damage of cellular DNA. After 24 h post
irradiation, we observed the shift of almost entire population of cells towards sub-G1 phase,
indicating an excellent phototoxic effect of TDC.
Mode of Cell Death. To understand the mode of phototoxic activity, flow- cytometric
analysis was performed on live SKOV-3 cells (both control and TDC treated cells) stained with
propidium iodide (PI). The control cells (untreated and dark control) showed negligible PI
fluorescence (population of cells in M2 window of ~7-9%), whereas the cells treated with TDC
and exposed to light displayed a very intense fluorescence and showed about ca. 80% of the cell
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Figure 4. Cell cycle analysis-time dependent phototoxicity of TDC (20 M) in SKOV-3 cells.
Flow cytometric analysis shows the percentage of Sub-G1 population (dead) of cells (shown in
red circles); A) control (2.2%), B) light control (7%), C) dark control (1%) and post- irradiation
(λirr 600-720 nm; dose 200 J/cm2 at 50 mW/cm2) of cells after D) 12 h (60%) and E) 24 h (77%).
F) Graphical representation of the cell cycle data
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population. These observations clearly demonstrated the loss of cell membrane integrity (Figure
5) and indicated necrosis to be the main mode of phototoxic killing effect of TDC. To have a
better understanding in generation of intracellular reactive oxygen species we have used a well-
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Figure 5. Live cell staining and flow cytometric analysis of control and TDC (20 M) treated
SKOV-3 cells with propidium iodide (PI) (10 μg/mL). PI-positive membrane-damaged cells are
seen only after photo-irradiation (λirr 600-720 nm; dose 200 J/cm2). The percentage of PI positive
cells in (denoted by arrows), A) control (7%), B) light control (7.5%), C) dark control (9%) and
D) after PDT (79%). E) Graphical representation of the live cell staining with Propidium Iodide.
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known fluorescent probe 5-(and-6)-chloromethyl-2′,7′-dichloro-dihydrofluorescein diacetate
acetyl ester (CM-H2DCFDA). To perform CM-H2DCFDA assay, the human ovarian cancer
SKOV-3 cells were treated with TDC and irradiated followed by the addition of CM-H2DCFDA
(10 μM) and examined through confocal microscopy. We observed appearance of green
fluorescence only in the TDC treated and light exposed cells indicating the generation of ROS,
while the untreated or dark control cells showed no fluorescence. A parallel flow cytometric
analysis under the same conditions also confirmed that ca. 73% of cell population displayed
ROS fluorescence only in the cells treated with TDC and exposed to light, while the control
cells exhibited negligible ROS levels (ca. < 1%) (Figure S2, Supporting Information).
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In SKOV-3 cells, the actin appears as circularly organized filaments, located near the
plasma membrane. Motivated from the live cell propidium iodide (PI) staining experiments,
which indicated loss of cell membrane integrity, we analyzed actin cytoskeletal integrity of the
SKOV-3 cells before and after the photodynamic treatment using tetramethylrhodamine (TRITC)
labeled Phalloidin, an F-actin specific fluorescent probe.43 The cells treated with TDC followed
by irradiation showed a complete collapse in F-actin filament network, whereas the control cells
remained intact (Figure 6). These observations demonstrated disruption of filamentous actin-
cytoskeleton organization in cells. Loss of cytoskeletal integrity could potentially disrupt several
physiological functions in the cell that ultimately trigger cell death.44
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Figure 6. Photodynamic destruction of actin filament organization in SKOV-3 cells. F-actin
stained by Phalloidin (red) and the nuclei stained with 4',6-diamidino-2-phenylindole (DAPI)
(blue); A) untreated cells, B) cells treated with TDC (20 M) but not exposed to light and C)
cells treated with TDC (20 M) and exposed to light (600-720 nm; 200 J/cm2). Corresponding
bright field images are shown in the lower panel.
To understand the mitochondrial integrity, TDC treated and light exposed SKOV-3 cells
were stained with MitoTracker Red (CMXRos), a fluorophore sensitive to the mitochondrial
membrane potential (ΔΨm) (Molecular Probes, OR, USA). The mitochondria of untreated
SKOV-3 cells showed typical fibrillar appearance with bright fluorescence denoting intact
mitochondrial membranes (Figure 7A). TDC treated and light exposed SKOV-3 cells showed a
loss of mitochondrial network, which can be clearly evidenced from Figure 7B. Damage to the
mitochondrial integrity can lead to cellular events such as cessation of adenosine 5'-triphosphate
(ATP) production, release of toxic proteins into cytoplasmic milieu that ultimately trigger cell
death.45,46 These results clearly demonstrate that the TDC photodynamically damages cell
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membrane, disrupts actin cytoskeletal network and result in the loss of mitochondrial membrane
potential (indicating mitochondrial damage). The photoxic killing of SKOV-3 cells by TDC is a
net destruction of critical cellular organelles and cell death is predominantly considered to be
through necrosis or necroptosis. Moreover, the TDC showed good photo-stability and its
photocytotoxic effects persisted over a period of time, thus probably minimizing the requirement
of multiple light exposures.
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Figure 7. Phototoxic destruction of mitochondria in SKOV-3 cells. Mitochondria was stained by
Mitotracker red fluorescent probe (red mesh like structures surrounding the nuclei) and the
nuclei were stained with Hoechst 33342 (blue); A) Untreated cells and B) cells treated with TDC
(20 M) and exposed to light (600-720 nm; 200 J/cm2).
Demonstration of In Vivo Photodynamic Activity. The excellent photodynamic
killing by TDC on SKOV-3 cells in culture encouraged us to establish its tumoricidal efficacy in
vivo using nude mice model. To analyze this, SKOV-3 cells (~3-5 X 106 cells, in a final volume
of 200 μL) were injected subcutaneously in the dorsal flank of the nude mice and kept for one
week in order to develop the tumors. After injecting SKOV-3 cells, the nude mice were divided
into two groups. The first group of six animals served as the tumor bearing controls, whereas the
second group of animals bearing tumor were treated with TDC followed by irradiation. The
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sensitizer, TDC (in physiological saline) was administered intravenously (20 mg/Kg body
weight) to the mice models, anesthetized and were exposed to light (100 J/cm2 and 100 mW/cm2)
for about 12-18 h post-TDC administration. The tumor volumes (TV; mm3) were monitored for
over a period of two weeks and are shown in Figure 8. As per the ethical animal practices,
animals with tumor volumes exceeding 10% of the body weight were sacrificed.
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Figure 8. Effect of TDC (20 mg/Kg body weight) on tumor progression in nude mice. A) Nude
mice bearing SKOV-3 tumors untreated (left) and treated with TDC (right) after measuring the
tumor volumes (TV) on day 1 and day 14, respectively. The corresponding Hematoxylin & Eosin
(H&E) stained tissue sections (biopsied at the site of injection) showing the elimination of
neoplastic cells in tumors of mice injected with TDC to show the clearance of neoplastic tissues.
B) Tumor volumes measured in controls (blue line) and animals (n=6) subjected to PDT (red
line) on various days.
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The tumor volumes measured on first day (before the administration of TDC) and
fourteenth day were monitored and are shown in Figure 8A. Interestingly, the tumor volumes
were not measurable after fourteen days in animal models when TDC treated and exposed to
irradiation. The in vivo biological experiments clearly indicate that the control animals those
shown in left panel exhibited enormous tumor growth, whereas TDC treated and light exposed
animals showed a dramatic reduction in tumor volumes to below measurable levels, sometimes
in a short span of < 14 days without any observable scar at the site of the tumor. For the post
experimentation, a small bit of the tissue at the tumor site was biopsied, processed for histology
and stained with Hematoxylin as well as Eosin. These tissue sections clearly showed the
complete replacement of neoplastic cells with fibrous and other cell types (Figure 8, H & E
stained panels on the right side). It can be observed that there is a minimal scarring at the site of
the tumor. Uniquely, the photodynamic treatment with the skin in peritumoral area of the animals
did not exhibit any sensitivity throughout the experimental period, thereby indicating that TDC
administration is safer compared to the commercially available photosensitizers.
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To determine the in vivo cellular localization, tumor bearing animals were injected with
TDC intra-tumorally and the tumors were biopsied for 12 h post administration, sectioned and
analyzed by fluorescence microscopy. We observed that the TDC fluorescence (red) is mainly
localized in the neoplastic cells (Figure 9), whereas the cells of fibrous tissue and that of stroma
(nuclei stained blue) did not show red fluorescence. Selective localization of TDC and other
photosensitizers in cancer cells is a subject of immense interest. It is hypothesized that
p-glycoproteins, which are responsible for drug efflux, play an important role in the selective
cellular retention of the photosensitizers.
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Figure 9. Intra-tumoral localization of TDC (20 mg/Kg body weight) in nude mice. Biopsies
from the tumors bearing animals injected with TDC intra-tumorally were sectioned and
visualized by fluorescence microscopy. A) DAPI stained section shows nuclei from both SKOV-
3 cancer cells and stromal tissue nuclei (blue) and B) TDC visualized as bright red fluorescence,
the sensitizer is localized in the neoplastic cells only.
To understand the effect of TDC on non-target organs, we examined the systemic effects
of PDT in biopsied and sectioned tissues of various body parts such as liver, kidney, spleen,
heart, lung of the animal models. The post-PDT treatment in these tissues under various
conditions such as untreated, dark control and with TDC were stained with standard
Hematoxylin and Eosin are shown in Figure 10. These studies clearly showed that the cellular
morphology of these organs remained intact without any detectable tissue pathology implying
that TDC does not affect the normal tissue, but uniquely localize only in the neoplastic tissue
and damage it selectively.
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Control
Dark Control
AfterPDT
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Liver
Kidney
Figure 10. The cytomorphology of liver and cytopathological changes of kidney tissues show
the photodynamic effect of TDC (20 mg of TDC/Kg body weight of nude mice). Representative
animal sections of the liver and kidney tissues under various conditions (control, dark control
and after PDT of treatment light dose; 100 J/cm2; 600-720 nm) are shown after staining with
standard Hematoxylin & Eosin.
CONCLUSION
In conclusion, the dihydroxychlorin, TDC described herein exhibited high solubility in
biological media and excellent quantum yields of triplet excited state (0.84 ± 0.02) and singlet
oxygen generation (0.80 ± 0.03). Our in vitro photodynamic investigations have indicated that
TDC localizes selectively in neoplastic cells and the biological activity involved predominantly
ROS as the reactive intermediates. Interestingly, TDC induces a severe intracellular oxidative
photodynamic damage in ovarian cancer cells through the destruction of cellular organelles such
as cell membrane, actin cytoskeletal and mitochondria. Moreover, the absence of any detectable
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changes in the apoptotic markers such as caspase-3 clearly indicates that the mechanism of cell
death is predominantly through a necrosis pathway. Uniquely, TDC exhibited negligible dark
cytotoxicity and excellent photodynamic activity both in cell culture and nude mice animal
models. The ease of synthesis, chemical purity, aqueous solubility, high optical absorption in the
photodynamic window, efficient generation of triplet and singlet oxygen species, excellent in
vitro and in vivo photodynamic activity and non-toxic effects on non-target tissues such as liver,
kidney, spleen, heart etc makes TDC a promising sensitizer for photodynamic therapy of cancer.
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EXPERIMENTAL SECTION
General Techniques. The general techniques and equipments employed were described
elsewhere.47-50 All the compounds synthesized were purified (≥95%) through re-crystallization
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and characterized based on elemental analysis and spectral evidence. H and C NMR spectra
were measured on a 300 or 500 MHz Bruker advanced DPX spectrometer. The IR spectra were
recorded on a Perkin Elmer Model 882 infrared spectrometer. The electronic absorption spectra
were recorded on a Shimadzu UV-VIS-NIR spectrophotometer and fluorescence spectra were
recorded on a SPEX-Fluorolog F112X spectrofluorimeter. The biological properties of the
sensitizer have been investigated using a human ovarian cancer cell line SKOV-3. For the
evaluation of phototoxicity, we have used a tunable, commercially available light source,
Waldman PDT 1200L (Germany) having the emission spectrum in the range of 600-750 nm. We
have used light dose of 200 J/cm2; 50 mW/cm2 for in vitro irradiation of cells in culture and 100
J/cm2; 100 mW/cm2 for in vivo irradiation, in animal model experiments. These cell culture
studies were performed on FACS Star (Becton Dickinson, USA) and Laser Scanning
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Microscopy (Leica SP8, Germany), cytotoxicity /phototoxicity analyses were done using Elisa
Plate Reader (Molecular Devices, USA).
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Materials. 3,4-Dimethoxybenzaldehyde, pyrrole, trifluoroacetic acid, DDQ, dulbecco's
modified eagle medium (DMEM), dimethyl sulphoxide (DMSO), propidium iodide, Hoechst
dye, and RNAse were purchased from Aldrich and used as received. 1,3-Diphenylisobenzofuran
(DPBF) was recrystallized from a mixture (1:3) of methanol and acetone. SKOV-3 cells were
procured from ATCC (USA). DMEM, fetal calf serum and antibiotics, 3-(4,5-dimethyl-thiazol-
2-yl)-2,5-diphenyltetrazolium bromide (MTT) and propidium Iodide were from Sigma-Aldrich,
USA, CM-H2DCFDA and Mitotracker Red CMXRos were procured from Molecular Probes,
USA.
Chemical Synthesis. Step 1. Synthesis of 3.4-Dimethoxyphenylporphyrin. The
synthesis of the porphyrin derivative was achieved through the modified procedure of Lindsey.36
3,4-Dimethoxybenzaldehyde (0.009 mol) and pyrrole (0.6 mol) were dissolved in distilled
dichloromethane (500 mL) and trifluoroacetic acid (~0.2 mL) was added and kept stirring for ~3-
5 h at room temperature and under argon atmosphere. 2,3-Dichlorodicyanobenzoquinone (1.2
eq) was then added and the reaction was allowed to continue for 2 h more. 3,4-
Dimethoxyphenylporphyrin was purified from the reaction mixture by sequentially passing
through basic alumina and silica (100-200 mesh) column using dichloromethane as the mobile
phase. The purple colored product was collected and concentrated by vacuum drying.
Step 2. Synthesis of 5,10,15,20-Tetrakis(3,4-dimethoxyphenyl)chlorin. 3,4-
Dimethoxyphenyl-porphyrin (3.2 mmol) and p-tosylhydrazine (32 mmol) were dissolved in dry
pyridine (500 mL) and activated potassium carbonate (1.3 mmol) was added. The reaction
mixture was allowed to reflux at 120 ºC under argon atmosphere for 24 h. After 2 h, the reaction
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was monitored by UV-visible spectroscopy (the peak at 650 nm corresponding to chlorin). After
completion of the reaction, the reaction mixture was cooled and extracted using
dichloromethane. The organic layer was concentrated, dried and was poured onto a column of
alumina (50 mm diameter, 150 mm length) and eluted with dichloromethane. The solvent was
removed under reduced pressure to give a black solid, which was then subjected to column
chromatography using over silica gel. Elution of the column with chloroform gave the chlorin
derivative. Yield: (83%), mp > 300 ºC, purity ≥95%, IR (KBr): νmax 3324, 2821, 1680, 1516,
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1261, 1246, 1230, 1138, 1028 cm-1. H NMR (300 MHz, CDCl3, TMS): δ (ppm) -1.44 (s, 2H),
3.95 (s, 12H), 4.11 (s, 6H), 4.14 (s, 6H), 4.2 (s, 4H), 7.16 (t, 4H, J=15 Hz), 7.39 (d, 4H, J= 5 Hz
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), 7.67 (d, 4H, J=5 Hz), 8.24 (d, 2H, J=15 Hz), 8.48 (s, 2H), 8.64 (d, 2H, J= 5 Hz). C NMR
(125 MHz, CDCl3): δ (ppm) 206.9, 167.7, 152.6, 148.7, 147.1, 140.97, 135.6, 134.7, 131.8,
128.0, 126.6, 124.6, 123.3, 122.3, 117.6, 109.4, 77.0, 56.1.FAB-MS: m/z calculated 857.4; found
857.35 (M+).
Step 3. Synthesis of 5,10,15,20-Tetrakis(3,4-dihydroxyphenyl)chlorin (TDC). Boron
tribromide (7.4 mmol) in dry dichloromethane (10 mL) was cooled to -78 ºC and was added
5,10,15,20-tetrakis(3,4-dimethoxyphenyl)chlorin (0.3 mmol) in dry dichloromethane (10 mL),
over a period of 20 min. The mixture was stirred for 2 h at -78 ºC and then for 12 h at 25 ºC.
After cooling to
0 ºC on an ice bath, methanol was added to quench any excess of boron
tribromide.Triethylamine was then added to neutralize the reaction mixture and concentrated
under reduced pressure to give an amorphous purple solid. The solid thus obtained was
recrystallized from a mixture of methanol and chloroform to obtain dihydroxychlorin derivative,
TDC. Yield: (85%), mp > 300 ºC, purity ≥95%, IR (KBr): νmax 3187, 2694, 1577, 1525, 1512,
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1500, 1431, 1354, 1255, 1112, 935 cm-1. H NMR (300 MHz, DMSO-d6, TMS): δ (ppm) -1.43
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(s, 2H), 4.2 (s, 4H), 7.12 (t, 4H, J=20 Hz), 7.17 (d, 2H, J=10 Hz), 7.28 (s, 2H), 7.39 (d, 2H, J=10
Hz), 7. 53 (s, 2H), 8.29 (d, 2H, J=5 Hz), 8.48 (s, 2H), 8.68 (d, 2H, J=5 Hz). 13C NMR (125 MHz,
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DMSO-d6):
δ (ppm) 167.8, 152.6, 145.0, 144.6, 140.7, 137.8, 135.3, 134.4, 131.2, 127.5,
125.8, 123.7, 122.3, 121.4, 119.3, 112.1, 99.8, 48.13. FAB-MS: m/z calculated: 745.2; found
745.31 (M+).
Cell Culture Analysis. The human ovarian carcinoma cell line SKOV-3 was maintained
in DMEM containing Penicillin, Streptomycin and Gentamycin (100 units, 100 μg and 50 μg/
mL respectively) supplemented with 10 % fetal calf serum under standard culture conditions (37
ºC , 5 % CO2 and 95% humidity). The cells were sub-cultured once in two days.
Cell Cycle Analysis by Flow Cytometry. SKOV-3 cells in T-25 flasks were treated with
TDC (20 M) and either exposed to light or left unexposed, were harvested at pre-determined
times and washed in ice-cold PBS. The cells were re-suspended in PBS and fixed in 1 mL ice-
cold 70% ethanol and left overnight at 4 ºC. After another PBS wash, cells were stained with
propidium iodide (60 μg/mL) and DNAse free ribonuclease A (0.2 mg/mL) and incubated for 30
min at 37 ºC. The cells were analyzed for propidium iodide fluorescence using a flow cytometer
equipped with an excitation laser at 488 nm. The cell cycle analysis was done as per the standard
procedure and the percent population of cells representing different phases of cell cycle (by
gating cells using width and area parameters) was estimated.
Flow Cytometric Analysis of Live SKOV-3 Cells Stained with Propidium Iodide.
Propidium iodide being membrane impermeant dye is excluded by living cells, but readily enters
dead and membrane-compromised cells and emits a strong fluorescent signal when bound to
DNA. SKOV-3 cells were subjected to different treatments as described, harvested and washed
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with phosphate buffered saline (PBS) and stained with propidium iodide (10 g/mL) for 20 min
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and analyzed by flow cytometry.
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Mitochondrial Membrane Potential. Control cells and cells after PDT were treated
with 200 nM of Mitotracker Red CMXRos and incubated at 37 ºC for 30 min. The cells were
then fixed in formaldehyde, counterstained with DAPI and visualized by confocal microscopy.
Nude Mice Xenograft Models of Ovarian Cancer. All experiments were carried out in
accordance with the guidelines of Institutional Animal Ethics Committee (CCMB, Hyderabad,
India). Approximately 3-4 million SKOV-3 cells were injected subcutaneously into the flank
region of 6-8 week old FoxN1 nude mice (20-25 g). Tumor dimensions were measured using a
digital calipers and the volumes were calculated using the formula V=LW2/2 (where V= volume;
L= length of the tumor; W=width of the tumor). When tumor volumes reached approximately
100 -150 mm3, the mice were randomly divided into four groups of six animals each, the
untreated control group, the light control group (only exposed to light), the dark control group
(TDC was administered, but not exposed to light) and the PDT group (administration of TDC
followed by irradiation for 24 h).
TDC was administered intravenously (dark control group and PDT group) at 20 mg/Kg
body weight. The animals (PDT group) were subjected to irradiation 24 h after administration of
TDC. For animal experiments, the irradiation parameters were 100 J/cm2 and 100 mW/cm2.
Prior to irradiation, the nude mice were anesthetized with ketamine (60 mg/Kg) and xylazine (8
mg/Kg) given intraperitoneally. Only the tumor region was exposed to light while the rest of the
animal was shielded. Tumor growth was assessed by monitoring its volume. For histopathology
studies, animals were euthanized according to IAEC protocols. Tumor, liver, spleen kidney,
heart and lung were collected, fixed in buffered formalin, dehydrated and paraffin-embedded.
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The tissues were sectioned at 5 m thickness and stained with Hematoxylin and eosin A
contiguous section was used to study the intratumoral localization of TDC.
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Staining of F-actin. Untreated dark control or photodynamically treated SKOV-3 cells
(grown on cover slips) were fixed with 4% paraformaldehyde in phosphate buffered saline
washed and permeabilized with 0.1% Triton X100. Following the permeabilization, cells were
treated with TRITC-Phalloidin (Sigma-Aldrich, USA) at a dilution of 1:50 for 1 h. Finally, the
cells were counterstained with DAPI and visualized by confocal microscopy.
Uptake and Intracellular Localization of TDC by SKOV-3 Cells. SKOV-3 cells,
grown in chambered glass slides were treated with 20 μM TDC for 1 h. The slides were then
rinsed with phosphate buffered saline (PBS), treated with Hoechst 33342 (1 μg/mL) and
visualized by confocal microscopy. TDC show red fluorescence and the nuclei appear blue.
ASSOCIATED CONTENT
Supporting Information Available: General methods, quantification of triplet states and
singlet oxygen quantum yields, Figures S1-S2 and Table S1 showing the photophysical and
photobiological properties of TDC are summarized. Molecular formula strings file uploaded
separately. This material is available free of charge via the internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
D. Ramaiah: ORCID # 0003-1388-4768
*E-mail: rama@neist.res.in or d.ramaiah@gmail.com.
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Notes
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The authors declare no competing financial interests.
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ACKNOWLEDGMENTS
The authors acknowledge the financial support from the Department of Biotechnology
(BMB/2015/53) and Department of Science and Technology (SR/S5/MBD-01C/2007),
Government of India. CMR and TKC acknowledge the DST, Sir JC Bose National Fellowship.
The authors acknowledge the help by Dr. Jerald Mahesh Kumar, Veterinary Pathologist and
Officer-in-charge, Animal House Facility, CCMB, for his inputs in histopathological analyses.
AUTHOR CONTRIBUTIONS
Dr. B. Marydasan and Dr. B. Madhuri have contributed equally to this manuscript. Dr. B.
Marydasan, Dr. M. Viji, Dr. S. C. Karunakaran, Dr. T. K. Chandrashekar and Dr. D. Ramaiah,
participated in the synthesis and photophysical characterization of TDC, while Dr. B. Madhuri, J.
Jose, S. Cherukommu, Dr. Ch Mohan Rao and Dr. K Sridhar Rao, participated in evaluation of
photobiological properties and animal experimentation. Dr. K. Sridhar Rao, Dr. Ch. Mohan Rao
and Dr. Ramaiah are also involved in collation of data, data analysis and manuscript preparation.
ABBREVIATIONS USED
PDT, photodynamic therapy; NIR, near-infrared; HPD, hematoporphyrin derivative;
TDC, 5,10,15,20-tetrakis(3,4-dihydroxyphenyl)chlorin; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide; PI, propidium iodide; DAPI, 4',6-diamidino-2-phenylindole;
DPBF, diphenylisobenzofuran; TRITC, tetramethylrhodamine; ROS, reactive oxygen species.
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REFERENCES
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