The in vitro photodynamic activity, photophysical and photochemical research of a novel chlorophyll-derived photosensitizer

Article abstract of DOI:10.1007/s00044-017-1962-2

Chlorophyll has always been used as the leading compound for photodynamic therapy drug development. In this paper, a novel methyl pyropheophorbide-a- derived photosensitizer, 3-acetyl-3-devinyl-13¹-dicyanomethylene-pyropheophorbide-a was synthesized through modifications at C-13¹, C-3, and C-17 positions of methyl pyropheophorbide-a. The compound exhibited a longer wavelength absorption at 713 nm (in methanol) than that of methyl pyropheophorbide-a (667 nm) due to the enlarged the aromatic conjugation system by dicyanomethylene, allowing it to be potential in deep tumor treatment. Moreover, benefiting from the carboxylic group at C-17 and the acetyl group at C-3, the title compound was endowed with better water solubility than that of methyl pyropheophorbide-a. Detailed in vitro photodynamic therapy research showed ADCPPa could be uptaken by cancer cells successfully and killed the cancer cells more efficiently than the leading compound methyl pyropheophorbide-a under light (light dose 10 J/cm²) due to the high singlet oxygen quantum yield (65.98%). The excellent anti-photobleaching ability (degradation rate 1.6% in 10 min) also boosted its potential in practical application. In addition, the research has disclosed that during photochemical processes of photodynamic therapy, the formation of singlet oxygen after photodynamic therapy treatment played a major role, comparing with the formation of superoxide anion and radicals. Finally, the real time quantitative polymerase chain reaction (RT-qPCR) experiments have showed that the target compound has important regulating effect on expression of CDK2 and Survivin, consequently leading to apoptosis and cell death.

Full text of DOI:10.1007/s00044-017-1962-2

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-017-1962-2
ORIGINAL RESEARCH
The in vitro photodynamic activity, photophysical and
photochemical research of a novel chlorophyll-derived
photosensitizer
1 ●
2 ●
2 ●
2 ●
2 ●
Guanghui Tan Qi Wang Hongyue Zhang Jianjun Cheng Zhiqiang Wang
Fengyu Qu
1
,2
1 ●
2
●
Changhong Guo Yingxue Jin
Received: 22 February 2017 / Accepted: 14 June 2017
Springer Science+Business Media, LLC 2017
©
Abstract Chlorophyll has always been used as the leading
compound for photodynamic therapy drug development. In
this paper, a novel methyl pyropheophorbide-a- derived
processes of photodynamic therapy, the formation of singlet
oxygen after photodynamic therapy treatment played a
major role, comparing with the formation of superoxide
anion and radicals. Finally, the real time quantitative
polymerase chain reaction (RT-qPCR) experiments have
showed that the target compound has important regulating
effect on expression of CDK2 and Survivin, consequently
leading to apoptosis and cell death.
1
photosensitizer, 3-acetyl-3-devinyl-13 -dicyanomethylene-
pyropheophorbide-a was synthesized through modifica-
1
tions at C-13 , C-3, and C-17 positions of methyl
pyropheophorbide-a. The compound exhibited a longer
wavelength absorption at 713 nm (in methanol) than that of
methyl pyropheophorbide-a (667 nm) due to the enlarged
the aromatic conjugation system by dicyanomethylene,
allowing it to be potential in deep tumor treatment. More-
over, benefiting from the carboxylic group at C-17 and the
acetyl group at C-3, the title compound was endowed
with better water solubility than that of methyl
pyropheophorbide-a. Detailed in vitro photodynamic ther-
apy research showed ADCPPa could be uptaken by cancer
cells successfully and killed the cancer cells more efficiently
than the leading compound methyl pyropheophorbide-a
●
Keywords Photodynamic therapy Methyl
●
●
pyropheophorbide-a Dicyanomethylene Antitumor
Introduction
Photodynamic therapy (PDT) is a clinically approved and
minimally invasive therapeutic procedure to cancer treat-
ment because of the low toxicity without light irradiation,
the selective toxicity to the multiple lesions upon irradiat-
ing, and the ability to retreat a tumor during various ther-
apeutic methods (Cabuy 2012). PDT involves the
administration of a photosensitizer (PS) followed by illu-
mination of the tissue with visible light. After administra-
tions of a drug and upon the absorption of appropriate light,
2
under light (light dose 10 J/cm ) due to the high singlet
oxygen quantum yield (65.98%). The excellent anti-
photobleaching ability (degradation rate 1.6% in 10 min)
also boosted its potential in practical application. In addi-
tion, the research has disclosed that during photochemical
*
*
*
Fengyu Qu
quprof@163.com
a PS is excited from ground state (S ) to the first excited
0
Changhong Guo
guoprof@163.com
state with the same spin multiplicity (S ). Then, the excited
1
PS can decay to the S state by losing its energy through
0
Yingxue Jin
emitting fluorescent light or converting into heat by
radiation-less internal conversion. Alternatively, the first
jyxprof@163.com
1
2
excited state S can be converted into electronic spin triplet
College of Life Science and Technology, Harbin Normal
University, Harbin 150025, China
1
(T ). From T state, the PS can return to the S state by
1
1
0
emitting phosphorescence. If this state (T ) has a sufficient
College of Chemistry & Chemical Engineering, Harbin Normal
University, Harbin 150025, China
1
long life and the process occurs in the presence of molecular

Med Chem Res
oxygen or water, it can generate superoxide anions and
PSs have attracted wide attention for medical application.
The natural porphyrins (chlorins) in plants, chlorophylls, are
extremely important biomolecules in photosynthesis, which
allow plants to absorb solar radiation, converting light
energy into chemical energy. The four pyrrole subunits
interconnected via methane bridges construct a conjugated
18-π electron macrocycle, which gives rise chlorophyll
strong absorption at the range of 600–700 nm and high
single oxygen quantum yield, making them ‘hot’ molecules
in PS research (Liu et al. 2015; Zhang et al. 2014; Asano
et al. 2013).
radical intermediate species (type I reaction) or transfer
3
energy to the oxygen ( O ) to produce active singlet oxygen
2
1
(
O ) (type II reaction) (Fig. 1) (Cabuy 2012; Phillips
2
2
010), which are responsible for damage to cancer cells. It
is generally accepted that type I reaction and type II reaction
1
can occur simultaneously and O generated from a type II
2
reaction is the principal cytotoxic component in PDT
(
Noimark et al. 2013).
Despite the advantages of PDT (minimum side effects,
low toxicity to tissues without light, non-invasive therapy
and high selectivity), its application in clinic is always
restricted to peripheral and endoscope accessible tissues
Because of inhomogeneous electric charge distribution
of the multi-π aromatic system caused by the asymmetrical
chemical structure of chlorophylls, and the active chemical
properties resulted from the exocyclic nonaromatic unsatu-
rated substituents, such as carbonyl or vinyl group, chlor-
ophylls are moderately easy and feasible for structural
modifications. The variety of chlorin derivatives can be
obtained by modifying peripheral substituted group, and C-
3, 5, 7, 10, 12, 13, 17 and 20 positions are highly active
reactive sites and are easily modified for development of
new PSs (Li et al. 2015; Smith et al. 1985; Han et al. 2001;
Li et al. 2016, 2014; Staron et al. 2015). Generally, good
PSs in PDT should possess large absorption wavelength and
rational water-soluble ability for practical use. Porphyrinic
methyl pyropheophorbide-a (MPPa), which is a semisyn-
thetic PS derived from chlorophyll a, has been used as the
leading compound in many structural modifications of
medicinal chemistry, due to the advantages of long
absorption wavelength (>667 nm), low dark cytotoxicity
and high singlet oxygen quantum yield (Eichwurzel et al.
2000; Sun and Leung 2002).
(
2
Agostinis et al. 2011; Lu et al. 2014; Sosnik and Carcaboso
014; Volgger and Betz 2016), because PDT for deep
tumors is limited by the tissue penetration depth of excita-
tion light (Yoon et al. 2014; Voon et al. 2014). Generally,
the reasonable tissue penetration depth is achieved with
irradiation light wavelengths in the range of 650–900 nm
(
NIR window) (Hu et al. 2015). It is acknowledged that
developing the PS with longer wavelength of maximum
absorption is of significance for treatment of deep tumor, for
light with longer wavelength will penetrate deeper into
tissues than the light with shorter wavelength (Payne et al.
1
996).
Many researchers have focused on developing novel PS
derivatives with large absorbance in the NIR window to
fight against deep tissue cancers (Wan et al. 2014; Guo et al.
2
014; Tuncel et al. 2013; Yang et al. 2013). Currently, the
most common PSs in clinic are porphyrins and their deri-
vatives, such as the first generation PSs hematoporphyrin
derivative (HpD) and Photofrin (with a maximal absorption
at 630 nm); the second generation PSs Foscan, Npe6, Lutex,
Verteporfin, Purlytin and Tookad (O’Connor et al. 2009).
Because of the low dark cytotoxicity, being prone to
accumulate in tumor lesions, strong absorption around the
NIR window and high singlet oxygen yield, the porphyrin
1
In the present study, a MPPa-derived PS, 3-acetyl-13 -
dicyanomethylene- pyropheophorbide-a (ADCPPa) was
1
synthesized through modification at C-13 position of Mppa
with dicyanomethylene to extend the absorption maximum
in the near-infrared spectral region (Qy absorption),
Fig. 1 Photochemical process
and the bioaction mechanism of
photosensitizer

Med Chem Res
allowing the PS to be potential in deep tumor treatment.
cells after drug treatment, were all investigated to evaluate
the title compound in support of PDT. In addition, the
photochemical mechanism in PDT was investigated by
using D-mannitol (DM) and sodium azide (SA) as
quenching agents of reactive oxygen species (ROS).
Finally, by using real time quantitative polymerase chain
reaction (RT-qPCR), the expression of CDK2 and Survivin
in drug-treated HeLa cells were investigated to explore the
PDT action of ADCPPa.
(
Scheme 1) (Vail et al. 2014). Meanwhile, the methyl pro-
pionate moiety at C-17 position was hydrolyzed into pro-
pionic acid group, and the vinyl group at C-3 group was
oxidized into acetyl group, both leading to a better water
solubility. Although modification by dicyanomethylene at
1
3
C
position has been reported (Wang et al. 2012), yet those
C-17 ester-hydrolyzed compounds of dicyanomethylene-
modified derivatives have hot been reported, and the
detailed photophysical and photochemical properties, the
antitumor activities and photochemical mechanism have not
been investigated systematically. In the present paper, a
comprehension research for ADCPPa, including the
UV–Vis and fluorescence spectral properties, the singlet
oxygen yield, the anti-photobleaching ability, the in vitro
photocytotoxicity against HeLa cells, the cancer cell
uptaking behavior, and the morphological changes of cancer
Materials and methods
General remarks
Chlorophyll paste was bought from Shandong Guangtong-
bao Pharmaceuticals Co., Ltd (Qingzhou city, Shandong,
Scheme 1 Synthesis of
ADCPPa: a 30% HBr (in acetic
acid); b LiOH, 4-
methylmorpholine-N-oxide,
tetrapropylammonium
perruthenate; c dicyanomethane,
triethylamine

Med Chem Res
China). SA and DM were bought from Sinopharm Chemi-
cal Reagent Co. Ltd (Shanghai, China). Dulbecco’s mod-
ified eagle medium, penicillin, fetal bovine serum (FBS),
and streptomycin were purchased from Beijing Dingguo
Biotechnology Co (Beijing, China). Dimethyl sulfoxide and
(s, 3H, 2-CH ), 3.38 (s, 3H, 12-CH ), 3.58 (s, 3H, 17-OCH ),
3
3
3
3.61~3.68 (m, 8-CH ), 4.18~4.23 (m, 1H, 17-CH), 4.23~4.44
2
2
(m, 1H, 18-CH), 5.06 (d, J = 17.2 Hz, 1H, 13 -CH ), 5.26 (d,
2
2
1
J = 19.6 Hz, 1H, 13 -CH ), 6.31~6.39 (m, 1H, 3 -CH), 8.49
2
(s, 1H, 20-H), 9.34 (s, 1H, 10-H), 9.61(s, 1H, 5-H).
3
-(4,5-dimethylthiazol-2-yl)-2,5-dipheny-ltetrazolium bro-
mide were purchased from Sigma (Shanghai, China).
Phosphate buffered saline purchased from Invitrogen
Synthesis of 3-acetyl-3-devinylpyropheophorbide-a
(APPa)
(
Beijing, China). Phosphate-buffered saline (PBS) used was
prepared by mixing stock solutions of NaH PO and
HMPPa (28 mg, 0.05 mmol) was dissolved into 20 mL of
25% methanol–water solution. Add 5 mL of 2 M LiOH
aqueous solution into the above mixture, which was then
stirred for 2 h under reflux conditions. After cooling, adjust
the pH value of the reaction mixture to 5 with 2 M HCl aq.
Then extract with 60 mL dichloromethane for three times.
The organic layer was dried and concentrated to remove the
solvent. The residue was dissolved in 4 mL dried dichlor-
omethane, and 4-methylmorpholine N-oxide (5 mg) was
then added. The mixture was stirred for 30 min, then add
tetrapropylammonium perruthenate in batches (11 mg).
After stirring for 3 h at r.t., 30 mLwater was added. The
organic layer was dried by anhydrous Na SO and then
2
4
Na HPO . RNApure Tissue Kit (DNase I) and Prime-
2
4
Script™ 1st strand cDNA Synthesis Kit were bought from
®
Tiangen Biotech (Beijing) Co. Ltd; SYBR Premix Ex
Taq™ (Tli RNaseH Plus) was bought from TAKARA
Biotechnology (DaLian) Co., Ltd.
All the chemical reactions were performed away from
1
sunshine under nitrogen atmosphere. H-NMR (400 MHz)
1
3
and C-NMR spectra (100 MHz) were recorded on an
AMX400 spectrometer (Bruker, Bremen, Germany) with
tetramethylsilane as an internal standard. Mass spectra were
recorded with a Hitachi VG-7070 spectrometer. UV–Vis
−
5
absorption in methanol (2.15 × 10 M) in the 300–800 nm
2
4
range were recorded using LAMBDA 25 spectrometer
concentrated. The residue was purified by gel column
chromatography separation with benzene/acetone (8:1) to
give a brownish-green solid (80%).
(
PerkinElmer). The emission spectra were recorded using
spectrofluoro-photometer with a 150 W xenon lamp as a
visible excitation light source (RF-5301PC, Shimadzu). All
spectra were obtained in a quartz cuvette (path length =
APPa
1
cm). The excitation and emission slit widths were both
1
0 nm, and photomultiplier tube voltage of 700 V.
UV–Vis (CH OH) λmax: 414, 513, 548, 623, 683; 1H-NMR
3
(
CDCl ): -2.07 (s, 1H, NH), -0.11 (s, 1H, NH), 1.69 (t, J =
3
Synthesis of 3-(1-hydroxyethyl)-3-
devinylmethylpyropheophorbide-a (HMPPa)
7.8 Hz, 8-CH ),1.78 (d, J = 7.2 Hz, 3H, 18-CH ), 2.20–2.25
3 3
2
1
(m, 2H, 17 -CH ), 2.59–2.77 (m, 2H, 17 -CH ), 3.20 (s,
2 2
1
3
H,3 -CH ), 3.26 (s, 3H, 1-CH ), 3.60–3.68 (m, 8H, 7-CH ,
3 3 3
According to the literature (Zhang et al. 2016), MPPa (78
mg, 0.14 mmol) was dissolved in 10 mL of 33% hydro-
bromic acid–ethanol solution, and stirred for 5 h at 50 °C.
The reaction mixture was then concentrated under vacuum
condition to remove the solvent. Then 30 mL of water was
added and the mixture was extracted with 30 mL dichlor-
omethane for three times. The organic layer was washed by
saturated sodium chloride solution, dried by anhydrous
Na SO , and concentrated to remove the solvent. The pro-
12-CH , 8-CH2), 4.34–4.36 (m, 1H,17-CH), 4.53–4.57 (m,
3
2
1H, 18-CH), 5.08 (d, J = 19.6 Hz, 1H, 13 -CH ), 5.26 (d, J
2
2
= 19.6 Hz, 1H, 13 -CH ), 8.77 (s, 1H, 20-H), 9.49 (s, 1H,
10-H), 9.93 (s, 1H, 5-H); C NMR(CDCl ): 11.1, 12.0,
2
1
3
3
13.2, 17.3, 19.4, 23.3, 29.3, 31.0, 33.4, 48.1, 49.2, 51.6,
94.3, 100.2, 103.5, 106.7, 129.5, 131.3, 134.1, 135.3,
137.3, 138.9,139.1, 144.8, 148.7, 151.8, 154.9, 161.3,
+
170.1, 173.3, 195.9, 199.2, 206.7; FAB-MS: 550 (M );
anal calcd for C H N O : C, 71.98; H, 6.22; N, 10.17;
2
4
33 34 4 4
duct was purified by gel column chromatography separation
with ethyl acetate/methanol (5:1) to give a bluish-green
solid (60%).
found C, 72.11; H, 6.30; N, 10.03;
Synthesis of the title compound ADCPPa
Dissolve APPa (25 mg, 0.05 mmol) in a mixture of ethanol
HMPPa
(2 mL) and dichlomethane (4 mL), then add dicyano-
1
UV–Vis(CH OH) λ : 405, 502, 542, 601, 664; H NMR
methane (110 mg, 1.65 mmol) and triethylamine (0.2 mL)
into the mixtures, which were stirred for 3 h at 85 °C. After
the reaction mixtures were cooled, pour them into 30 mL
water, and extract with 25 mL dichloromethane for three
times. The organic layer was dried by anhydrous Na SO
4
3
max
(
CDCl ) δ: -1.91(s, 1H, NH), -0.12 (s, 1H, NH), 1.67 (t, J =
3
7
=
.6 Hz, 8-CH ), 1.76 (d, J = 8.4 Hz, 3H, 18-CH ), 2.11 (d, J
3 3
2
6.4 Hz, 3H, 3 -CH ), 2.21~2.26 (m, 2H, 171-CH ),
3 2
2
2.60~2.71 (m, 2H, 17 -CH ), 3.21 (s, 3H, 7-CH ), 3.37
2 3
2

Med Chem Res
and concentrated. The residue was separated by gel column
chromatography with benzene/acetone (10:1) to give a dark
green solid (60%).
where A(sample) represents A values of the wells treated
with ADCPPa, and A(control) represents those of the wells
treated with DMEM + 10% FBS.
ADCPPa
Cellular uptake test
1
UV–Vis (CH OH) λmax: 403, 534, 570, 654, 713; H-NMR
Drug uptaking behavior in HeLa cancer was performed by
Leica DM IL LED fluorescent inverted microscope (FIM).
HeLa cells were incubated for 24 h. After discarding the
medium, cells were rinsed with PBS and treated with 20 μg/mL
ADCPPa for 15, 30, 45, 60 and 180 min, respectively.
Then, glutaraldehyde solution (1 mL, 2.5%) was used to fix
the cells (10 min, 37 °C). Then the glutaraldehyde solution
was removed and the cells were rinsed extensively with
PBS, subsequently stained with 1 mL of 1 μg/mL DAPI
probe for 10 min
3
(
CDCl ): 1.55–1.59 (t, J = 7.8 Hz, 8-CH ), 1.77 (d, J = 7.2
Hz, 3H, 18-CH ), 2.05–2.13 (m, 2H, 17 -CH ), 2.20–2.26
m, 2H, 17 -CH ), 2.86 (s, 3H, 3 -CH ), 3.00 (s, 3H, 7-
2 3
CH ), 3.10 (s, 3H, 2-CH ), 3.24 (s, 12H, 2-CH )3.39–3.45
m, 2H, 8 -CH ), 4.04–4.06 (m, 1H,17-CH), 4.23–4.27 (m,
2
H, 18-CH), 5.10–5.27 (dd, J = 20.8 Hz, 2H, 15 -CH ),
.20 (s, 1H, 20-H), 8.68 (s, 1H, 10-H), 9.05 (s, 1H, 5-H);
C NMR(CDCl ): 11.1, 12.0, 13.2, 17.3, 19.4, 23.3, 29.3,
3
3
1
3
2
2
1
(
3
3
3
1
(
1
1
8
2
1
3
3
3
1
1
1
1.0, 33.4, 48.1, 49.2, 51.6, 94.3, 100.2, 103.5, 105.0,
10.0, 116.0, 116.4 106.7, 129.5, 131.3, 134.1, 135.3,
37.3, 138.9,139.1, 144.8, 148.7, 151.8, 154.9, 161.3,
Morphological changes of HeLa cells after PDT
+
70.1, 173.3, 195.9, 199.2, 206.7; FAB-MS: 598 (M );
anal calcd for C H N O : C, 72.22; H, 5.72; N, 14.04;
found C, 72.16; H, 5.88; N, 13.94.
Morphological changes of HeLa cells after treatment of
ADCPPa were also analyzed by FIM. HeLa cells were
incubated for 24 h, the medium was then removed. The cells
were rinsed with PBS, and treated with 20 μg/mL ADCPPa
for 4 h, followed by exposure to infrared light (675–780 nm,
10 min, 10 J/cm ). The morphological changes were
observed after 1, 3, 6, 12 and 24 h, respectively, comparing
with the cells without light treatment.
3
6 34 6 3
Cell culture and in vitro cytotoxicity
2
The human cervical cancer cell line (HeLa) was cultured in
Dulbecco’s modified Eagle’s medium (DMEM, Gibco)
supplemented with 10%(v/v) FBS and 1% antibiotic
(
100 μg/mL penicillin–100 μg/mL streptomycin, Life
Technologies, USA) in an incubator (5% CO , 98%
Photochemical mechanism of ADCPPa in PDT
2
humidity, 37 °C). The cytotoxicity was investigated by
methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay.
HeLa cells were divided into experimental group (ADCPPa
group, L+) treated with different concentrations of
ADCPPa and exposed to light, and control group (blank
group, L-) treated with drugs without light.
SA and DM could selectively quench oxygen-free radicals
1
and singlet oxygen ( O ), respectively, making them inop-
2
erative. In this section, the cells were divided into three
groups: (1) L+ groups: treated with different concentrations
of ADCPPa and exposed to light; (2) SA groups: treated
with ADCPPa and SA (20 mmol/L), and exposed to light;
(3) DM groups: treated with ADCPPa and DM (40 mmol/
L), and exposed to light. Precisely, HeLa cells were incu-
bated in DMEM 24 h as described above. Hundred micro-
liters of different concentrations of sample (2.5, 5, 10, 15,
20, 25 and 30 μg/mL) were added to each well of L+ group,
SA groups and DM groups. To SA groups was added 20 μL
SA and to DM groups was added 40 μL DM. Then incu-
bated for 4 h followed by exposure to light for 10 min. Cell
viability was determined as previously mentioned.
Precisely, HeLa cells were seeded in a 96-well plate with
4
an initial density of 1 × 10 cells per well and incubated for
2
4 h (37 °C, 5% CO ). Then HeLa cells in the experimental
2
groups and the control groups were treated with different
concentrations of ADCPPa (2.5, 5, 10, 15, 20, 25 and 30 μg/
mL, respectively). Then the cells in the experimental groups
were incubated for 4 h followed by exposure to visible light
for 10 min, and then cultured in dark for additional 24 h in
DMEM as described above, while the control group was
incubated for the same time without light. Then MTT dyes
(
100 μL, 0.5 mg/mL) were added to the wells, and incubated
for another 4 h. Remove the MTT solutions and dissolve the
formazan crystals with 100 μL of DMSO. Finally, the
absorbance of each probe was read on a microplate reader at
Singlet oxygen quantum yield
In this paper, diphenylisobenzofuran (DPBF) was used as a
1
4
90 nm. Cell viability (%) was calculated according to the
O trapping reagent. Typically, 3 mL of a stock solution of
2
following equation:
DPBF in DMF (60 μM) was added 50 μL ADCPPa solution
(
2.8 μM), then the mixture was placed in a sealed quartz
Cell viability ð%Þ ¼ AðsampleÞ=AðcontrolÞ ꢀ 100%;
cuvette. A 5 mW Nd:YAG laser (675–780 nm) was used as

Med Chem Res
the light source to excite the sensitizer. The absorbance of
the solution at 415 nm was measured every 10 s for a 120 s
period with UV–Vis spectrophotometer. The decrease of the
absorbance resulted from DPBF was measured and cor-
Results and discussions
Synthesis and spectral properties
1
rected. The Singlet oxygen ( O ) quantum yield (Φ ) of
The inhomogeneous electric charge distribution of the
multi-π aromatic system caused by the asymmetrical che-
mical structure of MPPa lets it have many active sites in
periphery. The exocyclic nonaromatic unsaturated sub-
stituents, such as carbonyl or vinyl group are not involved
in the conjugation of multi-π aromatic system (Ivanov and
Boldyrev 2014), possessing the typical chemical char-
acteristics of carbonyl or vinyl group, based on which
structural modifications of MPPa are moderately feasible.
The Qy peak of MPPa in UV–Vis spectrum was at 667 nm.
In order to redshift the Qy peak (long wavelength absorp-
tion) and to improve penetration depths, strong electron-
withdrawing group dicyanomethylene moiety was intro-
2
Δ
ADCPPa is calculated using the following formula (Zimcik
et al. 2007):
S
R
Φ ðSÞ ¼ Φ ðRÞk I ðRÞ=k I ðSÞ
ð1Þ
ð2Þ
Δ
Δ
aT
aT
À
Á
ꢁ
2:3A
Ia ¼ I0 1 ꢁ e
:
Superscript S and R indicate the sample and DPBF,
respectively. I is the total amount absorbed light of the PS.
a
A is the absorbance at irradiation wavelength. A and A are
0
t
the absorbances of the DPBF at 415 nm before irradiation
and after irradiation time t, respectively, and k is a slope of a
plot of the dependence of ln(A /A ) on irradiation time t. The
1
duced on the 13 -position to expand the conjugation system
of the macrocycle. Scheme 1 shows the synthesis process of
ADCPPa. At first, the vinyl group at C-3 was converted into
hydroxyl group through electrophilic addition reaction in
hot HBr-acetic acid solution, meanwhile the ester group at
C-17 was hydrolyzed into carboxylic group. The obtained
intermediate HMPPa was oxidized by tetrapropyl ammo-
nium perruthenate to obtain the APPa, which reacted with
dicyanomethane under alkaline conditions to give the target
compound ADCPPa. As expected, the introduction of
dicyanomethylene moiety brought about obvious red shift
of its long wavelength absorption, showing Qy peak at 713
nm, with a 46 nm red-shift comparing with MPPa (667 nm)
(Fig. 2) Moreover, ADCPPa showed excellent fluorescence
characteristics, displaying a fluorescence emission peak at
near-infrared 723 nm under the excitation of 449 nm, which
means that the title compound has the prospects for tar-
geting fluorescence imaging in tumor diagnosis, because the
red fluorescence emitted by ADCPPa under light makes the
target cells more palpable (Silva et al. 2013).
0
t
1
O yield during the photochemical process was obtained
2
by a first-order linear least-squares model using the natural
logarithm values of DPBF absorption at 415 nm plotted
1
against the irradiation time. The O yield of ADCPPa was
2
calculated with methylene blue (MB) as a standard. The
slope for MB and ADCPPa was obtained after fitting with a
linear function.
Fluorescence quantitative polymerase chain reaction
(
PCR)
HeLa cells in logarithmic growth phase were seeded in a
5
six-well plate with an initial density of 1.5 × 10 cells per
well and incubated for 24 h (37 °C, 5% CO ). Then
2
ADCPPa (30 μg/mL) was added per well and incubated for
4
h and then exposed to light for 10 min. Then the cells
cultured with different time (0, 6, 12, 24 h) were collected
for later use. The total RNA extraction and cDNA synthesis
was performed in the usual way according to the literatures
(
Ramalho et al. 2004; Williams et al. 1992). The reverse
Photobleaching of ADCPPa
transcription (RT) reaction was carried out at 42 °C for 45
min, then RNase inhibitors were inactivated at 95 °C for 5
min. The fluorescence quantitative PCR was performed by
The photobleaching is an important property of a PS, and it
is a common phenomenon during PDT. Generally, PS will
interact with light to generate ROS, and can be reused or
recycled during PDT without being consumed. However,
during the same period, PS will suffer from serious of
photochemical reaction process, such as photodecomposi-
tion, photoionization, photoassciation, photoisomerization
or photo-oxidation/reduction, all of which will consume the
PS and cause the structural change of PS, leading to a
decrease of PDT effect (Yu et al. 2001). This is known as
photobleaching. Due to the negative effect of photo-
bleaching to PDT effect, it is necessary to determine to the
photobleaching rate of PS. For the irradiation wavelength
®
SYBR Premix Ex Taq™ (Tli RNaseH Plus) according to
the instruction using gene-specific primers and the internal
control GADPH primers. Precisely, 2 μL of cDNA solution
was added to 20 μL of PCR mixture (SYBRPremix Ex Taq
(
Tli RNaseH Plus) containing 10 μM each primer (Forward
primer and Reverse Primer) and ROX Reference Dye (50×)
for specific amplification as the follows: 40 cycles of
denaturation
(
95 °C for 30 s), primer annealing (56 °C for 30 s), and
extension (72 °C for 1 min). PCR products were analyzed
by electrophoresis in 1.5% agarose gel.

Med Chem Res
Fig. 2 The absorption and
fluorescence spectrum of
ADCPPa in CH OH
3
it was activated by an appropriate wavelength of light in the
presence of oxygen, producing ROS, which start a cascade
of molecular and biochemical events resulting in cell death
via apoptotic or necrotic mechanisms (Mroz et al. 2011;
Buytaert et al. 2007). Hence, prior the cytotoxicity research,
a research on the interaction of PS with plasma membrane
for cellular uptaking is needed. Most of the porphyrin PSs
are localized in mitochondria and cell nucleus. In the pre-
sent uptaking test, the nucleus was firstly stained by a DAPI
dye solution, after which the nucleus can be easily observed
due to the blue fluorescence. If the PS could be uptaken
successfully by cells, the corresponding red fluorescent
emitted from PS (such as ADCPPa) would make the cells
palpable. In the experimental group, the cells were incu-
bated with 20 μg/mL ADCPPa for 15, 30, 45, 60 and 180
min, respectively. Then cell imaging was observed on a
FIM.
As shown in Fig. 4, the cells in control group without
DAPI dye and light showed gray white without any changes
over the time, while in the experiment group after treatment
with ADCPPa and light, the cell nucleus showed obvious
blue fluorescence, and the brightness was increasing. After
incubation with ADCPPa for 15 min, weak red fluorescence
was observed from many of the cells, suggesting that the PS
began to enter the cells. After 30 min incubation, the
brightness of red fluorescence enhanced significantly, and
the signal intensity was stronger than that observed at 15
min. After incubation for 1 h, the red fluorescent signal can
be detected from almost all cells, and the signal intensity
was stronger. Longer incubation showed minor change,
suggesting that cell uptake has reached a point of saturation.
The above analysis has proved that ADCPPa can be suc-
cessfully and quickly uptaken by Hela cells, implying that
the PS could take effect more quickly in practical
application.
Fig. 3 Time dependence of the intensities of absorption maxima at
81 nm (20 °C, 20 μM) in PBS
6
used to excite the sensitizers was about 675–780 nm from a
Nd:YAG laser (675–780 nm), we here determine the time
dependence of the intensities of absorption maxima at 681
nm (20 °C, 20 μM) in PBS. The data on the time depen-
dence of absorption maxima is summarized in Fig. 3. At
2
0 °C, the photobleaching of band at 681 nm shows a gentle
and flat manner. Especially, at the stage of illumination time
less than 10 min, the decrease of intensity of absorption
maxima is approximately 1.6%, not significant. Even
though after extending the illumination time to 60 min, the
decrease of intensity of absorption maxima is about 9%. As
mentioned earlier, the PS could achieve good PDT effect
after being exposed to light for 10 min, therefore the pho-
tobleaching has a little influence on PDT effect of ADCPPa.
Cellular uptake
PSs should be able to enter the cells before taking effect,
hence PDT drugs need to be lipophilic to pass through
biomembranes, and reach subcellular sites for further oxi-
dative damage and subsequent cell-destroying (Bonnett and
Martínez 2002). Generally, PSs travel intravenously as
complexes of serum proteins (Wang et al. 2011) and are
taken up preferentially by rapidly proliferating tissue, such
as tumor tissue (Allison et al. 2008; Nowis et al. 2005), then
Photodynamic activities
A high photocytotoxicity within the aerobic tissues and low
dark toxicity are important for PS. In this part, Hela cells

Med Chem Res
Fig. 4 Fluorescence-inverted microscopic images of cellular uptake
test: HeLa cells were incubated with ADCPPa (1 mL, 20 μg/mL) at
stained with DAPI (blue), the red fluorescence was due to ADCPPa
(color figure online)
3
7 °C for 15, 30, 45, 60 and 180 min, respectively. The nucleus was
were used to detect the anticancer activity of our designed
compound. The viability of Hela cells incubated with dif-
ferent concentrations of ADCPPa and Mppa were analyzed
by MTT assay. The HeLa cells were divided into the
experimental group (ADCPPa group) and control group
control groups were treated with drugs and not exposed to
light.
Figure 5 shows the photodynamic activity of the
ADCPPa against Hela cells. Black columns stand for the
cell viability of ADCPPa experiment groups, red columns
stand for the cell viability without light. The control groups
(red columns) treated with different concentrations of
ADCPPa without light show high cell viability (>85%),
(
blank group). The experimental groups were treated with
different concentrations of ADCPPa (2.5, 5, 10, 15, 20, 25
and 30 μg/mL, respectively) and exposed to light; the

Med Chem Res
Fig. 5 The photo cytotoxicity and dark toxicity of ADCPPa against
HeLa cells. The black columns stand by the experimental groups, and
the red columns stand for the control groups (color figure online)
Fig. 6 Cell viability of three different PDT processing methods. L+
stands for the groups treated with ADCPPA under light. DM stands for
the groups treated with ADCPPA and D-mannitol under light; SA
stands for the groups treated with ADCPPA and sodium azide under
light
suggesting that ADCPPa caused low dark toxicity in the
present study. The experimental group test showed that with
the increasing concentrations of drugs, the cell viability rate
declined obviously. That’s to say, cell survival incubated
with ADCPPA with red-light irradiation was related to the
ADCPPA concentration, suggesting the inhibition ability of
ADCPPa was concentration-dependent. When the con-
centration of ADCPPA reached to 30 μg/mL, the cell via-
bility lowered to 7%, suggesting that the ADCPPa had
strong photodynamic effects on the HeLa cell at such
exposed to light; (3) DM groups: treated with ADCPPa and
DM (40 mmol/L), and exposed to light.
Figure 6 show the influences of three different PDT
processing methods on cytotoxicity effects. The cell viabi-
lity of SA groups was much higher than that of DM group at
various concentrations, especially at high concentrations
(40 μg/mL), suggesting that the key ROS (singlet oxygen)
have been quenched, resulting in low cytotoxicity to HeLa
cells, therefore Type II reaction played a prominent role
in vitro. Moreover, the cell viability of DM groups were
also slightly higher than that of L+ group, suggesting that
Type I photodynamic reactions also occupied a certain
proportion during PDT, but not essentially. Generally, Type
II reaction would take place in the presence of tissue oxy-
gen, yet in the present cell experiments, the cancer cells may
also provide some integral substrate for PS to complete the
Type I reaction, hence coexistence of Type I and Type II
was rational.
2
concentration under light (713 nm, 10 J/cm ). The IC of
5
0
ADCPPa against cancer cells was 6.21 μg/mL. Moreover, as
investigated in our previous study (Li et al. 2016), the MPPa
also showed antitumor activity with a cell viability of 55%
at the concentration of 5 μM (27 μg/mL), yet the present
compound led to a cell viability of 13% at the concentration
of 25 μg/mL, showing a much better activity compared with
the leading compound.
Mechanism of photochemical processes in PDT
Singlet oxygen quantum yield of ADCPPa
After a PS is administered to the body and exposed to light
for some time (drug-to-light interval), the inactive drug is
electronically excited after irradiated and carries on a type I
1
As mentioned earlier, production of singlet oxygen ( O ) is
based on the energy transfer from the triplet state of PSs to
2
reaction to form superoxide anion and radicals, or performs
ground state molecular oxygen and it has been demon-
1
1
a type II reaction to form singlet oxygen ( O ) (Fig. 1). Both
2
strated that generation of O is responsible for the initiation
2
1
of the ROS can cause cell death, yet the O generated
2
of cell death (Ashikaga et al. 2000). The above research has
disclosed that the generation of singlet oxygen plays a
major role in photochemical process of ADCPPa, therefore
the determination of singlet oxygen quantum yield of
ADCPPa is indispensable for mechanistic investigation and
through type II reaction is considered for the major lethal
material. To investigate the mechanism of photochemical
processes in PDT, SA and DM were utilized in our research.
SA and DM could selectively react with oxygen free radi-
cals and singlet oxygen respectively, making the corre-
sponding ROS invalid on cancer cells (Li et al. 2016;
Sparrow et al. 2003). HeLa cells in our experiments were
divided into three groups: (1) L+ groups: treated with dif-
ferent concentrations of ADCPPa exposed to light; (2) SA
groups: treated with ADCPPa and SA (20 mmol/L), and
PS development. In this study, the methods employed are
1
based on the reaction of O with a singlet oxygen quencher
2
and the time-dependent spectrofluorimetric determination of
quencher concentration (Berneburg et al. 1999). 1,3-
diphenylisobenzofuran (DPBF) has been reported as a good
1
candidate for O detecting probe (Belfield et al. 2005), its
2

Med Chem Res
2
(
20 μg/mL) and exposing with light (713 nm, 10 J/cm ) for
3, 6, 9, 12, 24 h, respectively, compared with the cancer
cells without any treatment.
As shown in Fig. 8a, normal HeLa cells are spindle-
shaped with steady cytoplasm. After treatment with PDT for
additional 3 h (Fig. 8c), the cells become rounder, and
shading degree decreases obviously. After 6 h (Fig. 7d),
dilation of intercellular space and injury of the membrane
were observed, meanwhile the cell adhesive ability
decreased. After prolonged incubation (Fig. 8e, f), the entire
membrane structure of the cells was destroyed and almost
all the cells died, emerging sporadically apoptotic body.
Morphological changes of HeLa cells clearly showed that
ADCPPA could cause cellular damage under light.
Although PDT can induce cell death by several mechan-
isms, including apoptosis, necrosis, mitotic catastrophe, and
autophagy, yet the mechanism of cell death caused by PS
was not single and immutable (Dewaele et al. 2010).
1
Fig. 7 a Photodecomposition of DPBF by
ADCPPa in DMF (monitoring the maximum absorption of DPBF at
15 nm). b The linear fitting curve comparison of ADCPPa and
2
O after irradiation of
4
methylene blue
conjugated structure can be destroyed after reacting with
singlet oxygen, which can be detected by spectro-
fluorimeter. The decay of the absorption of DPBF at 415 nm
corresponds to the ability of ADCPPa to produce singlet
oxygen. The changes in intensity of the emission peak
reflects the singlet oxygen production generation. Both the
of DPBF and ADCPPa solutions have fluorescence emis-
Preliminary antitumor action research of ADCPPa
The mechanism of the PDT-mediated cytotoxic effect is still
not fully understood. It was reported that PDT-induced
oxidative stress could increase the expression of early
response genes, the cellular stress protein and heme oxy-
genase (Luna et al. 1994). Moreover, it is widely acknowl-
edged that cell cycle arrest is an important cause of cell
apoptosis. Many porphyrin derivatives, such as proto-
porphyrin IX (Li et al. 2013), gold (III) porphyrin complexes
sion peak at 415 nm.
1
The O yield during the photochemical process was
2
obtained by a first-order linear least-squares model using the
natural logarithm values of DPBF absorption at 415 nm
plotted against the irradiation time. As shown in Fig. 7a, the
(Tu et al. 2009), TMPyP4 (Shammas et al. 2003), were
reported to be able to induce cell cycle arrest in cancer cells.
Generally, cyclin-dependent kinases (CDKs) and cyclin are
the core factors of endogenous regulation and control of cell
cycle, especially CDK2, playing an important role in tumor
cells proliferation (Malumbres and Barbacid 2009). The
reduction in CDK2-relative expressed genes would lead to
cell cycle arrest at G0–G1 phase, consequently the DNA
synthesis and cell proliferation would be inhibited, therefore
determination of the CDK2 expression is an effective way to
study the influence of drugs on cancer cells. If CDK2
expression is upgraded in drug-treated cancer cells, it may be
presumed that the PS can effectively induce cell apoptosis.
Moreover, Survivin, which exhibits divergent functions such
as regulating cell proliferation and cell death, is the strongest
inhibitor of apoptosis that has been found among inhibitor of
apoptosis protein family. (Garg et al. 2016). Generally,
Survivin is specially expressed in embryonic cells and tumor
cells, but is seldom expressed in normal adult cells. Over-
expression of Survivin inhibits both intrinsic and extrinsic
pathways of apoptosis. (Altieri 2001) Survivin expression in
tumor cells may promote tumor progression by various
pathways, i.e., dysregulation of apoptosis and cell division,
therefore, if obvious change in expression of Survivin is
absorption at 415 nm significantly decreased over the time.
1
The O quantum yield of ADCPPa in DMF was calculated
2
using MB as a standard, whose singlet oxygen quantum
yield (ΦΔ) in DMF is 49.1%. The slope for ADCPPa and
2
MB was obtained after fitting with a linear function (R å
0
.99752). The singlet oxygen quantum yield (Φ ) of
Δ
ADCPPa in DMF is 65.98% (Fig. 7b), suggesting the title
compound has remarkable capacity to generate singlet
oxygen during the photochemical process. However, the
singlet oxygen yield of PPa is only about 39.2% as reported
in our previous paper (Cheng et al. 2017), which has par-
tially explained why ADCPPa could efficiently inhibit the
growth of cancer cell in PDT experiments than the leading
compound.
Morphological changes of HeLa cells after PDT
The in vitro PDT test mentioned earlier showed that
ADCPPa possessed strong inhibition ability against tumor
growth. We here tried to visually examine the tumor cell
phenotype after treatment of ADCPPa by FIM. The cell
phenotype was analyzed after incubation with ADCPPa

Med Chem Res
Fig. 8 Morphological changes of HeLa cells incubated with 20 μg/mL ADCPPa under light: a 0 h; b 1 h; c 3 h; d 6 h; e 12 h; f 24 h
Fig. 9 The relative expressions
under ADCPPa treatment: a
CDK2 in HeLa cells; b Survivin
in HeLa cells; c CDK2 in MCF-
7
cells; d Survivin in MCF-7
cells
found in PS-treated cancer cells, it can be concluded that the
PS can influence the expression of Survivin, subsequently
resulting the cell death.
Therefore, in this paper, by using qRT-PCR, we tried
to investigate the expression CDK2 and Survivin in
drug-treated HeLa cells and MCF-7 cells to explore the

Med Chem Res
PDT action of ADCPPa. Figure 9a, b shows the expression
of CDK2 and Survivin in HeLa cells treated with
ADCPPa. It can be seen shat after treatment with
ADCPPa, the expression of CDK2 increased gradually
over the time, and peaked after 12 h, about 1.6 times as
high as that of the control group (Fig. 9a). After 24 h
treatment, expression of CDK2 was lower than that of the
control group, about 0.3 times, which suggested that the
expression of CDK2 was significantly inhibited by
ADCPPa at this time, leading to cell cycle arrest and
programmed cell death. In case of Survivin (Fig. 9b), the
expression of Survivin in ADCPPa-treated HeLa cells
showed an downward trend first, but then increased over
the time, however, the expressions were apparently less
than that of the control, about 0.57, 0.59 and 0.66 times at
ability. In vitro PDT study has shown that ADCPPa had
strong photodynamic effects and low dark toxicity against
the cancer cells. Cell uptaking test indicated ADCPPa could
quickly enter the cells in less than 1 h, suggesting that the
PS could take effect more quickly in practical application,
which also improved the feasibility in clinical application.
In addition, qRT-PCR has showed that the target compound
has important regulating effect on expression of CDK2 and
Survivin. All in all, our results indicated that ADCPPa was
an effective PS as a new drug candidate in PDT and was
worth for further study.
Acknowledgements Financial support of this research was provided
by National Natural Science Foundation of China (No. 20972036,
2
1272048, 21471041), the Natural Science Foundation of Hei-
longjiang Province (No.B20913), the Program for Scientific Techno-
logical Innovation Team Construction in the Universities of
Heilongjiang Province (No. 211TD010), the Scientific Research Fund
of Heilongjiang Provincial Education Department (No.12531194), the
Natural Science Youth Foundation of Heilongjiang Province (No.
QC2016011) and the Innovation Fund for Graduate Students of Harbin
Normal University
6
h, 12, 24, respectively, suggesting our compound could
degrade the expression of Survivin, displaying a negative
regulatory effect. As mentioned earlier, Survivin expres-
sion in tumor cells may promote tumor progression, the
negative regulation by ADCPPa would undoubtedly impel
cell death.
Compliance with ethical standards
Moreover, we have also investigate the expression
regulating effect on breast cancer MCF-7 cells to see if the
PS has an effect on the expression regulating of other
cells. As shown in Fig. 9c, the expression of CDK2 in
MCF-7 cells treated with ADCPPa decreases first, then
increases gradually over the time. The lowest expression
level is at 6 h, about 0.4 times as high as that of the control
group. With the passage of time the expression level rise
rapidly, reaching 3.7 times the size of control group,
which suggested ADCPPa would take effect after 6 h
treatment. The generous gene CDK2 expression at 24 h
was supposed to be a sort of cellular stress response to
external stimulus. For Survivin (Fig. 9d), the expression
in MCF-7 cells treated with ADCPPa decreases first,
reaching the minimal level after 12 h, about 0.1 time as
high as that of control. However, after 24 h, the expression
level rise rapidly, reaching 3.7 times the size of control
group, which was also considered as a sort of cellular
stress response, leading an obvious rebound of expres-
sion. All in all, the target compound has important reg-
ulating effect on expression of CDK2 and Survivin. It can
inhibit the HeLa cell cycle and induce apoptosis and kill
the tumor cells thoroughly.
Conflict of interest The authors declare that they have no competing
interests.
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