4-Hydroxyl-oxoisoaporphine, one small molecule as theranostic agent for simultaneous fluorescence imaging and photodynamic therapy as type II photosensitizer

Article abstract of DOI:10.1007/s43630-021-00030-0

Oxoisoaporphine (OA) is a plant phototoxin isolated from Menispermaceae, however, its weak fluorescence and low water solubility impede it for theranostics. We developed here 4-hydroxyl-oxoisoaporphine (OHOA), which has good singlet oxygen-generating ability (0.06), strong fluorescence (0.72) and improved water solubility. OHOA displays excellent fluorescence for cell imaging and exhibits light-induced cytotoxicity against cancer cell. In vitro model of human cervical carcinoma (HeLa) cell proved that singlet oxygen generated by OHOA triggered photosensitized oxidation reactions and exert toxic effect on tumor cells. The MTT assay using HeLa cells verified the low cytotoxicity of OHOA in the dark and high phototoxicity. Confocal experiment indicates that OHOA mainly distributes in mitochondria and western blotting demonstrated that OHOA induces cell apoptosis via the mitochondrial pathway in the presence of light. Our molecule provides an alternative choice as a theranostic agent against cancer cells which usually are in conflict with each other for most traditional theranostic agents. Graphic abstract: [Figure not available: see fulltext.]

Full text of DOI:10.1007/s43630-021-00030-0

Photochemical & Photobiological Sciences (2021) 20:501–512
https://doi.org/10.1007/s43630-021-00030-0
ORIGINAL PAPERS
4
‑Hydroxyl‑oxoisoaporphine, one small molecule as theranostic agent
for simultaneous ꢀuorescence imaging and photodynamic therapy
as type II photosensitizer
1
1
1
1,2
Qi Xu · Yunfan Ji · Meijun Chen · Xusheng Shao
Received: 18 November 2020 / Accepted: 17 February 2021 / Published online: 20 March 2021
The Author(s), under exclusive licence to European Photochemistry Association,European Society for Photobiology 2021
©
Abstract
Oxoisoaporphine (OA) is a plant phototoxin isolated from Menispermaceae, however, its weak ꢀuorescence and low water
solubility impede it for theranostics. We developed here 4-hydroxyl-oxoisoaporphine (OHOA), which has good singlet
oxygen-generating ability (0.06), strong ꢀuorescence (0.72) and improved water solubility. OHOA displays excellent ꢀuo-
rescence for cell imaging and exhibits light-induced cytotoxicity against cancer cell. In vitro model of human cervical car-
cinoma (HeLa) cell proved that singlet oxygen generated by OHOA triggered photosensitized oxidation reactions and exert
toxic eꢁect on tumor cells. The MTT assay using HeLa cells veriꢂed the low cytotoxicity of OHOA in the dark and high
phototoxicity. Confocal experiment indicates that OHOA mainly distributes in mitochondria and western blotting demon-
strated that OHOA induces cell apoptosis via the mitochondrial pathway in the presence of light. Our molecule provides an
alternative choice as a theranostic agent against cancer cells which usually are in conꢀict with each other for most traditional
theranostic agents.
Graphic abstract
Keywords Oxoisoaporphine · Photosensitizer · Cell imaging · Singlet oxygen · Cytotoxicity
*
Xusheng Shao
1
Introduction
shaoxusheng@ecust.edu.cn
1
Shanghai Key Laboratory of Chemical Biology,
School of Pharmacy, East China University of Science
and Technology, Shanghai 200237, China
Photodynamic therapy (PDT) has attracted tremendous
attention due to its non-invasive nature and high speciꢂc-
ity for cancer treatment [1–3]. This technology utilizes sin-
2
1
State Key Laboratory of Bioreactor Engineering, East China
University of Science and Technology, Shanghai 200237,
China
glet oxygen ( O ) generated by photosensitizers (PS) when
2
absorb speciꢂc wavelengths of light, causing oxidative stress
Vol.:(0
1
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3

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Photochemical & Photobiological Sciences (2021) 20:501–512
on tissues and abnormal proliferation of active cells [4–6].
This results in irreversible cellular damage and subsequent
cell death [7]. PS is the key for successful implementation
of PDT. The most attractive characteristic of PDT is its last-
ing selectivity as photodynamic eꢁect happens only where
structure that can stabilize the G-quadruplex structure by
π–π stacking and electrostatic interactions with G-quartet
plane of G-quadruplex DNA [25]. Oxoisoaporphine alka-
loids have been reported to have anticancer cells activities
through several mechanisms including reactive oxygen
species generation, DNA binding [26, 27], and telomerase
enzyme inhibition [26, 28, 29]. In addition, OAs also exhib-
ited variety of biological activities including anti-depressant
[30], anti-Alzheimer’s disease [30–32] and anti-leishmanial
activity [33]. Here, we found that the presence of the natu-
ral phenalenone skeleton in OAs gave them singlet oxygen
photosensitization capacity. However, there are few investi-
gations on their application in PDT. OA has poor water solu-
bility, weak ꢀuorescence and short absorption wavelength,
making it unsuitable for PDT. With the goal of developing
a novel high-eꢃciency photosensitizer, here we introduced
an electron donating group hydroxyl to OA to enhance the
ꢀuorescence quantum yield and water solubility. The gener-
ated 4-hydroxyl-oxoisoaporphine (OHOA), as a theranostic
agent, has excellent oxygen-generating ability, strong ꢀuo-
rescence and can kill various cancer cells upon irradiation
through intrinsic mitochondrial apoptosis.
the PSs accumulated and where the light is applied. How-
1
ever, the short-lived O (3 µs) will not migrate more than a
2
fraction of a micron from its site of formation (20 nm) [8].
Therefore, photosensitizers located in the organelles have
a more eꢁective photodynamic eꢁect [9–12]. Developing
a photosensitizer with excellent singlet oxygen-generating
ability, good photostability and biologically benign absorp-
tion band is urgent. Furthermore, some processes of photo-
sensitizer activation are accompanied by the generation of
ꢀ
uorescence and phosphorescence, which means a therag-
nostic eꢁect can be achieved on a single molecule [13–15].
Phenalenones (PNs) is a kind of phytoalexins synthesized
de novo by plants and accumulated rapidly at areas of patho-
gen infection, which is produced at the ꢂrst stage of black
sigatoka that Mycosphaerella fjensis infected banana trees.
PN is known to be an eꢃcient type II photosensitizer that
can produce equivalent singlet oxygen upon light irradia-
tion against mosquito and nematode [16–18]. Compounds
with a structure based on the PN skeleton are eꢁective type
II photosensitizers for fast killing of key oral pathogens, in
2 Materials and methods
particular, drug-resistant bacteria [19]. We previously found
1
that PN derivatives exhibited light-induced O -mediated
2.1 Chemicals and reagents
2
lethal activity against mosquito larvae and root-knot nema-
tode [20].
All chemical reagents or biological reagents and solvents
were purchased from commercial suppliers without further
Isoquinoline alkaloid oxoisoaporphines (OAs, Fig. 1) iso-
lated from Menispermum dauricum DC roots (family Men-
ispermaceae) [21–23]. Menispermum dauricum DC roots
as a perennial herb distributed in the northeast, north and
east of China, southern Japan, Korea and Russian Siberia
widely has been used as traditional medicine treatment for
analgesic and antipyretic [24]. OA is a kind of 1-azabenzan-
throne derivative with a large planar aromatic conjugated
1
13
puriꢂcation. H NMR and C NMR spectra were recorded
1
13
on Bruker AM-400 ( H at 400 MHz, C at 100 MHz)
spectrometer with CDCl or DMSO-d as the solvent and
3
6
TMS as the internal standard. High-resolution mass spec-
tra (HRMS) were collected in an XEVO G2 TOF mass
spectrometer (Waters, USA) using electrospray ionization
(ESI). The optical properties were tested by All Varian Cary
1
00 UV spectrophotometer and Varian Cary Eclipse FL
spectrophotometer.
2.2 Synthesis of phenalenone (PN)
Naphthalene (0.64 g, 5 mmol) and anhydrous aluminum
chloride (1.4 g, 5.5 mmol) were put into 10 mL of dichlo-
romethane, stirred for 5 min in an ice bath, cinnamoyl
chloride (0.83 g, 5 mmol) was dissolved in 2.5-mL dichlo-
romethane and then dropped into the ꢀask during 30 min,
reacted at room temperature for 20 min, the reaction solu-
tion was rotary evaporated under vacuum condition puriꢂed
with silica gel chromatography using petroleum ether/ethyl
acetate (10:1) as eluting solvent to aꢁord phenalenone as
1
a yellow solid with a yield of 76% (0.40 g) [34]. H NMR
Fig. 1 Synthesis routes for OHOA
(400 MHz, Chloroform-d) δ 8.59 (d, J=7.6 Hz, 1H), 8.16
1
3

Photochemical & Photobiological Sciences (2021) 20:501–512
503
(
d, J = 8.0 Hz, 1H), 7.98 (d, J = 8.0 Hz, 1H), 7.77–7.67
m, 3H), 7.55 (t, J=7.6 Hz, 1H), 6.71 (d, J=9.8 Hz, 1H).
(s, 1H), 8.83 (d, J=7.6 Hz, 1H), 8.77 (d, J=5.6 Hz, 1H),
8.48 (d, J=8.4 Hz, 1H), 8.28 (dd, J=7.8, 1.4 Hz, 1H), 8.10
(d, J=5.6 Hz, 1H), 7.90–7.83 (m, 1H), 7.76–7.70 (m, 1H),
(
1
3
C NMR (100 MHz, Chloroform-d) δ 185.14, 137.17,
1
3
1
1
32.90, 132.63, 131.84, 131.61, 131.51, 130.24, 129.30,
29.25, 127.89, 127.15, 126.61. HRMS (ESI): m/z calcd
7.33 (d, J=8.0 Hz, 1H). C NMR (100 MHz, DMSO-d )
6
δ 180.57, 160.44, 146.81, 142.88, 136.00, 133.29, 132.93,
+
+
for C H O [M+H] : 181.0653; found: 181.0654.
132.20, 130.28, 126.63, 126.14, 124.76, 123.36, 119.65,
1
3
9
+
1
16.10, 113.03. HRMS (ESI): m/z calcd for C H NO
16 10 2
+
2
.3 Synthesis of oxoisoaporphines (OA)
[M+H] : 248.0713; found: 248.0712.
A mixture of phthalic anhydride (15 g, 100 mmol) and
b-phenylethylamine (12.1 g, 100 mmol) in anhydrous etha-
nol was reꢀuxed for 6 h. After cooling to 0–5 °C, crystals of
phenylethylphthalimide (19 g, 78%) separated. Then ꢂltered
oꢁ to give a solid, washed twice with ethanol. The product is
pure enough for the next reaction. To a mixture of anhydrous
aluminum chloride (50 g, 0.38 mol) and sodium chloride
2
.5 Cell culture and treatment
Human cell lines HeLa (human cervical carcinoma cell)
were purchased from the Cell Bank of Type Culture Col-
lection of the Chinese Academy of Sciences (Shanghai,
China). The cells were cultured in Dulbecco’s modiꢂed
Eagle’s medium (DMEM, Hyclone, Logan, Utah, USA) sup-
plemented with 10% of heat-inactivated fetal bovine serum
(
(
9.9 g, 0.17 mol) was slowly added phenylethylphthalimide
25.1 g, 0.1 mol) at 180 °C for 30 min. The reaction was
−
1
(
FBS, Gibco, Grand Island, NY, USA), 100 U mL of
allowed to continue at 220–230 °C for 2 h. The product was
cooled, ꢂnely ground and poured slowly into concentrated
sulfuric acid (550 mL) at 90 °C. The mixture was stirred
and heated at 230–240 °C for 2 h. After being cooled, the
solution was poured into ice. Sodium hydroxide was added
until pH=3 was obtained and the resultant precipitate was
ꢂltered oꢁ and washed in turn with dilute aqueous sodium
hydroxide and water to give the crude product, which was
extracted with acetic acid. The extract was condensed under
reduced pressure and the resultant precipitate was washed
oꢁ, dried, puriꢂed with silica gel chromatography using
dichloromethane as eluting solvent to aꢁord as a light yel-
−
1
penicillin and 100 μg mL of streptomycin (Gibco, USA),
and then put in a 5% CO atmosphere hatching incubator at
2
3
7 °C. The cells were regularly subcultured to maintain them
in a logarithmic phase of growth.
2.6 Singlet oxygen detection
1
To detect the irradiation-induced singlet oxygen ( O ), a
2
singlet oxygen trap 9,10-anthracenediylbis (methylene)
dimalonic acid (ABDA) was used in air-saturated MilliQ
1
water to monitor the O generation. Water-soluble ABDA
2
1
low solid with a yield of 28% (9 g) [35]. H NMR (400 MHz,
exhibits photobleaching when oxidized by singlet oxygen
to endoperoxide, which causes the degradation of ABDA at
400 nm. Compounds were dissolved in MilliQ water (0.5%
DMSO as cosolvent) and then added to equivalent ABDA
Chloroform-d) δ 8.87 (d, J=8.0 Hz, 1H), 8.74 (d, J=5.2 Hz,
1
8
1
H), 8.62 (d, J=7.2 Hz, 1H), 8.39 (dd, J=7.8, 1.4 Hz, 1H),
.11 (d, J=8.4 Hz, 1H), 7.88 (t, J=7.8 Hz, 1H), 7.80 (m,
1
3
−2
H), 7.71 (d, J=5.6 Hz, 1H), 7.64 (t, J=7.6 Hz, 1H).
C
stock solution. After light (470 nm, 5 mW cm ) irradia-
NMR (100 MHz, CDCl ) δ 183.31, 148.70, 143.97, 136.69,
tion, the absorbance of ABDA at 400 nm was recorded
every 5 min. Phenalenones was utilized as a control. The
control experiment was carried out using 10 mM ABDA
without photosensitizer. The decomposition of ABDA by
OA, OHOA and PN with diꢁerent irradiation time in water
3
1
1
35.12, 134.01, 133.35, 132.34, 130.53, 130.38, 129.85,
29.06, 127.55, 125.30, 122.86, 120.89. HRMS (ESI): m/z
+
+
calcd for C H NO [M+H] : 232.0762; found: 232.0761.
16
10
2.4 Synthesis of oxoisoaporphines‑OH (OHOA)
is compared in Fig. 2c. A is the absorbance of ABDA at
0
4
00 nm before irradiation, A is the absorbance of ABDA
A mixture of oxoisoaporphines (0.50 g, 2.2 mmol) and
hydrazine hydrate (1 mL) was stirred in diethylene gly-
col (15 mL) for 10 min. Then, 2 mL of 40% NaOH solu-
tion was added dropwise, and the mixture was heated at
at 400 nm under diꢁerent irradiation time. The direct evi-
1
dence of O could be obtained by EPR technique [37–40],
2
the experimental data were shown in supporting informa-
tion. Photostability experiment was carried out as follow-
–
5
1
40 °C for 3 h. The reaction mixture was cooled to room
ing description: samples dissolved in MilliQ water (10 M,
0.5% DMSO as cosolvent) was irradiated with a LED light
temperature, and poured into water. The hydrochloric acid
was added to adjust the solution to pH=3. The precipitate
was ꢂltered oꢁ and puriꢂed with silica gel chromatogra-
phy using chloroform/methanol (100:5) as eluting solvent
−
2
(460–470 nm, 5 mW cm ) for 3 h, then absorbances were
collected every 30 min during irradiation. The changes of
optical density at λ with irradiation time under blue light
max
to aꢁord compound 3a as a yellow solid with a yield of
were recorded in Fig. 2c. During irradiation, the samples
1
7
6% (0.40 g) [36]. H NMR (400 MHz, DMSO-d ) δ 12.12
were stirred and the temperature was kept constant. Singlet
6
1
3

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Photochemical & Photobiological Sciences (2021) 20:501–512
Fig. 2 a UV–Vis absorption spectra and b ꢀuorescent emission spec-
tra of targeted compounds in H O (λ =490 nm, λ =528 nm). c
ance at the maximum absorption wavelength before irradiation, A is
the absorbance at the maximum absorption wavelength after diꢁer-
ent irradiation durations. d The changes of absorbance at 400 nm of
ABDA as a function of irradiation time
2
ex
em
Absorption spectrum changes of compounds after irradiation with
−
2
blue light (5 mW cm ) for diꢁerent periods of time. A is the absorb-
0
oxygen quantum yields of test compounds were obtained
based on following equation:
in ethanol), Grad is the gradient form the plot of integrated
ꢀuorescence intensity versus absorbance, η is the refractive
index of the solvent.
k
PS
ϕ (PS) = ϕ (ST)
Δ
.
Δ
k
ST
2.8 Photoinduced cytotoxicity test
When ϕ means the singlet oxygen quantum yields of
∆
PDT eꢃcacy of OHOA was determined by MTT assay using
photosensitizer and PN as a reference (ϕ =1 in water), k is
∆
1
HeLa cancer cell lines. PDT utilizes O generated by photo-
2
the plot of the logarithm of change in ABDA absorbance at
sensitizers when absorb speciꢂc wavelengths of light, caus-
ing oxidative stress on tissues and abnormal proliferation of
active cells. In this respect, PDT is applicable to all kinds
of tumor cancer cells. We choose three other cancer cells
besides HeLa cells including A549 (human lung adenocarci-
noma cell), HepG2 (human hepatocellular carcinomas cell)
and MCF7 (human breast cancer cells). The cytotoxicity
of OAs were evaluated towards diꢁerent cancer cells in the
light stimulation and dark condition and the half maximal
4
00 nm versus irradiation time.
2
.7 Fluorescence quantum yield calculation
OHOA was formulated with MilliQ water (5% DMSO as
−5
−6
cosolvent) to 10 –10 M, the absorbance spectra were col-
lected on Varian Cary 100 UV–Vis spectrophotometer and
the corresponding ꢀuorescence spectra were obtained on a
Varian Cary Eclipse Fluorescence spectrophotometer. The
inhibitory concentration (IC ) values against diꢁerent cell
50
lines were summarized in supplementary information. Cells
ꢀ
uorescent quantum yields of test compounds were obtained
were digested with trypsin to make a single cell suspension
based on following equation:
5
−1
(
1 × 10 cells mL ) and were seeded onto 96-well plates
2
PS
GradPS
GradST
η
η
(100 μL per well) for 12 h. Two 96-well plates were set as
control trails. One plate was accepted with light stimulation,
while the other one was kept in dark condition. Compounds
were formulated with culture medium to diꢁerent concentra-
tion including 0.1% DMSO as cosolvent. After removing the
ϕ (PS) = ϕ (ST)
F
.
F
2
ST
When ϕ means the ꢀuorescent quantum yields of photo-
F
sensitizer and naphthalimide as a reference (ϕ (ST)=0.66
F
1
3

Photochemical & Photobiological Sciences (2021) 20:501–512
505
5
−1
original culture medium, chemicals of series of concentra-
tions (200 μL per well) was added to 96-well plates. After
cells (1×10 cells mL ) were seeded on a glass bottom cell
culture dish and co-incubated with OHOA (2.5 μM) for 6 h.
After 15 min of PDT treatment, to explore the subcellular
localization of OHOA, one dish of cells was washed with
PBS for three times and incubated in culture medium with
100 nm MitoTracker Red (invitrogen™) for 20 min, another
dish of cells was incubated with 50 nm Lysosome Tracker
Red (invitrogen™) for 30 min. Then, stained live cells were
observed by confocal laser scanning microscope (Nikon Inc.,
Melville, NY, USA). Emission was collected at 530± 20 nm
upon excitation at 488 nm for OHOA. MitoTracker Red and
Lysosome Tracker Red were excited at 561 nm and emis-
2
4
2
4 h incubation in darkness, the samples were subjected to a
−2
70–480 nm LED light at power density of 20 mW cm for
−2
0 min (ꢀuence, 40 J cm ). After 24 h incubation, 20 μL of
−1
MTT (5 mg mL ) was added to each well. After 4 h incuba-
tion at 37 °C, culture medium was discarded and the residue
was added 150 μL DMSO to dissolve the purple formazan
crystals. Absorbance was measured at 570 nm and 630 nm
by a Synergy H1 microplate reader (Bio-Teck, Winooski,
VT, USA). The inhibitory rates of the cells were calculated
by the following formula:
ꢀ
ꢁ
ꢂꢃ
mean OD570nm(sample) − OD630nm(sample)
ꢁ
%
inhibitory rate = 1 −
ꢂ
× 100%,
mean OD570nm(control) − OD630nm(control)
where OD (sample) and OD (control) were denoted as the
absorbance of the sample solution and control wells, respec-
tively. The MTT assay was performed in triplicate, the mean
and standard deviation was calculated for each group.
sions were collected at 599± 20 nm.
2.11 Intracellular ROS detection by confocal
microscope
2
.9 Clonogenic assay
2′,7′-Dichlorofluorescin diacetate (DCFH-DA) (Sigma-
Aldrich, USA) was selected to detect the intracellular ROS.
5
−1
The eꢁectiveness cytotoxicity of OHOA with or without
light irradiation was compared by employing clonogenic
assay. Clonogenic assay is an in vitro cell survival assay to
determine the ability of a single cell to grow into a colony.
Here, a colony is deꢂned as a cluster of at least 50 cells. Cells
HeLa cells (5×10 cell mL ) were seeded to glass bottom
cell culture dish for overnight, then treated with diꢁerent
concentrations (dark control, light control, OHOA in dark,
OHOA plus with light irradiation for 5 min, OHOA plus with
light irradiation for 15 min, OHOA plus with light irradia-
tion for 30 min). After that, cells were incubated with 10 μM
3
−1
were seeded in 6 cm dish at a density of 1×10 cells mL
and cultivated in incubator for 12 h. The medium in the
dish (4 mL) was then replaced with fresh medium contain-
ing OHOA (10 μM) and the light group was subjected to
light stimulation, then the cells were incubated continued
for 2 weeks with culture medium that was refreshed every
H DCFDA for 20 min. Then, cells were washed three times
2
with PBS, analyzed immediately by confocal laser scanning
microscope (Nikon Inc., Melville, NY, USA). Emission
was collected at 530 ± 20 nm upon excitation at 488 nm.
Flow cytometric analysis was utilized to detect intracellular
6
−1
2
days. Colonies were rinsed with PBS, fixed with 4%
reactive oxygen quantitatively. HeLa cells (10 cell mL )
were seeded in a 6-well culture plates and were treated with
diꢁerent conditions as described before, then digested and
collected in 1.5-mL centrifuge tube, washed with PBS for
paraformaldehyde for 1 h at room temperature and stained
with 10% Giemsa staining solution for 1 h. After cells were
washed with PBS and air-dried, colonies consisting of more
than 50 cells were counted. Each experiment was done in
triplicate and colony number in the absence of OHOA was
used as a control. Six experiments groups (dark control,
light control, OHOA in dark, OHOA plus with 5-min light
irradiation, OHOA plus with 15-min light irradiation and
OHOA plus with 30-min light irradiation) were conducted
simultaneously.
three times (1500 rpm). H DCFDA (10 μM ꢂnal concen-
2
tration) was added to the cell and then incubated at 37 °C
for 20 min. Cells were washed with PBS for three times
before they were analyzed on a FAC Scan ꢀow cytometer
(BD, FACS Calibur). The ꢀuorescence intensity of cells was
measured with excitation 488 nm and emission at 530 nm.
Green mean ꢀuorescence intensities were analyzed using
FlowJo 7.6 software.
2.10 Localization of OHOA by ꢀuorescence
observation
2.12 Mitochondrial membrane potential (△Ψm)
analysis
Cellular uptake and intracellular imaging eꢃciency were
performed by confocal laser scanning microscope on Human
Cervical Cancer cell line, HeLa cells, as model system. HeLa
JC-1 can easily penetrates cells and healthy mitochondria.
In normal mitochondria with high membrane potential JC-1
1
3

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Photochemical & Photobiological Sciences (2021) 20:501–512
keeps aggregated and the ꢀuorescence is red, while in dys-
functional mitochondria with decreased membrane potential,
JC-1 keeps monomeric and ꢀuoresces green. Therefore, the
red/green ꢀuorescence ratio represented the status of mito-
2.15 Western blotting analysis
Total protein from HeLa cells was extracted in RIPA lysis
buꢁer (Sigma-Aldrich, St. Louis, MO, USA) with 1 mM
phenylmethylsulfonyl ꢀuoride (PMSF, Sigma-Aldrich, St.
Louis, MO, USA), and quantiꢂed by BCA method. Cellular
extracts were separated on 10–15% SDS-PAGE and trans-
ferred to a polyvinylidene diꢀuoride (PVDF) membrane
(Millipore Corp, Atlanta, GA, USA) from gel. Then blocked
in Tris-buꢁered saline-Tween (TBST; 10 mM Tris–HCl,
pH 7.5/150 mM NaCl/0.1% Tween-20) with 5% non-fat dry
milk for 1 h at room temperature. These was then incubated
with antibodies for caspase 3 (1:1000), cleaved-caspase 3
(1:1000), PARP (1:1000) and β-actin (1:1000) overnight at
4 °C, subsequently incubated with HRP conjugated second-
ary antibodies for 2 h at room temperature. Signals were
visualized after treatment by enhanced chemiluminescence
(ECL) reagent (Pierce, Rockford, IL, USA). All protein
bands were scanned.
5
−1
chondria to some extent. HeLa cells (5×10 cell mL ) were
seeded to glass bottom cell culture dishes for overnight, then
treated with diꢁerent conditions as described before. After
treatment, the cells were incubated at 37 °C for 30 min with
−
1
5
mg L JC-1 (Invitrogen™), washed twice with PBS and
placed in fresh medium without serum. Finally, images were
viewed and scanned by confocal laser scanning microscope
(
Nikon Inc., Melville, NY, USA) at 488 nm excitation and
emission at 530 nm for green, and at 561 nm excitation and
emission at 590 nm for red. Confocal images of JC-1 stain-
ing were processed as red/green signal ratio, relative inten-
sity was calculated using ImageJ software. Mitochondrial
depolarization was indicated by an increase in the ratio of
green/red ꢀuorescence intensity.
2.13 Chromatin morphology assay
2
.16 Statistical analysis
Cells were treated as described before. Hoechst 33342
nuclear staining procedure was chosen to observe the
changes of cells morphology which were induced by OHOA.
After the HeLa cells were treated with diꢁerent conditions,
cells were washed three times with serum-free medium, then
ꢂxed with paraformaldehyde under 4 °C for 15 min, wash
twice with PBS, and incubated with Hoechst 33342 (10 μM)
at 37 °C for 10 min, after that washed three times with PBS
All experiments were performed in triplicate and repeated
at least three times. Data were shown as means ± standard
deviations (SDs). The results were analyzed by one-way
analysis of variance (ANOVA) followed by post hoc statis-
tical tests, using the Tukey test for each pair of compared
groups. Statistical signiꢂcance was deꢂned as *p < 0.05,
**p<0.01, ***p<0.001.
(
pH 7.4). The morphology of Hela cells was observed
and photographed by confocal laser scanning microscope
Nikon Inc., Melville, NY, USA). Emission was collected at
60± 20 nm upon excitation at 346 nm for blue, at 488 nm
excitation and emission at 530 nm for green.
(
3 Results and discussion
4
3.1 Synthesis
2
.14 Apoptosis analysis by Annexin V‑FITC/PI
The synthetic routes of oxoisoaporphine derivative was
depicted in Fig. 1. OA and OHOA were synthesized by
a four-step reaction. O-phthalic anhydride 1 reacted with
amine 2 gave phthalimide 3. Treatment of 3 with anhydrous
aluminum chloride at 220–230 °C provided intermediate 4.
Finally, intermediate 4 reacted with sulfuric acid aꢁording
the target compound OHOA. Reaction of OA with hydroxy-
lamine hydrochloride at 140 °C oꢁered the target compound
OHOA.
apoptosis kit
To explore the apoptosis-induced eꢁects of OHOA, the per-
centage of apoptotic cells by ꢀow cytometry with Annexin
V-FITC/PI apoptosis kit (Invitrogen™) were analyzed. In
brief, HeLa cells cultured in 6-well plates were treated with
various conditions (OHOA in dark, OHOA with light irra-
diation for 5 min, OHOA with light irradiation for 15 min,
6
OHOA with light irradiation for 30 min) for 2 h, 1×10 cells
were washed, trypsinized and collected in 1.5-mL centrifuge
tube. Then, the cells were washed with PBS (pH 7.6) for
three times (2000 rpm). After centrifugation, the cells col-
lected and resuspended in 500 μL of buꢁer solutions loaded
with Annexin V-FITC and PI (with 5 μL Annexin V-FITC
and 10 μL PI) for 15 min. Finally, the Annexin V-FITC-
stained cells were tested on ꢀow cytometer (BD, FACS Cali-
bur) and the data were analyzed using FlowJo 7.6 software.
3.2 Photophysiochemical properties
To determine the performance of OA and OHOA, their pho-
tophysiochemical properties, such as maximum absorption
and emission wavelength, and photostability and singlet
oxygen-generating ability were investigated. OA has two
absorption bands in acetonitrile centered at 250 nm and
390 nm, respectively. The molar extinction coeꢃcients of
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Photochemical & Photobiological Sciences (2021) 20:501–512
507
OA and OHOA are 11,012 M−1 cm and 14,997 M cm ,
respectively. In PBS buꢁer, a bathochromic shift occurred
for OHOA with the appearance of a new absorption band
around 490 nm (Fig. 2a). This can be attributed to deprotoni-
zation of the hydroxyl group. A high ꢀuorescence quantum
yield facilitates cell imaging and diagnosis for a photosensi-
tizer. OA was weakly emissive, while OHOA showed strong
−1
−1
−1
to light, cytotoxicity increased by tenfold for OA. OHOA
exhibited signiꢂcant activity enhancement after light irra-
diation, with an increase in cytotoxicity of about 150-fold.
The lack of cytotoxicity in dark veriꢂed that OHOA is safe
for normal tissues in PDT treatment. The degree of light
irradiation on the anticancer eꢁects of OHOA is shown in
Fig. 3b. The increasing cytotoxicity correlated closely with
the increasing duration of light irradiation, as the viability
of HeLa cells decreased from 43.88 to 8.23%. Similar results
were also observed in colony growth. When the irradiation
time increased from 5 to 30 min, colony growth decreased
by 57.75%, 85.56% and 97.69%, respectively (Fig. 3c), in
comparison with the dark control.
ꢀ
uorescence emission with quantum yields approaching
0
.72 (PBS buꢁer, naphthalimide as reference, Φ =0.66 in
F
ethanol) and an emission maximum wavelength occurring
at around 535 nm (Fig. 2b). 9,10-Anthracenediylbis (meth-
ylene) dimalonic acid (ABDA) was used to detect the ability
of singlet oxygen generation by OAs in water under irradia-
tion. The reaction of ABDA and singlet oxygen cause the
decrease of ABDA absorbance at a wavelength of 400 nm.
Figure 2d shows linear relationship between irradiation
time and the logarithm of change in ABDA absorbance at
3.4 Intracellular singlet oxygen detection
After the photosensitizer is absorbed by cells, large amount
of reactive oxygen species (ROS) including singlet oxygen
produced rapidly after illumination, which caused the imbal-
ance of intracellular reactive oxygen species and oxidation
stress [41]. To identify the light-induced mechanism behind
the light-responsive toxicity, 2′,7′-dichloroꢀuorescin diac-
etate (DCFH-DA) was used as an indicator to detect ROS
produced in HeLa cells. The HeLa cells were subjected to
six diꢁerent conditions: no treatment (negative control),
light treatment only, OHOA in dark, and OHOA plus dif-
ferent irradiation times (5, 15 or 30 min). Fluorescence
microscopy images were collected at 530 nm upon exci-
tation at 488 nm. For HeLa cells treated with OHOA and
4
00 nm. Phenalenone produced singlet oxygen at a faster
rate than OA and OHOA. The photodegradation processes
of PN, OA and OHOA were monitored by optical density
−
2
changes at λ upon blue light (470 nm, 5 mW cm ) irra-
max
diation. No obvious photodegradation was detected when
OA and OHOA were subjected to irradiation at 470 nm for
3
h in PBS buꢁer (Fig. 2c), this demonstrated their stabil-
ity during of biological testing and in the presence of light.
3.3 Phototoxicity evaluation
The cytotoxicity of OAs towards HeLa cells was evaluated
in light stimulation and dark conditions. A summary of half
maximal inhibitory concentration (IC ) values is shown in
−
2
light (470 nm, 20 mW cm ), a signiꢂcant ꢀuorescence
increase was observed (Fig. 4a). When OHOA was irradi-
ated for 5 min, the mean ꢀuorescence intensity quickly rose
to 101, which is approximately double that of OHOA in
dark conditions. Similar results also were obtained by ꢀow
cytometric analysis (Fig. 4b). These ꢂndings suggest that
OHOA treatment together with light can eꢃciently produce
high cytotoxic ROS levels in HeLa cells.
5
0
Fig. 3a. In general, all compounds exhibited higher toxicity
upon irradiation in comparison with dark conditions, indi-
cating that OA and OHOA have obvious photo-dependent
activity towards HeLa cells. OA has moderate dark toxicity
to HeLa cells with IC values of 41.04 μM, which corre-
5
0
sponds well to previously reported results. When exposed
Fig. 3 a In vitro dark toxicity and phototoxicity of IC values for
genic assay performed in six-well plates, with clones produced by
HeLa cells under diꢁerent conditions. All the data are presented as
mean± SD (n=3 per group). Signiꢂcant diꢁerences from dark con-
trol: *p<0.05; **p<0.01; ***p<0.001
5
0
PN, OA and OHOA in HeLa cells at 48 h post-treatment. b Eꢁects
of light dose on HeLa cells viability, OA2 concentration: 10 μM. c-a
Colony numbers counted using Image J. b Digital images of clono-
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Photochemical & Photobiological Sciences (2021) 20:501–512
Fig. 4 a Confocal microscopic images and ꢀow cytometric quantiꢂ-
cation of cellular ROS levels detected by DCFH-DA staining. Scale
bar: 20 μM. MFI mean ꢀuorescence intensity. b Confocal microscopy
images of HeLa cells incubated with OHOA (2.5 μM) and treated
with commercial organelle trackers. Overlay images and colocali-
zation analysis of cells stained with mitochondria (Pearson coeꢃ-
cient=0.52), and lysosome (Pearson coeꢃcient=0.54). Scale bar:
20 μM. The representative ꢀuorescence line proꢂles of the white
arrows show colocalization eꢃciencies
3
.5 Subcellular localization
OHOA in cells, ꢀuorescent localization probes of mito-
chondria and lysosome were applied for colocalization
inside cells (Fig. 4b). The merged images demonstrated
that OHOA mainly accumulated in the lysosome with a
Pearson coeꢃcient of 0.54 and in mitochondria with a
Pearson coeꢃcient of 0.52. These phenomena indicated
that OHOA could optimize the imaging results for moni-
toring therapy processes to ensure anticancer therapeutic
eꢁects.
Since the OHOA has high ꢀuorescence quantum yield, the
intracellular imaging eꢃciency of OHOA during therapy
on HeLa cells was studied via confocal laser scanning
microscopy. OHOA could easily pass through the cell
membrane and visualize cells morphology before or after
treatment. Images indicated that OHOA predominantly
distributed in the cytoplasm rather than in the nucleus.
Subsequently, to explore the subcellular localization of
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509
Fig. 5 a Eꢁects of OHOA in dark or irradiated with blue light on the
green ratio). Data are expressed as mean± SD of three separate sets
of independent experiments. Signiꢂcant diꢁerences from OHOA in
dark: *p<0.05; **p<0.01; ***p<0.001
△
of Hela cells. Cells were treated with OHOA under various con-
Ψm
ditions (OHOA in dark, OHOA with 5/15/30 min light irradiation).
Scale bar: 20 μM. b Quantitative ꢀuorescence analysis of JC-1 (red/
3
.6 OHOA induces cell apoptosis
with OHOA under diꢁerent conditions. Red ꢀuorescence
appeared when no irradiation was performed (Fig. 5a).
Green ꢀuorescence became stronger and stronger when irra-
diation time was increased from 5 to 15 min. The ratio of
red/green ꢀuorescence intensity representing mitochondrial
membranes decreased from 5.46 (OHOA in dark) to 0.03
(irradiation with light for 30 min), as shown in Fig. 5b. This
observation indicates that light can trigger the generation of
ROS in mitochondria in situ. ROS damaged mitochondria
and led to a signiꢂcant decrease in membrane potential.
To further determine the cell apoptosis mechanism of
OHOA in the PDT process, we investigated the expression
Furthermore, on the basis of mitochondrial location behav-
ior of ꢀuorescence confocal imaging, a series of trials were
designed and conducted on HeLa cells to conꢂrm the detec-
tion of OHOA-PDT-induced cell apoptosis. Generation of
singlet oxygen in mitochondria can trigger a rapid apop-
totic response in cell. Mitochondrial membrane potential
is a marker of mitochondrial disorder associated closely
with apoptosis. We chose JC-1 dye as the sensor to detect
mitochondrial membrane potential of HeLa cells incubated
Fig. 6 Western blot assays of OHOA-PDT on expression of caspase 3 and cleaved-caspase 3, PARP, cleaved-PARP. GAPDH was determined as
an internal reference protein
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510
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Fig. 7 a DNA fragmentation and chromatin condensation were
observed under diꢁerent conditions. OHOA-PDT-induced HeLa cell
apoptosis in a light dose-dependent manner. b OHOA with light-
induced apoptosis of HeLa cells analyzed by ꢀow cytometry after
Annexin V-FITC and PI staining
levels of the caspase family, which plays a key role in
the apoptotic process. The protein levels in the apoptotic
pathway, caspase 3 and PARP (poly ADP-ribose polymer-
facilitate downstream activation of caspase 3, which sub-
sequently leads to the cleavage of PARP and triggered the
caspase-3-dependent apoptotic pathway. The cleaved-cas-
pase 3 and cleaved-PARP were increased in an irradiation
time-dependent manner (Fig. 6).
ase) were assayed for the OHOA- and light-treated HeLa
1
cells. After receiving an apoptotic O stimulus generated
2
in the intracellular matrix or mitochondria, cytochrome
c is released from the intermembrane space of the mito-
chondria into the cytoplasm. Then, cytochrome c in the
cytoplasm executes a series of functions to recruit and
ROS overproduction can induce DNA destruction in
the nuclei of cancer cells [42], we subsequently selected
Hoechst 33342 nuclear staining to observe the changes
in cell morphologies induced by OHOA. Morphologies
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Photochemical & Photobiological Sciences (2021) 20:501–512
511
of HeLa cells were imaged by ꢀuorescence microscope
Acknowledgements This work was financial supported by
National Key Research and Development Program of China
(
Fig. 7a). The chromatin ꢀuorescence of OHOA treated
(
(
2018YFD0200100), National Natural Science Foundation of China
No. 21877039), Science and Technology Commission of Shanghai
cells in dark conditions stained dimly and was observed
in the majority of the cells, indicating that OHOA treat-
ment without illumination did not cause any damage to
HeLa cells. HeLa cells incubated with OHOA and light
stimulation underwent signiꢂcant chromatin condensa-
tion, shrinkage, formed bubbles and DNA fragmenta-
tion. In addition, the chromatin ꢀuorescence intensity
was notably enhanced. This phenomenon indicated that
ROS generated in the intracellular matrix by OHOA-
PDT increased the level of DNA damage. To explore the
light-induced apoptosis eꢁects of OHOA, the percentage
of apoptotic cells was analyzed by ꢀow cytometry using
an Annexin V–FITC/PI apoptosis kit (Invitrogen™). In
Municipality (16391902300) and Innovation Program of Shanghai
Municipal Education Commission (2017-01-07-00-02-E00037).
Author contributions XS designed experiments; QX and YJ carried
out experiments; MC analyzed experimental results. QX and XS wrote
the manuscript.
Data availability All data generated or used during the study appear
in the submitted article and supplementary information. The data used
to support the ꢂndings of this study are also available from the cor-
responding author upon request.
Declarations
ꢀ
ow cytometry assays, Annexin V–FITC–/PI– (viable
Conflict of interest The authors declare that they have no conꢀict of
cells), Annexin V–FITC+/PI– (early apoptotic cells), and
Annexin V–FITC+/PI+ (necrotic or late-stage apoptotic
cells) were used to determine cell populations at diꢁerent
stages of cell death. A high eꢃciency of OHOA-mediated
PDT was observed. An increased proportion of the apop-
totic cells were observed following prolonged irradiation
times (Fig. 7b). When the irradiation time is extended from
interest.
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In summary, we synthesized and characterized a phen-
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improved water solubility and relatively longer absorption
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