Multiple Functions Integrated inside a Single Molecule for Amplification of Photodynamic Therapy Activity

Article abstract of DOI:10.1021/acs.molpharmaceut.9b00893

Nitric oxide (NO) can play both prosurvival and prodeath roles in photodynamic therapy (PDT). The generation efficiency of peroxynitrite anions (ONOO^-), by NO and superoxide anions (O₂ ^?-), significantly influenced the outcome. Reports indicated that such efficiency is closely related to the distance between NO and O₂ ^?-. Thus, in this manuscript, l-arginine (Arg) ethyl ester-modified zinc phthalocyanine (Arg-ZnPc) was designed and synthesized as a photosensitizer (PS) and NO donor. Post light irradiation, the guanido of Arg-ZnPc can be effectively oxidized by the generated reactive oxygen species (ROS) in the PDT process to release NO. Such a strategy could ensure O₂ ^?- and NO generation in the same place at the same time to guarantee effective ONOO^- formation. In addition, NO has other multiple synergistic cancer treatment functions, including tumor tissue vasodilatation for drug extravasation promotion, P-glycoprotein (P-gp) downregulation for drug efflux inhibition, and glutathione depletion for cancer cell endogenous antioxidant defense destruction. In vitro and in vivo results indicated that the effective ONOO^- formation and multiple functions of Arg-ZnPc could synergistically enhance its PDT activity and ensure satisfactory cancer treatment outcome.

Full text of DOI:10.1021/acs.molpharmaceut.9b00893

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Article
Multiple functions integrated inside a single molecule
for amplification of photodynamic therapy activity
Xianqing Shi, Qichen Zhan, Yanqing Li, Lin Zhou, and Shaohua Wei
Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/
acs.molpharmaceut.9b00893 • Publication Date (Web): 05 Dec 2019
Downloaded from pubs.acs.org on December 5, 2019
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Multiple functions integrated inside a single molecule for amplification of photodynamic
therapy activity
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Xianqing Shi1, Qichen Zhan1, Yanqing Li1, Lin Zhou1*, Shaohua Wei1,2*
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1
College of Chemistry and Materials Science, Jiangsu Key Laboratory of Biofunctional
Materials, Jiangsu Collaborative Innovation Centre of Biomedical Functional Materials, Key
Laboratory of Applied Photochemistry, Nanjing Normal University, Nanjing, Jiangsu 210023,
China.
2 School of Chemistry and Chemical Engineering, Yancheng Institute of Technology, Yancheng,
Jiangsu 224051, China.
E-mail: zhoulin@njnu.edu.cn (L. Zhou), shwei@njnu.edu.cn (S.H. Wei).
X.Q. Shi and Q.C. Zhan contributed equally to the work.
ABSTRACT: Nitric oxide (NO) can play both pro-survival and pro-death roles in photodynamic
therapy (PDT). The generation efficiency of peroxynitrite anions (ONOO−), by NO and
superoxide anions (O2˙−), significantly influenced the outcome. Reports indicated that such
efficiency is closely related to the distance between NO and O2˙−. Thus, in this manuscript, L-
arginine (Arg) ethyl ester modified zinc phthalocyanine (Arg-ZnPc) was designed and
synthesized as a photosensitizer (PS) and NO donor. Post light irradiation, the guanido of Arg-
ZnPc can be effectively oxidized by the generated reactive oxygen species (ROS) in PDT
process to release NO. Such strategy could ensure O2˙− and NO generation in the same place
at the same time to guarantee the effective ONOO− formation. In addition, NO has other multiple
synergistic cancer treatment functions, including tumor tissue vasodilatation for drug
extravasation promotion, P-glycoprotein (P-gp) down-regulation for drug efflux inhibition and
glutathione (GSH) depletion for cancer cell endogenous anti-oxidant defense destruction. In
vitro and in vivo results indicated that the effective ONOO− formation and multiple functions of
Arg-ZnPc could synergistically enhance its PDT activity and ensure its satisfactory cancer
treatment outcome.
Keywords: Zinc phthalocyanine; nitric oxide; photodynamic therapy; synergistic treatment.
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INTRODUCTION
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PDT utilizes non-toxic PS, light and O2 to generate cytotoxic ROS, including singlet oxygen
(1O2), O2˙−, hydroxyl radicals (HO˙), hydrogen peroxide (H2O2), et al, to kill cancer. 1 PDT has
been authorized by the U.S. food and drug administration for oncotherapy because of its low
system toxicity and slight trauma. 2
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In recent years, gas, such as NO, 3 CO, 4 SO2, 5 H2S, 6 et al., attracted great attention in
disease treatment. Under normal physiological condition, above gases are necessary
physiological messengers with important biological function and negligible toxicity. However,
elevated concentrations of these gases can kill cancer cells directly. 5 And they also can play
synergistic tumor suppression function with other treatment modality, such as chemotherapy,
3, 7
photothermal therapy, et al.
Using these gases to influence diverse physiological and
pathophysiological processes for direct or assistant disease treatment was denoted as gas
therapy. 8
NO is a gaseous molecular messenger among the gas transmitter family. Several of its
functions, such as down-regulating P-gp expression to inhibit drug efflux, 9 reacting with O2˙− to
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generate high biocidal ONOO−,
promoting vasodilatation to enhance drug tumor
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accumulation and damaging GSH to break anti-oxidant defense, are beneficial to tumor
therapy in PDT. However, different researches indicated that NO could play both negative (pro-
survival) 13-16 and positive (pro-death) 17-19 roles in PDT treatment. 20 These controversial results
closely related to PDT dose 19 and NO treatment timing, location or concentration. 21
In addition, the generation efficiency of ONOO−, a reactive nitrogen species (RNS) formed
by O2˙− and NO, 21 is the predominant pro-cell death mechanism in NO treatment on account
of its high oxidative damage activity. However, if the distance between O2˙− and NO source is
22
not close enough, ONOO− formation efficiency would be ultra-low. In addition, the half-life
time of NO is extremely short (~ 1 s) and it is rapidly converted into nitrate by oxyhemoglobin
in blood. 23 Therefore, it is significant to control the release of NO inside tumor cells. Co-loading
PS and NO donor (with tumor microenvironment or ROS response release function) inside one
24-27
nanoscale carrier is a classical way to meet the above requirements.
As an alternative
option, integration ROS responsive NO donor within PS could be an ideal method to utilize NO
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in PDT process because such strategy could ensure O2˙− and NO generation in the same place
at the same time to guarantee the effective ONOO− formation.
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Guanido of arginine could be oxidized by ROS to release NO. 28 Thus, in this manuscript,
L-arginine ethyl ester modified ZnPc (Arg-ZnPc) was designed and synthesized as a PS with
ROS responsive NO donor. As shown in Scheme 1, post trapping by cancer cells and irradiating
by light, Arg-ZnPc could generate abundant ROS (including O2˙−) to play PDT activity and
trigger NO release in the same place at the same time. Thus, the effective ONOO− generation
could guarantee the synergistic oxidative damage of ROS and RNS. Furthermore, NO could
promote tumor vasodilatation and down-regulate cancer cells P-gp expression to enhance drug
extravasation and retention at tumor tissue. Reduced GSH builds endogenous anti-oxidant
defense inside cells. 29 And many recent researches indicated that the high concentrations of
reduced GSH in cancer cells could consume ROS to reduce PDT activity. 30-32 NO could deplete
GSH to avoid ROS or RNS nontherapeutic consumption and further enhance their oxidative
damage efficiency. Furthermore, L-lysine (Lys) ethyl ester modified ZnPc (Lys-ZnPc), with
similar structure to Arg-ZnPc but without guanido, was synthesized as a control drug to prove
the released NO from guanido of Arg-ZnPc dominated above synergistic functions. All
researches indicated that integration ROS responsive NO donor within PS was an ideal method
to utilize NO in PDT process.
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Scheme 1. Mechanism diagram of multiple functions integrated inside Arg-ZnPc for its PDT
activity amplification (I: NO and react with O2•- to regenerate ONOO- to kill cancer cells directly;
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II: tumor tissue vasodilatation can promote PS extravasation to enhance its accumulation at
tumor tissue; III: P-gp down-regulation can inhibit intracellular PS efflux to elevate its
concentration inside cancer cells; IV: GSH depletion destroy cancer cell endogenous anti-
oxidant defense to avoid ROS consumption).
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EXPERIMENT SECTION
Synthesis of Arg-ZnPc, Lys-ZnPc and Two-photon-excitable and NIR-emissive
Fluorescence Probe (TPNIR-FP). The synthetic routes of Arg-ZnPc and Lys-ZnPc were
shown in Scheme 2. Synthesis of TPNIR-FP was shown in Scheme 3. These compounds were
identified by infrared absorption spectra (IR), H NMR spectra, 13C NMR spectra and Mass
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spectra (MS) spectrometer.
Synthesis of ZnPc. The 4-(3, 4-dicyanophenoxy) benzoic acid was synthesized according
to our previous report. 33 Then above product (586.3 mg, 2.22 mmol) and Zn(OAc)2 (241.7 mg,
1.32 mmol) were dissolved in 1-pentanol (8 mL). Under the protection of N2, the reaction
temperature was heated to 90 °C for 1 h. Later, 1, 8-Diazabicyclo [5, 4, 0] undec-7-ene (DBU,
300 µL) was added and the temperature continued heating to 140 °C for reacting 12 h. Later,
when cooled down to room temperature, residual 1-pentanol was dislodged via vacuum
distillation. Then crude green product was treated via column chromatography, and ethyl
acetate/methyl alcohol = 1/3 and DMF/alkaline water = 2/1 (v/v) were chosen as developing
solvent, successively. The ZnPc could precipitate out by adjusting pH = 4, and then, dissolved
in alkali water to remove insoluble impurities. The green product was treated with distilled water
and acetone several times. Finally, pure product was received after vacuum dried (113.8 mg,
18.3%). M. P. ＞ 200 °C. IR (KBr, cm-1): 3460, 1700 (C = O), 1603, 1467, 1401, 1236, 1163.
1H NMR (400 MHz, d6-DMSO): δ (ppm) 8.88 (br, 4H, Pc-H), 8.49 (br, 4H, Pc-H), 8.22-8.11 (m,
8H, Ar-H), 7.83-7.78 (m, 4H, Pc-H), 7.58-7.48 (m, 8H, Ar-H). 13C NMR (100 MHz, d6-DMSO): δ
(ppm) 167.41, 161.48, 157.30, 151.35, 139.70, 133.87, 132.41, 126.87, 124.29, 121.40, 118.81,
118.30, 1112.63. MS-MALDI-TOF (m/z): calcd for C60H32N8O12Zn: 1122.3. Found [M + H]+
1122.3.
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Synthesis of Arg-ZnPc and Lys-ZnPc. ZnPc (52.7 mg, 46.96 μmol), 1-(3-
Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl, 68.3 mg, 356.40 μmol)
and 1-Hydroxybenzotriazole (HOBt, 51.1 mg, 378.20 μmol) were added into 6 mL N, N-
Dimethylformamide (DMF) and kept reacting for 1 h at 20 °C. In the ice-water bath, above
mixture was dropped into the L-Arginine ethyl ester hydrochloride (105.4 mg, 383.02 μmol) and
N, N-Diisopropylethylamine (DIPEA, 0.5 mL, 3.02 mmol) solution (4 mL DMF). After then, the
reaction was kept for another 1 h at 0 °C. Next, the mixture was added into CH2Cl2 (60 mL) and
the sediment was collected via centrifugation. The crude solid was dissolved in acidic aqueous
solution (pH = 4). After centrifugation, the supernatant was obtained, and adjusted pH to 10 by
10% (wt%) NaOH. The solid product was purified via centrifugation again, then rinsed with ethyl
acetate several times. Then, Arg-ZnPc (21.4 mg, 25.8 %) was afforded by vacuum drying. M.
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P. ＞ 200 °C. IR (KBr, cm-1): 3361, 1733 (ROC = O), 1646 (C = O), 1484, 1229, 1093. 1H NMR
(400 MHz, d6-DMSO): δ (ppm) 9.23 (dd, 4H, J1 = 6.92 Hz, J2 = 16.08 Hz, CONH), 8.95-8.80
(m, 8H, H of guanidino), 8.17-7.79 (m, 16H, Pc-H, Ar-H), 7.52-7.41 (m, 12H, Pc-H, Ar-H), 4.47
(s, 4H, CH), 4.16 (t, 8H, J = 6.68 Hz, CH2), 3.17 (s, 8H, CH2), 1.89 (s, 8H, CH2), 1.66 (s, 8H,
CH2), 1.25-1.16 (m, 12H, CH3). MS-MALDI-TOF (m/z): calcd for C92H96N24O16Zn: 1859.28.
Found: [M + H]+ 1859.30.
The synthesis of Lys-ZnPc was similar to Arg-ZnPc. M. P. ＞ 200 °C. IR (KBr, cm-1): 3414,
1726 (ROC=O), 1639 (C=O), 1480, 1236, 1083. 1H NMR (400 MHz, d6-DMSO): δ (ppm) 8.66-
8.46 (m, 8H, Ar-H), 8.27-8.08 (m, 8H, Pc-H), 8.05-7.95 (m, 4H, Pc-H), 7.59-7.46 (m, 4H, Ar-H),
7.42-7.27 (m, 4H, Ar-H), 4.53 (s, 4H, CH), 4.14-4.10 (m, 8H, CH2), 1.85 (s, 8H, CH2), 1.57 (s,
8H, CH2), 1.26-1.16 (m, 20H, CH2, CH3). MS-MALDI-TOF (m/z): calcd for C92H96N16O16Zn:
1747.2. Found: [M + H]+ 1747.2.
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R1
R1
N
OH
DBU
Zn(OAc)2
NC
NC
O
N
N
N
N
NC
NC
NO2
K2CO3
DMF
N
+
N
Zn
N
n-pentanol
COOH
COOH
4-(3,4-dicyanophenoxy)benzoic acid
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R1
R1
O
COOH
R2
R1
=
ZnPc
R2
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N
NH2HCl
NH
EDC HCl, HOBT
·
HN
DIPEA
N
N
ClHH2N
C2H5OOC
ZnPc
+
N
N
Zn
N
DMF
N
N
NH2
NH
O
C
HN
O
NH
R2=
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R3
R2
Arg-ZnPc
C2H5OOC
R3
N
EDC HCl, HOBT
·
OCH2CH3
O
DIPEA
N
N
N
N
ZnPc
ClHH2N
+
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Zn
N
DMF
NH2HCl
O
C
NH2
O
NH
R3
=
R3
R3
Lys-ZnPc
C2H5OOC
Scheme 2. Synthesis route of ZnPc derivatives.
Synthesis of TPNIR-FP. Two-photon-excitable and NIR-emissive amidogen (TPNIR-NH2)
was synthesized according to previous literature. 34 TPNIR-FP was used to detect ONOO− and
it was also synthesized according to previous literature. 35
O
N
HO
O
H2SO4
HClO4
O
O
N
+
H2N
H2N
TPNIR-NH2
OH
O
N
O
NH
O
O
TPNIR-NH2
Oxalyl chloride
DMF, CH2Cl2
O
+
H2N
N
TPNIR-FP
NO2
NO2
Scheme 3. Synthetic routes of TPNIR-FP.
Cell and Animals. Murine mammary carcinoma (4T1) and human cervical carcinoma
(Hela) cells were bought from the Cell Bank of Chinese Academy of Science (Shanghai, China).
The cells were incubated in Dulbecco’s modified eagle medium (DMEM) with 10% (v/v) fetal
bovine serum FBS in the incubator (5 % CO2, 37 °C). All animals were purchased from
Yangzhou University, the center for comparative medicine. The animal experiments were
conducted according to the Guide for the Care and Use of Laboratory Animals, which was the
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guidelines of Nanjing Normal University.
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Analysis of Anticancer Activity in Vitro. 3-(4, 5-dimethylthiazol-2-yl)-2, 5-
diphenyltetrazolium bromide (MTT) colorimetric assay was used to detect the dark and light
cytotoxicity, Hela cells were incubated on 96-well plates for 24 h. Arg-ZnPc or Lys-ZnPc was
treated with the concentration ranging from 0 to 10 μM for incubating in dark condition. Besides,
the drugs with the concentration of 2 μM were added inside cells respectively. After 48 h, the
cells were in dark or irradiated under 665 nm light emitting diode (LED, 5 W, light dose for in
vitro experiment was 0.4 W/cm2) for 4 min. Next, the cell survival ability was detected with MTT
assay using microplate reader.
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Cell cytotoxicity behaviors of Arg-ZnPc and Lys-ZnPc were also studied by flow cytometry
(FCM), respectively. The cells, treated with Arg-ZnPc or Lys-ZnPc, were illuminated by light for
4 min. After incubating for 24 h, the apoptosis properties were detected using the apoptosis
assays kit (Annexin V/FITC-PI). Besides, the Hoechst 33342, which the concentration was 25
μg mL-1 in DMEM, was put into the irradiated cells and cultured in dark for another 0.5 h. Then,
blue fluorescence in above cells was observed with the confocal laser scanning microscopy
(CLSM, 405 nm laser, 100 mW, 5% strength).
In Vivo Anticancer Activity. The BALB/c mice (female, ~20 g) bearing tumor model was
built via hypodermic injection with 4T1 cells. These mice were grouped 3 teams randomly when
the tumor size was reached about 100 mm3. Then the mice were intravenously injected with
saline, Arg-ZnPc or Lys-ZnPc (the dosage of drugs was 2 mg kg-1), respectively. Then, tumor
sizes of mice were recorded every day with dark condition or irradiated by 665 nm LED (5 W,
light dose for in vivo experiment was 0.9 W/cm2) for 30 min. The tumor length (L), width (W)
and the weight of mice were recorded during the treatment. Then the above mice were
sacrificed after 14 days and dissected to obtain the main organs, which included heart, liver,
spleen, lung, kidney and brain. The calculation formula of tumor volume (V) was: V (mm3) =
L×W2 / 2. And the tumor suppression effect of the two ZnPc was evaluated by comparing their
Vt/V0 value during 14 days’ treatment (Vt: represented the tumor volume post t days treatment;
V0: represented tumor volume at day 0)
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ROS and RNS Detection in PDT Process. The fluorescence intensity of ROS and
reactive nitrogen species (RNS) could be detected via chemical process with 9, 10-
anthracenedip-ropionic acid (ADPA), dihydroethidium (DHE), 3-Amino, 4-aminomethyl-2′, 7′-
difluorescein, diacetate (DAF-FM DA) and TPNIR-FP as the probe to monitor the fluorescence
changes. Extracellular 1O2 generation was detected by ADPA. The interaction between ADPA
and 1O2 could produce its endoperoxide, inducing the decrease of its absorbance intensity (the
λmax of ADPA was 378 nm). 18 μL ADPA (6 mM) was mixed with 3 mL aqueous of Arg-ZnPc or
Lys-ZnPc. Immediately, the above samples were irradiated with a 665 nm irradiation. The
above spectra were recorded from 0 to 3 min.
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Then, the formation of NO was detected with DAF-FM DA test kits. 3 μL of DAF-FM DA in
DMSO was hydrolyzed to DAF-FM by 0.01 M NaOH (3 mL) for 20 min at room temperature.
And the reaction was stopped with 3 mL phosphate buffered saline. DAF-FM (2.5 μM) was
mixed with aqueous of Arg-ZnPc or Lys-ZnPc. The fluorescence intensity was detected from 0
to 54 min, which the excitation wavelength was 495 nm. The fluorescence changes of
superoxide anions and ONOO− with time were measured respectively.
Besides, DHE or TPNIR-FP were mixed with aqueous solution, respectively. Then the
drugs of Arg-ZnPc or Lys-ZnPc were put into the solution. Immediately, the solution was
irradiated by light with different time (DHE: from 0 to 6 min; TPNIR-FP: from 0 to 12 min).
The corresponding ROS and RNS probes, including singlet oxygen sensor green reagent
(SOSG), DHE, DAF-FM DA, TPNIR-FP, 2’ 7’-Dichlorofluorescin diacetate (DCFH-DA), were
1
served to monitor O2, O2˙−, NO, ONOO− and H2O2 generation in cells post treating by drugs
and light. Cells were cultured in confocal dishes and treated with drugs (2 μM). After 48 h and
the samples were irradiated by light for 4 min, the probes (5 μM) as an indicator were added
into the dishes for 1 h. Then washing cells with phosphate buffered saline, the fluorescence
inside cells was observed via CLSM. The parameter settings of CLSM for ROS/RNS detection
were listed as follows: (1) For 1O2 detection using SOSG: 488 nm laser, 150 mW, 15% strength;
(2) For O2˙− detection using DHE: 561 nm laser, 150 mW, 10% strength; (3) For NO detection
using DAF-FM DA: 488 nm laser, 15% strength; (4) For ONOO- detection using TPNIR-FP: 561
nm laser, 150 mW, 5% strength; (5) For ROS/RNS detection using DCFH-DA: 488 nm laser,
150 mW, 15% strength.
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Besides, the NO, ROS and RNS generation in cells using their specific scavengers.
Carboxy-PTIO (c-PTIO), ADPA, tempol and L-cysteine were selected as the scavenger of NO
1O2, O2˙− and ONOO-, respectively. Briefly, c-PTIO (100 μM), ADPA (500 μM), tempol (1500
μM) or L-cysteine (500 μM) was added in cells for incubation 1 h in advance. Then Arg-ZnPc
was treated and their corresponding probes were added as described above, respectively,
while the other sample was directly treated Arg-ZnPc to compare their fluorescence intensity.
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Detection of GSH Content in Vitro. GSH content was measured by GSH and oxidized
glutathione (GSSG) assay kit. Briefly, the preparation of total glutathione content was tested as
follows. Cells were cultured in 6-well plates and treated by Arg-ZnPc or Lys-ZnPc. Then the
one kind of above samples were incubated in dark and another incubated with 4 min light
irradiation. After that, the cells were washed and centrifuged. After removing the supernatant,
the protein removal reagent was added and vortexed sufficiently, which the content was three
times than the cells volume. The samples were frozen and thawed twice by liquid nitrogen and
37 °C water bath, and placed in 4 °C for 5 min. After that, the samples were centrifuged at
10000 g (4 °C) for 10 min. The obtained supernatant was used for the determination of total
glutathione. The preparation of GSSG content was detected as follows. Part of the above
samples were added into the diluted GSH scavenging auxiliary solution at the rate of 20 μL per
100 μL, then vortex immediately. GSH scavenging working fluid was added at the rate of 4 μL
per 100 μL, then vortex immediately and reacted at 25 °C for 60 min. The above treatment
could be used for the measurement of GSH levels.
Besides, GSH probe was synthesized to detect the GSH levels in cells. Cells were
incubated in confocal dishes and treated with different drugs. After 4 min light irradiation, GSH
probe (5 μM) was added and the green fluorescence was observed by CLSM (488 nm laser,
150 mW, 5% strength) after 30 min incubation.
P-gp Expression and Uptake of Drugs in Vitro. P-gp expression was quantified by
western blot assay. Cells were cultured in 6-well plates and treated by Arg-ZnPc or Lys-ZnPc.
After 48 h, the cells were irradiated by light for 4 min and incubated for another 4 h. Later, the
cells were lysed by radio immune precipitation assay (RIPA) and collected after centrifugation
to obtain the total protein. Also, the protein quantification was detected by bicinchoninc acid
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protein assay reagent. The protein samples were resolved using sodium dodecyl sulfate
polyacrylamide gel electrophoresis. Next, the above samples were transferred to polyvinylidene
fluoride membrane, and ponceaux was used to stain them for about 1 min and washed by tris-
buffered saline with Tween (TBST). After that, the membranes were sealed using 5% nonfat
milk and incubated with antibodies of P-gp (1:500) and glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) (0.8:3000) at 4 °C. Overnight, the secondary antibody was added for
incubating at room temperature. P-gp protein was detected by enhanced chemiluminescence
system (ECL).
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Cells were cultured in confocal dishes and the drugs were treated for incubating different
time, including 8 h, 48 h, 72 h and 96 h, and red fluorescence was observed with CLSM (647
nm laser, 200 mW, 10% strength). Meanwhile, cell uptake amount could be calculated by UV-
Vis spectrum. After seeded cells in 24-well plates, the Arg-ZnPc or Lys-ZnPc was treated and
incubated another 8 h, 24h, 45h, 48 h, 72 h and 96 h using colorless DMEM with 1% FBS. Then
the supernatant was removed and measured the UV absorbance value.
Fluorescence Imaging and Enhanced Permeability and Retention in Vivo. The
BALB/c mice bearing tumor model were established by subcutaneous injection using 4T1 cells.
2 mg kg-1 of drugs were administrated by intravenous injection three times and the above mice
were sacrificed, dissected them to achieve visceral organs, including heart, liver, spleen, lung
and kidney, and tumors for the fluorescence imaging.
Evans blue assay was used to verify the NO generation inducing the enhancement of
vascular permeability. 10 mg kg-1 of evans blue was intravenous injected to the mice.
Immediately the drugs were subcutaneous and intravenous injected (2 mg kg-1 of drugs have
been treatment for 3 days) to the mice and irradiated for 10 min. After 2 h, the mice were
dissected for obtaining the target skin and tumor, then the above skin and tumor were immersed
with methanamide (100 mg mL-1) in 60 °C for 24 h so as to detect the value of the evans blue
extravasation at the 470 nm absorbance.
The Analysis of Data. The statistical analysis was performed by ANOVA and the value of
p<0.05 was considered meaningful. The value of *p<0.05, **p<0.01 and ***p<0.001 were the
drugs treated groups versus control and the value of #p<0.05, # #p<0.01 and # # #p<0.001 were
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Lys-ZnPc treated group versus Arg-ZnPc treated group.
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RESULTS AND DISCUSSION
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Synthesis and Characterization of Arg-ZnPc and Lys-ZnPc. To prove the released NO
from guanido of Arg-ZnPc guarantee the synergistic anticancer effect, Lys-ZnPc, with similar
structure to Arg-ZnPc but without guanido, was synthesized and its PDT activity was studied
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and compared with Arg-ZnPc. A mixture of four possible structural isomers, C4h, C2v, Cs, and
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D2h, is obtained from the synthesis of tetra-substituted phthalocyanines.
However, the
In this study,
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isomers are difficult to separate. But, generally, C4h is the major isomer.
Arg-ZnPc and Lys-ZnPc were obtained as isomer mixtures. But Arg-ZnPc and Lys-ZnPc are
modified from a tetra-carboxyl substituted ZnPc. Thus, we proposed that they could have similar
isomer form ratio. The chemical structure (C4h isomer form) of Arg-ZnPc and Lys-ZnPc were
presented in Figure 1A and Figure S1. Furthermore, Arg-ZnPc and Lys-ZnPc were verified by
IR, 1H NMR spectra, and MS spectrometer. The molar extinction coefficients of Arg-ZnPc and
Lys-ZnPc at various wavelength (εmonomer, εaggregator and ε665nm of light source) via the Lambert-Beer
law were listed in Table S1 and Figure S2.
In Vitro Anticancer Activity. In vitro toxicity of ZnPcs were assessed by MTT assay.
Without light irradiation, the two ZnPcs showed weak toxicity in cancer cells at low
concentration (< 4 μM, Figure S3). Thus, we chose the drug concertation of 2 μM for further
phototoxicity and in vitro anticancer mechanism analysis to avoid their dark toxicity influence.
However, after light irradiation, the viability of Arg-ZnPc (2 μM) treated cells rapidly decreased
but the viability of Lys-ZnPc (2 μM) treated cells only showed slightly decreased (Figure 1B).
In addition, the cancer cell apoptosis by the two ZnPcs post light irradiation were studied
by FCM assay. 41 The apoptotic cells percent (Q2 and Q3 zones) in Arg-ZnPc treated cells post
light irradiation was about 66.9%. However, only 11.0% of apoptotic cells could be observed in
Lys-ZnPc treated cells post same treatment (Figure 1C and 1D).
The nuclei of the cells could be stained for DNA with Hoechst 33342 dye with blue
fluorescence signal. 42 The fluorescent signal in control cells for Hoechst 33342 were dimmed
with uniform distribution pattern. In apoptotic cells, their DNA condensed and their Hoechst
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33342 signal should be bright with concentrated distribution pattern. Thus, apoptosis induced
nuclear condensation can therefore be used to distinguish Hoechst 33342 signal changes. As
shown in Figure 1E and Figure S4, DNA condensing degree of Arg-ZnPc group was more
significant comparing with Lys-ZnPc group post light irradiation. All of above data proved the
superior light cytotoxicity of Arg-ZnPc rather than Lys-ZnPc.
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Figure 1. The chemical structure of Arg-ZnPc and Lys-ZnPc (A, only the major C4h isomer is
shown for Arg-ZnPc and Lys-ZnPc, which likely contains other isomers); light toxicity
comparison of Arg-ZnPc and Lys-ZnPc post 665 nm light irradiation (B); cell apoptosis by Arg-
ZnPc and Lys-ZnPc by FCM assay (C) and their relative events percent comparison (D);
Hoechst 33342 staining pattern in control, Arg-ZnPc and Lys-ZnPc post light irradiation (E).
In Vivo Antitumor Activity. As shown in above in vitro results, without light irradiation, the
toxicity of Arg-ZnPc and Lys-ZnPc were very weak, which could be sure their safety in vivo
treatment. However, after irradiating the PS accumulated tumor tissue, they could show tumor
suppression effect. To verify this hypothesis and compare the safety in vivo and tumor
suppression activity of Arg-ZnPc and Lys-ZnPc, bilateral subcutaneous transplantation tumor
model was used.
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As shown in Figure 2A, during 14 days’ treatment, no significant weight loss in all groups.
And the obvious pathological changes were detected in main organs slices by Hematoxylin &
eosin (H&E) stain method (Figure 2B) (purple hematoxylin label nucleic acids and pink eosin
label inside and outside cell proteins 43) after 14 days’ treatment. These results proved the good
safety of the two ZnPcs.
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During 14 days’ treatment by Arg-ZnPc or Lys-ZnPc and light irradiation at tumor site, their
tumor volumes were recorded every day. As shown in Figure 2C, both Arg-ZnPc and Lys-ZnPc
could suppress tumor growth in comparison with the control mice. And treatment outcome of
Arg-ZnPc group was superior to Lys-ZnPc one. Besides, as shown in Figure S5, no obvious
tumor suppression effect was detected in Arg-ZnPc or Lys-ZnPc treated mice without light
irradiation. In addition, serious nuclear damage was detected in Arg-ZnPc group, which further
proved the effective tumor cell death post treatment (Figure 2D).
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Figure 2. The weight (A) and relative tumor volume (C) changes of the mice during 14 days’
treatment by various drugs and light irradiation at tumor tissue; H&E labeled main organs (B)
and tumor tissue (D) sections post 14 days’ treatment (Bar = 50 μm).
Synergistic Anticancer Mechanism of Arg-ZnPc
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RNS Synergistic Effect with ROS Based on NO Generation by Arg-ZnPc. O2 is a
predominant ROS in PDT. O2˙− and NO are key factors for ONOO− generation. Thus,
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extracellular O2, O2˙−, NO and ONOO- formation by Arg-ZnPc and Lys-ZnPc were studied
using ADPA, DHE, DAF-FM and TPNIR-FP probes by spectra method, separately.
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ADPA would convert to its endoperoxide by O2, which could induce its absorbance
intensity decreasing. Figure 3A showed the decrease in absorbance intensity of ADPA by Arg-
ZnPc or Lys-ZnPc, as a function of light exposure time, indicating their effective 1O2 generation
post light irradiation. The decrease degree of Arg-ZnPc was higher than that of Lys-ZnPc,
indicating that the 1O2 production capability of Arg-ZnPc was stronger than Lys-ZnPc.
DHE reacts with O2˙− to form a fluorescent product ethidium. As shown in Figure 3B, both
Arg-ZnPc and Lys-ZnPc could induce DHE fluorescence intensity enhancing, indicating their
effective O2˙− generation post light irradiation.
The guanido of Arg-ZnPc can effectively release NO via oxidization by ROS in PDT
process. DAF-FM could interact with NO to generate benzotriazole derivative with strong
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fluorescence.
It was observed that the fluorescence was significantly enhanced post the
treatment of Arg-ZnPc (Figure 3C) and light irradiation, indicating NO release. On the contrary,
no NO generation could be detected in Lys-ZnPc treated group.
O2˙− could react with NO to generate ONOO−, a RNS with higher biocidal activity than
ROS.18 According to previous reports, TPNIR-FP could react ONOO− to generate a product
(TPNIR-NH2) with strong red fluorescence. Thus, TPNIR-FP was used to detected ONOO−
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generation post light irradiation. As obviously observed in Figure 3D, the Arg-ZnPc could
effectively generate the ONOO−.
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Many researchers suggested that these isomers have similar physical and chemical
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properties.
And some researches indicated that the isomers of some phthalocyanines
could have different UV-visible absorption property, such as different monomer/aggregator
peak position, strength and ratio, which could influence their ROS generation ability and PDT
activity. 47-49 As shown in Figure S2, the monomer and aggregator peak for Arg-ZnPc and Lys-
ZnPc were similar but their monomer/aggregator peak strength and ratio were different. The
monomer form of ZnPc was more conducive for ROS generation. Even if Lys-ZnPc has more
monomer than Arg-ZnPc, the ROS production capability of Lys-ZnPc was lower than that of
Arg-ZnPc. Thus, the ROS generation capability enhancement of Arg-ZnPc comparing with Lys-
ZnPc was not due to its aggregation states and isomers existence.
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Figure 3. (A) O2 probe bleaching effect by Arg-ZnPc (A1) and Lys-ZnPc (A2) post light
irradiation, and (A3) absorbance intensity change degree as a function of irradiation time (A0
means the absorbance intensity at the 0 min and At means the absorbance intensity at the
other time); (B) O2˙− probe fluorescence enhancing effect by Arg-ZnPc (B1) and Lys-ZnPc (B2)
post light irradiation, and (B3) fluorescence intensity change degree as a function of irradiation
time (F0 means the fluorescence intensity at the 0 min and Ft means the fluorescence intensity
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at the other time); (C) NO probe fluorescence enhancing effect by Arg-ZnPc (C1) and Lys-ZnPc
(C2) post light irradiation, and (C3) fluorescence intensity change degree as a function of
irradiation time; (D) ONOO− probe fluorescence enhancing effect by Arg-ZnPc (D1) and Lys-
ZnPc (D2) post light irradiation, and (D3) fluorescence intensity change degree as a function of
irradiation time.
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In addition, intracellular 1O2, O2˙−, NO, ONOO− and ROS+RNS were detected by SOSG,
DHE, DAF-FM DA, TPNIR-FP and DCFH-DA probes using CLSM (Figure 4 and Figure S6-
S10). And most the intracellular detection obtained similar results as above extracellular
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detection. However, Arg-ZnPc is more efficient in O2 generation but less efficient in O2˙−
generation than Lys-ZnPc in aqueous solution. In contrast, for ROS detection inside cancer
cells, Arg-ZnPc is more efficient in 1O2 and O2˙− generation than Lys-ZnPc possibly because of
the difference between pure water and complicated cytoplasm. DCFH-DA could response to
both ROS and RNS to generate fluorescent DCF. 50 And Arg-ZnPc has higher total ROS and
RNS production ability than Lys-ZnPc to ensure its ROS-RNS synergistic oxidative damage.
Furthermore, under our experiment condition for ROS/RNS detection (drug incubation time
was 48 h), there were no obvious difference of cellular uptake efficiency between Arg-ZnPc
(the uptake percent was 88.76% ± 2.84%) and Lys-ZnPc (the uptake percent was 86.74% ±
7.75%) (Figure S11). Thus, the ROS/RNS generation differences between Arg-ZnPc and Lys-
ZnPc is due to the compound, but not the variability in cellular uptake. In addition, the NO, ROS
and RNS generation experiments were validated using their specific quenchers. c-PTIO (Figure
S12), ADPA (Figure S13), tempol (Figure S14) and L-cysteine (Figure S15) were selected as
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the scavenger of NO, O2, O2˙− and ONOO-, respectively. The NO, ROS and RNS probes
fluorescence intensity by Arg-ZnPc treated alone was higher than adding their corresponding
scavengers, which further validated NO, ROS and RNS generation.
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Figure 4. Fluorescence images and intensity comparison of intracellular 1O2, O2˙−, NO, ONOO−
and total ROS+RNS were detected by SOSG, DHE, DAF-FM DA, TPNIR-FP and DCFH-DA
probes (Bar = 10 μm).
GSH Depletion Function. GSH is an important antioxidant defense of cells and its
concentration in cancer cells is much higher than that in normal cells. ROS, NO and GSH are
mutual constraints. GSH could consume ROS to reduce PDT efficiency. Similarly, GSH is also
responsible for detoxifying NO-mediated cell damage. 32, 51 In turn, GSH could be depleted by
ROS and NO through oxidation or nitrosation mechanism. 12 And GSH depletion could promote
ROS oxidative damage efficiency and NO induced toxicity to enhance PDT and NO gas
treatment effect. Thus, during repeated in vivo treatment, the front therapy induced GSH
depletion could be helpful for subsequent PDT and NO combination treatment, which could
obtain satisfied tumor suppression effect finally. Intracellular GSH depletion by the two ZnPcs
were studied by GSH assay kit and GSH fluorescence probe. 52 After light irradiation, comparing
with Lys-ZnPc, GSH depletion degree by Arg-ZnPc was aggravated possibly due to the
combination contribution from ROS and NO (Figure 5 and Figure S16).
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Figure 5. (A) Intracellular GSH concentration change in Arg-ZnPc or Lys-ZnPc treated cells
before (A) and after (B) light irradiation from the detection results by GSH and GSSG assay kit;
(C) intracellular GSH probe fluorescence intensity change in Arg-ZnPc or Lys-ZnPc treated
cells before and after light irradiation (Bar = 10 μm).
P-gp Inhibition by NO to Avoid Drug Efflux. P-gp, a cell membrane protein, contributing
to the development of drug resistance in cancer by promoting drug efflux, which could induce
intracellular drug concentration decreasing to reduce activity. Reports indicated that NO can
inhibit the P-gp expression at ultra-low concentration. 53 Thus, we proposed that Arg-ZnPc also
has the P-gp expression inhibition function post NO release to avoid ZnPc efflux. The P-gp
expression in cancer cells and tumor tissue were effectively inhibited in Arg-ZnPc treated group,
which could be helpful for drug retention inside cancer cells. On the contrary, no obvious P-gp
expression changing was detected in control and Lys-ZnPc treated group (Figure 6A and 6B),
verifying P-gp expression was inhibited by NO.
To verify P-gp inhibition could avoid ZnPc efflux, cellular uptake effect of Arg-ZnPc or Lys-
ZnPc was studied and compared with the function of prolonged incubation time. As shown in
Figure 6C and Figure S17-S18, the cellular uptake percent of the two ZnPc were gradually
increased from 8 to 48 h. After 48 h, obviously drug efflux could be detected. However, the drug
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efflux effect of Arg-ZnPc was lower than Lys-ZnPc. These results indicated that P-gp down-
regulation by NO could inhibit drug efflux, which could helpful for the enhanced PDT activity.
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The drug efflux experiments were carried out in the dark. There was intrinsic ROS in cancer
cells. Arginine modified compound can respond to intrinsic ROS to generate NO (Figure S19).
54 However, the amount was not enough for directly killing cancer cells since Arg-ZnPc was not
induced effective cell death without light irradiation under our experiment condition ([Arg-ZnPc]
= 2 μM). But, under such condition, P-gp down-regulation can be detected.
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Figure 6. Western blot (A1) and quantitative analysis (A2) for the detection of P-gp expression
in cancer cells post various drugs and light irradiation; western blot (B1) and quantitative
analysis (B2) for the detection of P-gp expression in tumor tissue post various treatment; (C)
detection of drug efflux effect with the prolonged incubation time (Bar = 10 μm).
Relaxation of Blood Vessel to Promote Drug Accumulation at Tumor. NO could
promote tumor vasodilatation to enhance blood vessel permeability, and therefore promote
drug (including nano-sized and molecular medicine) extravasation at tumor tissue. 55 To verify
above hypothesis, the influence of NO from Arg-ZnPc on vascular permeability was studied in
mouse dorsal skin model and subcutaneous transplantation tumor model. Evans blue assay is
a classic method to measure vascular permeability. The subcutaneous injection of Arg-ZnPc at
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dorsal skin of mice and light irradiation induced obvious evans blue leaking around the injection
position (Figure 7A and 7B). On the contrary, evans blue leaking increasing in Lys-ZnPc treated
one was weak. Similar results were obtained in subcutaneous transplantation tumor model.
Obvious evans blue extravasation could be observed in Arg-ZnPc and light treated (at tumor
position) post tail vein injection comparing with Lys-ZnPc or control group (Figure 7C and 7D).
The results suggested that NO release from Arg-ZnPc could enhance tumor vascular
permeability and improve the extravasation at tumor tissue.
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In order to confirm the enhanced drug extravasation at tumor by NO, in vivo distribution of
Arg-ZnPc and Lys-ZnPc was studied and compared. As shown in Figure 7E and 7F, tumor
accumulation ability of Arg-ZnPc was obviously superior to Lys-ZnPc. In addition, a recent
research indicated that the isomers of some phthalocyanines could have different
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pharmacokinetic property in a patient.
distribution of various ZnPc isomers.
And we proposed such property would influence
Figure 7. Experiments in skin (A) and tumor (C) for illustrating the enhanced vascular
permeability effect using the evans blue assay; the quantification of the evans blue
extravasation by methanamide from skin (B) and tumor (D); (E) in vivo imaging of tumor bearing
mice after intravenous injection of saline, Arg-ZnPc and Lys-ZnPc post 3 days treatment and
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the mean fluorescence intensity of their major organs and tumor (F).
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PDT, a FDA approved cancer treatment modality, utilizes non-toxic PS, light and O2 to
generate cytotoxic ROS to kills cancer cells. Gas therapy uses various gases to influence
diverse physiological and pathophysiological processes for disease treatment. Many
researches indicated that the combination of gas therapy and other traditional cancer therapy
modality was an effective way to obtain satisfied cancer treatment outcome. However,
combination of NO and PDT could obtain positive or negative effect for cancer treatment since
NO can play either pro-survival or pro-death roles in PDT. Reports indicated that the high
biocidal ONOO- formation efficiency by NO and O2˙− determined the synergistic treatment
outcome. And the distance between NO and O2˙− can decide ONOO- generation performance.
Thus, here, an arginine modified ZnPc was designed and synthesized as bifunctional drug
(photosensitizer and NO donor) and a lysine modified ZnPc was synthesized as a control drug.
PDT process generates various ROS, including O2˙−, efficiently. Simultaneously, the ROS can
oxidize guanido of Arg-ZnPc to release NO. NO and O2˙− generation at the same place and
time can ensure effective ONOO- formation. Furthermore, NO can promote tumor vasodilatation
and down-regulate cancer cells P-gp expression to enhance drug extravasation and retention
at tumor tissue. NO also can deplete reduced GSH inside cancer cells to avoid its ROS
consuming. These synergistic functions of NO further improve PDT treatment activity. In vitro
and in vivo results all indicated that the anticancer activity of Arg-ZnPc was superior to Lys-
ZnPc. Thus, integrating ROS response NO donor within PS was an ideal strategy to utilize NO
for gas therapy and PDT combination for cancer treatment.
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ASSOCIATED CONTENT
Supporting Information.
The Supporting Information is available free of charge on the ACS Publications website.
Supplemental data including materials and instrumentation, MS-MALDI-TOF spectrometer,
molar extinction coefficients and UV-Vis spectra, dark toxicity, fluorescent images and intensity,
uptake percent and references.
ACKNOWLEDGEMENTS
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This work was supported by the National Natural Science Foundation of China (grant
number 21671105), the project (grant number BK20161554) supported by NSF of Jiangsu
Province of China, the Priority Academic Program Development of Jiangsu Higher Education
Institutions and the Foundation of Jiangsu Collaborative Innovation Centre of Biomedical
Functional Materials (grant number 161090H001), the New Technologies and Methods of
Scientific Instrument and Equipment Sharing Service Platform in Jiangsu province and the
Postgraduate innovation Project of Jiangsu Province (grant number 1812000006477).
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ABBREVIATIONS: NO, nitric oxide; PDT, photodynamic therapy; ONOO−, oxidizing
peroxynitrite anions; O2˙−, superoxide anions; Arg, L-arginine; ZnPc, zinc phthalocyanine; Arg-
ZnPc, L-arginine ethyl ester modified ZnPc; ROS, reactive oxygen species; P-gp, P-
glycoprotein; GSH, reduced glutathione; PS, photosensitizers; 1O2, singlet oxygen; HO˙,
hydroxyl radicals; H2O2, hydrogen peroxide; RNS, reactive nitrogen species; Lys-ZnPc, L-lysine
ethyl ester linked ZnPc; TPNIR-FP, two-photon-excitable and NIR-emissive fluorescence probe;
DBU, 1, 8-diazabicyclo [5, 4, 0]-undec-7-ene; DMSO, dimethyl sulfoxide; EDC·HCl, 1-(3-
Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; HOBt, 1-Hydroxybenzotriazole; DMF,
N, N-dimethyl-formamide; DIPEA N, N-Diisopropylethylamine; TPNIR-NH2, two-photon-
excitable and NIR-emissive amidogen; DMEM, dulbecco's modified eagle media; FBS, fetal
bovine serum; MTT, 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide; LED, light
emitting diode; FCM, flow cytometry; CLSM, confocal laser scanning microscope; ADPA, 9, 10-
anthracenedip-ropionic acid; DHE, dihydroethidium; DAF-FM DA, 3-Amino, 4-aminomethyl-2′,
7′-difluorescein, diacetate; SOSG, singlet oxygen sensor green reagent; DCFH-DA, 2, 7-
dichlorofluorescin diacetate; c-PTIO, carboxy-PTIO; GSSG, oxidized glutathione; RIPA, radio
immune precipitation assay; TBST, Tris-HCl buffer saline and tween; GAPDH, glyceraldehyde-
3-phosphate dehydrogenase; ECL, enhanced chemiluminescence; H&E, hematoxylin & eosin.
REFERENCES
1. Li, X.; Lee, S.; Yoon, J. Supramolecular photosensitizers rejuvenate photodynamic therapy.
Chem. Soc. Rev. 2018, 47, 1174-1188.
2. Agostinis, P.; Berg, K.; Cengel, K. A.; Foster, T. H.; Girotti, A. W.; Gollnick, S. O.; Hahn,
ACS Paragon Plus Environment

Page 23 of 29
Molecular Pharmaceutics
1
2
3
4
5
6
7
8
S. M.; Hamblin, M. R.; Juzeniene, A.; Kessel, D.; Korbelik, M.; Moan, J.; Mroz, P.; Nowis, D.;
Piette, J.; Wilson, B. C.; Golab, J. Photodynamic therapy of cancer: an update. CA Cancer J.
Clin. 2011, 61, 250-281.
9
3. Fan, W.; Yung, B. C.; Chen, X. Stimuli-responsive NO release for on-demand gas-
sensitized synergistic cancer therapy. Angew. Chem. Int. Ed. 2018, 57, 8383-8394.
4. Wang, C.; Li, Y.; Shi, X.; Zhou, J.; Zhou, L.; Wei, S. Use of an NIR-light-responsive CO
nanodonor to improve the EPR effect in photothermal cancer treatment. Chem. Commun. 2018,
54, 13403-13406.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
5. Li, S.; Liu, R.; Jiang, X.; Qiu, Y.; Song, X.; Huang, G.; Fu, N.; Lin, L.; Song, J.; Chen, X.;
Yang, H. Near-infrared light-triggered sulfur dioxide gas therapy of cancer. ACS Nano 2019,
13, 2103-2113.
6. Kang, J.; Li, Z.; Organ, C. L.; Park, C. M.; Yang, C. T.; Pacheco, A.; Wang, D.; Lefer, D.
J.; Xian, M. pH-Controlled hydrogen sulfide release for myocardial ischemia-reperfusion injury.
J. Am. Chem. Soc. 2016, 138, 6336-6339.
7. Huang, X.; Xu, F.; Hou, H.; Hou, J.; Wang, Y.; Zhou, S. Stimuli-responsive nitric oxide
generator for light-triggered synergistic cancer photothermal/gas therapy. Nano Res. 2019, 12,
1361-1370.
8. Yu, L.; Hu, P.; Chen, Y. Gas-generating nanoplatforms: material chemistry,
multifunctionality, and gas therapy. Adv. Mater. 2018, 30, DOI: 1801964.
9. Zhang, X.; Tian, G.; Yin, W.; Wang, L.; Zheng, X.; Yan, L.; Li, J.; Su, H.; Chen, C.; Gu, Z.;
Zhao, Y. Controllable generation of nitric oxide by near-infrared-sensitized upconversion
nanoparticles for tumor therapy. Adv. Funct. Mater. 2015, 25, 3049-3056.
10. Wu, J.; Akaike, T.; Hayashida, K.; Okamoto, T.; Okuyama, A.; Maeda, H. Enhanced
vascular permeability in solid tumor involving peroxynitrite and matrix metalloproteinases. Jpn.
J. Cancer Res. 2001, 92, 439-451.
11. Maeda, H.; Wu, J.; Sawa, T.; Matsumura, Y.; Hori, K. Tumor vascular permeability and the
EPR effect in macromolecular therapeutics: a review. J Control. Release 2000, 65, 271-284.
12. Jourd'heuil, D.; Jourd'heuil, F. L.; Feelisch, M. Oxidation and nitrosation of thiols at low
micromolar exposure to nitric oxide. Evidence for a free radical mechanism. J. Biol. Chem. 2003,
278, 15720-15726.
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 24 of 29
1
2
3
4
5
6
7
8
13. Fahey, J. M.; Girotti, A. W. Nitric oxide-mediated resistance to photodynamic therapy in a
human breast tumor xenograft model: improved outcome with NOS2 inhibitors. Nitric Oxide
2017, 62, 52-61.
9
14. Girotti, A. W. Modulation of the anti-tumor efficacy of photodynamic therapy by nitric oxide.
Cancers 2016, 8, DOI: 10.3390/cancers8100096.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
15. Bhowmick, R.; Girotti, A. W. Pro-survival and pro-growth effects of stress-induced nitric
oxide in a prostate cancer photodynamic therapy model. Cancer Lett. 2014, 343, 115-122.
16. Bhowmick, R.; Girotti, A. W. Cytoprotective signaling associated with nitric oxide
upregulation in tumor cells subjected to photodynamic therapy-like oxidative stress. Free Radic.
Biol. Med. 2013, 57, 39-48.
17. Xiang, H. J.; Deng, Q.; An, L.; Guo, M.; Yang, S. P.; Liu, J. G. Tumor cell specific and
lysosome-targeted delivery of nitric oxide for enhanced photodynamic therapy triggered by 808
nm near-infrared light. Chem. Commun. 2016, 52, 148-151.
18. Deng, Y.; Jia, F.; Chen, S.; Shen, Z.; Jin, Q.; Fu, G.; Ji, J. Nitric oxide as an all-rounder for
enhanced photodynamic therapy: hypoxia relief, glutathione depletion and reactive nitrogen
species generation. Biomaterials 2018, 187, 55-65.
19. Rapozzi, V.; Della Pietra, E.; Zorzet, S.; Zacchigna, M.; Bonavida, B.; Xodo, L. E. Nitric
oxide-mediated activity in anti-cancer photodynamic therapy. Nitric Oxide 2013, 30, 26-35.
20. Rapozzi, V.; Della Pietra, E.; Bonavida, B. Dual roles of nitric oxide in the regulation of
tumor cell response and resistance to photodynamic therapy. Redox Biol. 2015, 6, 311-317.
21. Wink, D. A.; Mitchell, J. B. Chemical biology of nitric oxide: insights into regulatory,
cytotoxic, and cytoprotective mechanisms of nitric oxide. Free Radic. Biol. Med. 1998, 25, 434-
456.
22. Beckman, J. S.; Carson, M.; Smith, C. D.; Koppenol, W. H. ALS, SOD and peroxynitrite.
Nature 1993, 364, 584-584.
23. Deepagan, V. G.; Ko, H.; Kwon, S.; Rao, N. V.; Kim, S. K.; Um, W.; Lee, S.; Min, J.; Lee,
J.; Choi, K. Y.; Shin, S.; Suh, M.; Park, J. H. Intracellularly activatable nanovasodilators to
enhance passive cancer targeting regime. Nano Lett. 2018, 18, 2637-2644.
24. Quinn, J. F.; Whittaker, M. R.; Davis, T. P. Delivering nitric oxide with nanoparticles. J.
Control. Release 2015, 205, 190-205.
ACS Paragon Plus Environment

Page 25 of 29
Molecular Pharmaceutics
1
2
3
4
5
6
7
8
9
25. Chandrawati, R.; Chang, J. Y. H.; Reina-Torres, E.; Jumeaux, C.; Sherwood, J. M.; Stamer,
W. D.; Zelikin, A. N.; Overby, D. R.; Stevens, M. M. Localized and controlled delivery of nitric
oxide to the conventional outflow pathway via enzyme biocatalysis: toward therapy for
glaucoma. Adv. Mater. 2017, 29, DOI: 10.1002/adma.201604932.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
26. Jin, H.; Yang, L.; Ahonen, M. J. R.; Schoenfisch, M. H. Nitric oxide-releasing cyclodextrins.
J. Am. Chem. Soc. 2018, 140, 14178-14184.
27. Hou, J.; Pan, Y.; Zhu, D.; Fan, Y.; Feng, G.; Wei, Y.; Wang, H.; Qin, K.; Zhao, T.; Yang,
Q.; Zhu, Y.; Che, Y.; Liu, Y.; Cheng, J.; Kong, D.; Wang, P. G.; Shen, J.; Zhao, Q. Targeted
delivery of nitric oxide via a 'bump-and-hole'-based enzyme-prodrug pair. Nat. Chem. Biol. 2019,
15, 151-160.
28. Fan, W.; Lu, N.; Huang, P.; Liu, Y.; Yang, Z.; Wang, S.; Yu, G.; Liu, Y.; Hu, J.; He, Q.; Qu,
J.; Wang, T.; Chen, X. Glucose-responsive sequential generation of hydrogen peroxide and
nitric oxide for synergistic cancer starving-like/gas therapy. Angew. Chem. Int. Ed. 2017, 56,
1229-1233.
29. Birben, E.; Sahiner, U. M.; Sackesen, C.; Erzurum, S.; Kalayci, O. Oxidative stress and
antioxidant defense. World Allergy Organ. J. 2012, 5, 9-19.
30. Russo, A.; DeGraff, W.; Friedman, N.; Mitchell, J. B. Selective modulation of glutathione
levels in human normal versus tumor cells and subsequent differential response to
chemotherapy drugs. Cancer Res. 1986, 46, 2845-2848.
31. Zhang, W.; Lu, J.; Gao, X.; Li, P.; Zhang, W.; Ma, Y.; Wang, H.; Tang, B. Enhanced
photodynamic therapy by reduced levels of intracellular glutathione obtained by employing a
nano-MOF with Cu(II) as the active center. Angew. Chem. Int. Ed. 2018, 57, 4891-4896.
32. Ju, E.; Dong, K.; Chen, Z.; Liu, Z.; Liu, C.; Huang, Y.; Wang, Z.; Pu, F.; Ren, J.; Qu, X.
Copper(II)-graphitic carbon nitride triggered synergy: improved ROS generation and reduced
glutathione levels for enhanced photodynamic therapy. Angew. Chem. Int. Ed. 2016, 128,
11639-11643.
33. Wang, A.; Zhou, R.; Zhou, L.; Sun, K.; Zhou, J.; Wei, S.; Jiang, J. Arginine-substituted
phthalocyanine with concentration-driven self-disaggregation performance: synthesis,
properties and mechanistic study. Chem. Asian J. 2016, 11, 3008-3013.
34. Shang, H.; Chen, H.; Tang, Y.; Ma, Y.; Lin, W. Development of a two-photon fluorescent
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 26 of 29
1
2
3
4
5
6
turn-on probe with far-red emission for thiophenols and its bioimaging application in living
tissues. Biosens Bioelectron 2017, 95, 81-86.
7
8
9
35. Xie, X.; Tang, F.; Liu, G.; Li, Y.; Su, X.; Jiao, X.; Wang, X.; Tang, B. Mitochondrial
peroxynitrite mediation of anthracycline-induced cardiotoxicity as visualized by a two-photon
near-infrared fluorescent probe. Anal. Chem. 2018, 90, 11629-11635.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
36. Sarı, S.; Durmuş, M.; Bulut, M. Microwave assisted synthesis of novel zinc(II)
phthalocyanines bearing 1,3-diazido-2-propanoxy functional groups and investigation of their
photochemical properties. Tetrahedron Lett. 2016, 57, 1124-1128.
37. Nyokong, T. Effects of substituents on the photochemical and photophysical properties of
main group metal phthalocyanines. Coordin. Chem. Rev. 2007, 251, 1707-1722.
38. Li, X. S.; Ke, M. R.; Zhang, M. F.; Tang, Q. Q.; Zheng, B. Y.; Huang, J. D. A non-aggregated
and tumour-associated macrophage-targeted photosensitiser for photodynamic therapy: a
novel zinc(II) phthalocyanine containing octa-sulphonates. Chem. Commun. 2015, 51, 4704-
4707.
39. Li, X.; Peng, X. H.; Zheng, B. D.; Tang, J.; Zhao, Y.; Zheng, B. Y.; Ke, M. R.; Huang, J. D.
New application of phthalocyanine molecules: from photodynamic therapy to photothermal
therapy by means of structural regulation rather than formation of aggregates. Chem. Sci. 2018,
9, 2098-2104.
40. Zorlu, Y.; Ermeydan, M. A.; Dumoulin, F.; Ahsen, V.; Savoie, H.; Boyle, R. W. Glycerol and
galactose substituted zinc phthalocyanines. Synthesis and photodynamic activity. Photochem.
Photobiol. Sci. 2009, 8, 312-318.
41. Crowley, L. C.; Marfell, B. J.; Scott, A. P.; Waterhouse, N. J. Quantitation of apoptosis and
necrosis by annexin V binding, propidium iodide uptake, and flow cytometry. Cold Spring Harb.
Protoc. 2016, DOI: 10.1101/pdb.prot087288.
42. Lukinavicius, G.; Blaukopf, C.; Pershagen, E.; Schena, A.; Reymond, L.; Derivery, E.;
Gonzalez-Gaitan, M.; D'Este, E.; Hell, S. W.; Wolfram Gerlich, D.; Johnsson, K. SiR-hoechst is
a far-red DNA stain for live-cell nanoscopy. Nat. Commun. 2015, 6, 8497-8503.
43. Fischer, A. H.; Jacobson, K. A.; Rose, J.; Zeller, R. Cutting sections of paraffin-embedded
tissues. CSH Protoc. 2008, DOI: 10.1101/pdb prot4987.
44. Duong, H. T.; Kamarudin, Z. M.; Erlich, R. B.; Li, Y.; Jones, M. W.; Kavallaris, M.; Boyer,
ACS Paragon Plus Environment

Page 27 of 29
Molecular Pharmaceutics
1
2
3
4
5
6
C.; Davis, T. P. Intracellular nitric oxide delivery from stable NO-polymeric nanoparticle carriers.
Chem. Commun. 2013, 49, 4190-4192.
7
8
9
45. Li, X.; Zheng, B.-D.; Peng, X.-H.; Li, S.-Z.; Ying, J.-W.; Zhao, Y.; Huang, J.-D.; Yoon, J.
Phthalocyanines as medicinal photosensitizers: developments in the last five years. Coordin.
Chem. Rev. 2019, 379, 147-160.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
46. Zhang, X.-F.; Zheng, H. Tetra(β-phenothiazinyl) zinc phthalocyanine: An easily prepared
D4–A system for efficient photoinduced electron transfer. Inorg. Chim. Acta 2010, 363, 2259-
2264.
47. George, R. D.; Snow, A. W.; Shirk, J. S.; Barger, W. R. The alpha substitution effect on
phthalocyanine aggregation. J. Porphyr. Phthalocya. 1998, 2, 1-7.
48. Ngubeni, G. N.; Britton, J.; Mack, J.; New, E.; Hancox, I.; Walker, M.; Nyokong, T.; Jones,
T. S.; Khene, S. Spectroscopic and nonlinear optical properties of the four positional isomers
of 4α-(4-tert-butylphenoxy)phthalocyanine. J. Mater. Chem. C 2015, 3, 10705-10714.
49. Chen, Y.; Fang, W.; Wang, K.; Liu, W.; Jiang, J. Nonperipheral
tetrakis(dibutylamino)phthalocyanines.
new
types
of
1,8,15,22-
tetrakis(substituted)phthalocyanine isomers. Inorg. Chem. 2016, 55, 9289-9296.
50. Rastogi, R. P.; Singh, S. P.; Häder, D.-P.; Sinha, R. P. Detection of reactive oxygen
species (ROS) by the oxidant-sensing probe 2′,7′-dichlorodihydrofluorescein diacetate in the
cyanobacterium anabaena variabilis PCC 7937. Biochem. Bioph. Res. Commun. 2010, 397,
603-607.
51. Yin, S. Y.; Song, G.; Yang, Y.; Zhao, Y.; Wang, P.; Zhu, L. M.; Yin, X.; Zhang, X. B.
Persistent regulation of tumor microenvironment via circulating catalysis of MnFe2O4@metal–
organic frameworks for enhanced photodynamic therapy. Adv. Funct. Mater. 2019, DOI:
10.1002/adfm.201901417.
52. Zhang, Q.; Yu, D.; Ding, S.; Feng, G. A low dose, highly selective and sensitive colorimetric
and fluorescent probe for biothiols and its application in bioimaging. Chem. Commun. 2014, 50,
14002-14005.
53. Guo, R.; Tian, Y.; Wang, Y.; Yang, W. Near-infrared laser-triggered nitric oxide
nanogenerators for the reversal of multidrug resistance in cancer. Adv. Funct. Mater. 2017, 27,
DOI: 10.1002/adfm.201606398.
ACS Paragon Plus Environment

Molecular Pharmaceutics
Page 28 of 29
1
2
3
4
5
6
7
8
9
54. Wan, M.; Chen, H.; Wang, Q.; Niu, Q.; Xu, P.; Yu, Y.; Zhu, T.; Mao, C.; Shen, J. Bio-
inspired nitric-oxide-driven nanomotor. Nat. Commun. 2019, 10, DOI: 10.1038/s41467.
55. Fang, J.; Nakamura, H.; Maeda, H. The EPR effect: unique features of tumor blood vessels
for drug delivery, factors involved, and limitations and augmentation of the effect. Adv. Drug
Deliv. Rev. 2011, 63, 136-151.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
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41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
56. Zheng, X.; Cui, X.; Yu, H.; Jiang, J. Development of a quantitative method for four
photocyanine isomers using differential ion mobility and tandem mass spectrometry and its
application in a preliminary pharmacokinetics investigation. J. Chromatogr. A 2018, 1577, 109-
119.
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Mechanism diagram of multiple functions integrated inside Arg-ZnPc for its PDT activity amplification
82x44mm (220 x 220 DPI)
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