A smart copper-phthalocyanine framework nanoparticle for enhancing photodynamic therapy in hypoxic conditions by weakening cells through ATP depletion

Article abstract of DOI:10.1039/c8tb00334c

Hypoxia in solid tumors hinders the efficacy of photodynamic therapy (PDT). The route of generation of adenosine triphosphate (ATP) in hypoxic tumor cells comprises ineffective glycolysis processes, but ATP is crucial for rapid and uncontrolled growth and division. ATP depletion would inhibit DNA replication and glutathione (GSH) biosynthesis, which could inhibit the proliferation of cells and make them sensitive to PDT treatment. In this context, a nanoscale metal-organic framework nanoparticle consisting of Cu²⁺ and carboxyl-modified zinc phthalocyanine (ZnPc-(COOH)₈) ({Cu₈(ZnPc-(COOH)₈)}_n, ZPCN) was designed and prepared. In the dark cycle, ATP could remove Cu²⁺ from ZPCN and form a copper-ATP complex. ATP in the above complex could be destroyed by a copper-mediated Fenton reaction, which could finally induce ATP depletion to inhibit the proliferation of cells and make them sensitive to PDT treatment. In the light cycle, ZPCN could generate abundant cytotoxic reactive oxygen species to suppress tumor growth. These results demonstrated the potential applicability of the new strategy for achieving tumor treatment in hypoxic conditions by PDT.

Full text of DOI:10.1039/c8tb00334c

View Article Online
View Journal
Journal of
Materials Chemistry B
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: L. Gui, J. zhou, L.
Zhou and S. Wei, J. Mater. Chem. B, 2018, DOI: 10.1039/C8TB00334C.
This is an Accepted Manuscript, which has been through the
Volume
4 Number 1 7 January 2016 Pages 1–178
Royal Society of Chemistry peer review process and has been
accepted for publication.
Journal of
Materials Chemistry B
Accepted Manuscripts are published online shortly after
Materials for biology and medicine
www.rsc.org/MaterialsB
acceptance, before technical editing, formatting and proof reading.
Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. We will replace this Accepted Manuscript with the edited
and formatted Advance Article as soon as it is available.
You can find more information about Accepted Manuscripts in the
author guidelines.
Please note that technical editing may introduce minor changes
to the text and/or graphics, which may alter content. The journal’s
standard Terms & Conditions and the ethical guidelines, outlined
in our author and reviewer resource centre, still apply. In no
event shall the Royal Society of Chemistry be held responsible
for any errors or omissions in this Accepted Manuscript or any
consequences arising from the use of any information it contains.
ISSN 2050-750X
PAPER
Guoping Chen et al.
Regulating the stemness of mesenchymal stem cells by tuning
micropattern features
rsc.li/materials-b

Please do not adjust margins
Journal of Materials Chemistry B
Page 1 of 10
View Article Online
DOI: 10.1039/C8TB00334C
Journal Name
ARTICLE
A smart Copper-phthalocyanine framework nanoparticle for
enhance hypoxic photodynamic therapy by weakening cell
through ATP depletion
Received 00th January 20xx,
Accepted 00th January 20xx
Li Gui, Jiahong Zhou, Lin Zhou*, Shaohua Wei*
DOI: 10.1039/x0xx00000x
www.rsc.org/
Solid tumor hypoxia hinder photodynamic therapy (PDT) efficacy. Adenosine triphosphate (ATP) generation route of
hypoxia tumor cells was an ineffective glycolysis processes, but ATP is crucial for rapid and uncontrolled growing and
dividing. ATP depletion would inhibit DNA replication and glutathione (GSH) biosynthesis, which could inhibit its
proliferation and make it sensitive to PDT treatment. In this regard, a nanoscale metal-organic framework nanoparticle,
consisted of Cu2+ and carboxyl modified zinc phthalocyanine (ZnPc-(COOH)8) ({Cu8(ZnPc-(COOH)8)}n, ZPCN), was design and
prepared. In the dark cycle, ATP could deprive Cu2+ from ZPCN and form copper-ATP complex. ATP in above complex could
be destroyed by copper mediated Fenton reaction and finally induce ATP depletion to inhibit its proliferation and make it
sensitive to PDT treatment. In the light cycle, ZPCN could generate abundant cytotoxic reactive oxygen species to suppress
tumor growth. These results demonstrated the potential applicability of the new strategy to overcome hypoxic tumor
treatment
by
PDT.
15 16
outcome of PDT is strong O2 dependent.
In light of this,
Introduction
integration of a PDT system with high activity under hypoxia
condition could be promising for enhanced anticancer therapy.
To solve this problem, several different strategies were
exploited to overcome tumor hypoxia and to improve the
therapeutic effect. The first effective strategy is increasing O2
concentration to relieve hypoxia by in situ catalase H2O2 to
As a noninvasive treatment for cancer, PDT has received
considerable attention in recent years. PDT employs light-
excited photosensitizers (PS) to activate the transformation of
surrounding O2 into cytotoxic reactive oxygen species (ROS),
such as singlet oxygen (1O2), H2O2, superoxide radical (O2
)
·
-
4, 17-19
generate O2 or directly delivering O2 to hypoxic tumor.
and hydroxyl radical (·OH), to cause irreversible damage to
1,
malignant cells.
2
Another very useful strategy is combining PS with a hypoxia-
Furthermore, under appropriate light
activated prodrug to contribute a highly effective synergistic
20, 21
irradiation position control at tumor site, PDT offers the
advantage of an effective and selective method of destroying
diseased tissues without damaging adjacent healthy ones for
cancer therapy.
Beyond that, using vascular-disrupting
agents (VDA) to effectively starve the tumor to death, by
depriving it of vital nutrients and oxygen, leading to tumor
necrosis, is another good option to obtain satisfied treatment
efficacy. 22
3
tumor targeting treatment. Hypoxia, which is caused by an
inadequate oxygen supply, is increasingly being recognized as
4-6
a characteristic feature of solid tumors. Hypoxia is believed
ATP, a nucleoside triphosphate, is classified as the most
important energy source for most of the physiological
biochemical process. Hypoxic cancer cells ATP generation rely
on glycolysis processes which is an ineffective ATP generation
route comparing with well-oxygenated conditions. Cancer cells
12 are strong ATP dependent to support their unique
physiological processes, such as active DNA replication and
high levels of GSH to build a strong antioxidant defence, to
to be one of the major causes for the failure of many kinds of
cancer treatment, including chemotherapy (by up-regulating
drug efflux P-glycoprotein expression to enhance drug
7, 8
resistance),
9
effect),
radiotherapy (by decreasing ionizing radiation
photothermal therapy (by up-regulating heat
10, 11
resistance heat shock proteins expression)
and PDT.
PDT depends on the ability of light irradiated PS to transfer
energy from light to the surrounding O2 to generate cytotoxic
23-
13, 14
maintain their rapid and uncontrolled growing and dividing.
26
DNA replication is the cornerstone for cancer cell
ROS to kill tumor cells.
In other words, the therapeutic
proliferation and GSH could consume ROS and sharply reduce
PDT activity. Therefore, we proposed that if we deplete ATP in
hypoxic cancer cells, they would weak and be more sensitive
to PDT treatment to obtain the highly effective cancer therapy
under hypoxic condition.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 1
Please do not adjust margins

Please do not adjust margins
Journal of Materials Chemistry B
Page 2 of 10
View Article Online
DOI: 10.1039/C8TB00334C
ARTICLE
Journal Name
Sijiqing. ATP Assay Kit, 3-[4,5-dimethylthiazol-2-yl]-2,5-
diphenyl tetrazoliumbromide (MTT), Hematoxylin and Eosin
Staining Kit(H&E), 2,7-dichlorodi-hydrofluorescein diacetate
(DCFH-DA), Dihydroethidium (DHE), Rhodamine 123, Cell Cycle
Analysis Kit, Annexin V-FITC Apoptosis Detection Kit, GSH and
GSSG Assay Kit were obtained from Beyotime. Mitotracker
green, Lysotracker blue and singlet oxygen sensor green
(SOSG) were from thermo fisher. BES-H2O2-Ac was from Wako
Chemicals. The necessary solvents were used after purification
according to the reported literature. Chromatographic
purifications were performed on silica gel columns (300-400
mesh, Qingdao Haiyang Chemical Co., Ltd, China). All chemical
reagents were obtained from commercial suppliers and used
as received unless otherwise stated.
Characterization
Ultraviolet-Visible (UV-Vis) absorption spectra were obtained
by a Varian Cary 50 spectrophotometer. Fluorescence spectra
were recorded on a Cary Eclipse fluorometer. Infrared spectra
were obtained by Bruker Tensor infrared spectrometer. Zeta
potential was detected with Malvern Zetasizer Nano 90
measurements in aqueous system. Transmission Electron
Microscope (TEM) images were got by Hitachi H7650
transmission electron microscope. Morphology and
Scheme 1 Schematic presentation of anticancer mechanism of ZPCN.
To verify this hypothesis, herein, a new nanoscale metal-
organic framework nanoparticle, consisted of Cu2+ and
carboxyl modified ZnPc-(COOH)8 based on the bond between
Cu2+ and COO ({Cu8(ZnPc-(COOH)8)}n, ZPCN), 27, 28 was prepared
and used for hypoxic cancer treatment. As shown in scheme 1,
ZPCN maintain inactive state during the delivery process in
blood because during this process, it maintains in aggregation
form. After been entrapped by cancer cells, ATP could deprive
Cu2+ from ZPCN and make its disaggregation because of the
fluorescence inside cells was monitored by
a Nikon Ti
microscope and Nikon Ti-E-A1R confocal laser scanning
microscope. 1H NMR and 13C NMR spectra were recorded using
a Bruker Advance 400 MHz NMR spectrometer. Mass spectra
were obtained on UltrafleXreme MALDI-TOF-MS spectrometer.
Elemental analysis was measured by Vario MICRO Elementar.
Fluorescence lifetime and fluorescence quantum yield were
obtained using a Horiba Jobin Yvon FM-4P-TCSPC system. Flow
Cytometry (FCM) analysis was carried on a Beckman XL flow
cytometer. Differential thermal analysis (TG-DTA) was taken
on Perkin Elmer Diamond TG-DTA analyser in N2 atmosphere
at a heating rate of 10 °C min-1. A 665 nm LED (5 W) was used
as light source.
higher affinity of phosphate to Cu2+ than that of COO and
29
the active ZnPc-(COOH)8 was released. Similar like Fe3+, Cu2+ is
also an effective Fenton regent to generate hydroxyl radical. 30,
This fearsomely-reactive hydroxyl radical, once generated,
31
attacks whatever it is next to. Its lifetime inside cell is
vanishingly small, so, it reacts at its site of formation.
32
Therefore, during the dark cycle of the treatment, the Fenton-
type reaction, in which copper ion in its complex with ATP
reacts with H2O2 (cancer cells would produce excessive
amounts of H2O2 and cause significant increase of H2O2 level in
OH
COOCH3
NC
NC
NO2
NC
NC
O
K2CO3
+
13, 33, 34
·
the tumor microenvironment)
ATP (because they are close enough) and induce its depletion
to form OH to damage
H3COOC
COOCH3
COOCH3
1
35
inside cells. ATP depletion could inhibit DNA replication and
decrease GSH level to provide a weak and sensitive cell state
for PDT. 36, 37 During the light cycle of treatment, ROS could be
generated after light irradiation. Finally, the synergistic of
above cycles could cause mitochondrial membrane potential
hyperpolarization or depolarization and induce effective cell
apoptosis under hypoxic condition. Therefore, the potential
applicability of a new strategy to overcome hypoxic tumor
treatment by PDT is successfully demonstrated.
Zn(OAc)2
DBU
n-pentanol
R
R
R
R
N
N
N
N
N
N
N
N
N
THF
NaOH, H2O
N
N
N
N
Zn
Zn
N
N
N
2
R
R
R
R
ZnPc-(COOH)8
Experimental
COOH
COOCH3
COOCH3
Chemicals
O
O
R=
R=
The 1, 8-diazabicyclo [5, 4, 0]-undec-7-ene (DBU), 4-
Nitrophthalonitrile, dimethyl 5-hydroxyisophthalate, 9, 10-
anthracenedip-ropionic acid (ADPA) and ATP was obtained
from Sigma-Aldrich. Dulbecco’s minimum essential medium
(DMEM) was from Gibco. Fetal bovine serum (FBS) was from
COOH
Scheme 2 The synthetic route of ZnPc-(COOH)8.
2 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Please do not adjust margins
Journal of Materials Chemistry B
Page 3 of 10
View Article Online
DOI: 10.1039/C8TB00334C
Journal Name
ARTICLE
Synthesis of ZnPc-(COOH)8
Chemical method to detect 1O2
38
ZnPc-(COOH)8 was synthesized using our previous methods.
1O2 was detected using ADPA because it could interact with
All the compounds were detected by melting point (M.P.), 1O2 to generate its endoperoxide, which induce its absorbance
1
infrared spectra (IR), H Nuclear Magnetic Resonance Spectra intensity decreasing. The mixture of 3 mL drugs solutions and
(1H NMR) and UltraflexXreme MALDI-TOF-MS. ZnPc-(COOH)8 150 μL ADPA (5.5 mM) was irradiated by 665 nm light and
synthetic route was shown in Scheme 2.
their UV-Vis spectra changing were recorded. The decay of
Compound 1 was synthesized as following process. Under [ADPA] follows first order kinetics as the following Eq.
N2 atmosphere, 4-nitrophthalonitrile (0.25 g, 1.45 mmol),
dimethyl 5-hydroxyisophthalate (0.30 g, 1.45 mmol) and
potassium carbonate (0.29 g, 2.89 mmol) were mixed in 8 mL
ꢀꢁ([ꢂꢃꢄꢂ]ꢅ/[ꢂꢃꢄꢂ]ꢆ) = −ꢇꢅ
DMF was stirred at 75 °C for 8 h. Then, 50 mL water was added Where [ADPA]t and [ADPA]0 are the concentrations of ADPA
into the solution after it cooling to room temperature and the after and before irradiation, respectively. Therefore, the
product was extracted by ethyl acetate. The impurities in ethyl relative rate constant k can be extrapolated by a linear fit using
acetate were removed by washing ethyl acetate solution using the experimental points and calculated.
water and saturated sodium chloride for 3 times. Ethyl acetate
Cell culture
was removed by vacuum distillation. The obtained product
Human lung alveolar basal carcinoma epithelial cells (A549
was further purified by silica gel column chromatography using
cells) were cultured in DMEM containing FBS (10%, v/v),
ethyl acetate / petroleum ether (1:2, v/v). Finally, pure
penicillin (100 U/mL) and streptomycin (100 μg/mL) in an
compound
1 as white solid was obtained (454.9 mg, 92.6%).
M.P. was 125 °C; IR (KBr, νmax/cm-1): 3425, 3084, 2233, 1735,
1584, 1493, 1316, 1264, 1008, 837, 765. 1H-NMR (400 MHz,
DMSO-d6): δ/ppm: 8.32 (t, 1H, J=1.6 Hz, Ar), 8.13 (d, 1H, J=8.8
incubator (Thermo Scientific) at 37 °C under an atmosphere of
5% CO2 and 5% O2 to simulate hypoxia microenvironment of
solid tumour. 39
Hz, Ar), 7.90 (d, 2H, J=1.6 Hz, Ar), 7.86 (d, 1H, J=2.8 Hz, Ar), Cellular intracellular distribution
7.54 -7.51 (m, 1H, Ar). 13C-NMR (100 MHz, DMSO-d6): δ /ppm:
A549 cells were seeded onto glass-bottom dishes at a density
of 1×105 cells per dish in 1 mL of medium and incubated for 24
h before treatment of ZPCN, and ZnPc-(COOH)8 separately.
After 4 h incubation, DMEM with LysoTracker blue (100 nM)
and Mitotracker green (100 nM) were replaced and incubated
for 30 min before observation. LysoTracker was observed
using a 405 nm laser; the emission wavelength was 425 to 475
nm and appeared blue. Mitotracker was observed using a 488
nm laser; the emission wavelength was 510 to 540 nm and
appeared green. ZnPc-(COOH)8 and ZPCN were observed using
a 647 nm laser; the emission wavelength was 700 nm and
appeared red.
165, 160.7, 154.9, 136.8, 133.0, 126.9, 125.7, 123.6, 123.3,
117.3, 116.2, 115.7, 109.7. Anal. Calcd for C18H12N2O5: C,
64.29; H, 3.60; N, 8.33. Found: C, 64.31; H, 3.57; N, 8.29.
ZnPc-(COOH)8 was synthesized by following protocols.
Under N2 atmosphere, compound
1 (0.25 g, 0.75 mmol) and
Zn(OAc)2 (0.09 g, 0.47 mmol) were added to 8 mL dried n-
pentanol and stirred at 90 °C for 1 h. Then, DBU (0.1 mL, 0.67
mmol) was added and the mixture was heated to 130 °C and
stirred for another 12 h. Then, the solvents were removed
under reduced pressure. Silica gel column chromatography
using ethyl acetate / petroleum ether (1:2, v/v) and methanol/
dichloromethane (1:5, v/v) was used to purify the product. The
vacuum-dried product was washed by NaOH and methanol
Apoptosis assay by MTT and flow cytometry
(1:4, v/v, 50 mL) at 60 °C for 12 h. The impurities were The apoptosis of A549 cells induced by ZPCN, ZnPc-(COOH)8 or
removed by filtration. Then, 10% HCl was added to above free Cu2+ was studied by MTT and flow cytometry assay.
solution drop by drop until many dark green solid precipitates. Untreated A549 cells served as the control. To detect the dark
Above purification process was repeated 3 times to obtain toxicity, after exposure to various drug formulations, Cu2+,
pure ZnPc-(COOH)8 (0.06 g, 24.3%). M.P.>200 °C, IR (KBr, ZPCN, and ZnPc-(COOH)8, at equivalent concentrations (ZnPc-
νmax/cm-1): 3436, 3083, 2922, 1710, 1593, 1463, 1265, 1227, (COOH)8: 7.5 μM; Cu2+: 60 μM) for 48 hours in DMEM (10%
1116, 974, 838, 758.1H-NMR (400 MHz, DMSO-d6): δ/ppm: FBS), MTT solution was added to each well to detect cell
13.37 (br, 8H, COOH), 8.93-8.54 (m, 4H, Pc-H), 8.34-7.76 (m, viability based on the absorption of the formazan product
12H, Ar), 7.47-7.24(m, 8H, Pc-H). HRMS (MALDI-TOF) m/z: dissolved in DMSO. To detect the light induced toxicity, after
1296.682, (Calculated. For C64H32N8O20Zn: 1296.102). Anal. exposure to various drug formulations, Cu2+, ZPCN, and ZnPc-
Calcd for C64H32N8O20Zn: C, 59.20; H, 2.48; N, 8.63. Found: C, (COOH)8, at equivalent concentrations (ZnPc-(COOH)8: 7.5 μM;
59.19; H, 2.47; N, 8.65.
Cu2+: 60 μM) for 24 hours, the cells were irradiated by light for
5 min. Then, the cells were re-incubated in incubator for 24 h
and cell viability was detected using MTT assay. In addition,
after re-incubation, cells were thoroughly washed with cold
PBS and collected for treatment using Annexin V-FITC
Apoptosis Detection Kit according to the manufacturer’s
protocol to detected cell apoptosis.
{Cu8(ZnPc-(COOH)8)}n (ZPCN) preparation
100 μL of ZnPc-(COOH)8 (10 mM) and 100 μL of CuCl2 solution
(160 mM) were mixed in 10 mL Tris-HCl buffer (pH =7.4). The
mixture was stirred for 30 min to form ZPCN. The result ZPCN
was purified by dialysis the solution using a 12-14 kDa cut-off
cellulose membrane for 24 h to water.
In vitro ATP deprivation detection
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3
Please do not adjust margins

Please do not adjust margins
Journal of Materials Chemistry B
Page 4 of 10
View Article Online
DOI: 10.1039/C8TB00334C
ARTICLE
Journal Name
For determination of intracellular ATP concentration, A549 animal experiments were carried out in accordance
cells were seeded in 6-well plates at a density of 1×107 /well. with regulations of the Nanjing Committee of Use and Care of
After incubated in incubator for 12 h, the cells were treated by Laboratory Animals, and all experimental protocols were
various drug formulations, Cu2+, ZPCN, and ZnPc-(COOH)8, at approved by the Animal Ethics Committee of Nanjing Normal
equivalent concentrations (ZnPc-(COOH)8: 7.5 μM; Cu2+: 60 μM) University. For biocompatibility study, female Kunming mice
for 48 hours in DMEM (10% FBS) incubated overnight. After were divided into 3 groups and intravenously injected through
that, cells were washed with cold PBS and lysed with ATP lysis tail vein for saline, ZnPc-(COOH)8 and ZPCN (ZnPc-(COOH)8 per
buffer. The ATP levels were measured according to the administration was 20 mg/kg, Cu2+ per administration was 160
protocol of the ATP assay kit.
mg/kg). Their body weight was recorded every day and after
20 days, the mice were sacrificed and their organs (heart, liver,
spleen, lung, kidney, and brain) were taken out for the
assessment using H&E staining for histological analysis.
For anti-tumor growth study, female BALB/c nude mice
were subcutaneously injected with A549 cells to establish the
tumor xenograft (tumor volume about 100 mm3). The mice
were divided into 3 groups including control group, ZnPc-
(COOH)8 and ZPCN (ZnPc-(COOH)8 per administration was 1.5
mg/kg, Cu2+ per administration was 12 mg/kg). The drugs were
intravenously injected through tail vein to nude mice 1 time
every two days. 665 nm LED was used to irradiate the tumor
position carried out after injection of drugs for 12 h. The body
weight and the tumor volume were recorded every day. After
20 days, the mice were sacrificed and their tumor tissue were
taken out for the assessment using H&E staining for
histological analysis.
In vitro GSH deprivation detection
For determination of intracellular GSH concentration, A549
cells were seeded in 6-well plates at a density of 1×107 /well.
After incubated in incubator for 12 h, the cells were treated by
various drug formulations, Cu2+, ZPCN, and ZnPc-(COOH)8, at
equivalent concentrations (ZnPc-(COOH)8: 7.5 μM; Cu2+: 60 μM)
for 48 hours in DMEM (10% FBS) incubated overnight. After
that, cells were washed with cold PBS and their GSH
concentrations were measured according to the protocol of
the GSH and GSSG Assay Kit.
In vitro ROS generation detection
Intracellular ROS generation was detected using following
method. A549 cells were cultured in DMEM with 10% FBS with
the density of 1×106 cells/mL in incubator for 24 h. Then,
DMEM (10% FBS) with various drug formulations, Cu2+, ZPCN,
and ZnPc-(COOH)8, at equivalent concentrations (ZnPc- Statistical analysis
(COOH)8: 7.5 μM; Cu2+: 60 μM) was replaced, separately. After
All biochemical experiments were performed in duplicate and
24 h incubation, DMEM with DCFH-DA (10 μM), DHE (5 μM),
SOSG (10 μM) and BES-H2O2-Ac (5 μM) were replaced and
incubated respectively for 30 min. Then, cells were washed
with PBS for three times and irradiated for 5 min. Finally, cells
were monitored by fluorescence microscopy.
an average of the results were used. Statistical analysis was
done and the mean, standard deviation, standard error, and
the significant changes were expressed as the mean ± SD. P <
0.05 was considered to be statistically significant.
Cell cycle analysis by flow cytometry
Results and discussion
Flow cytometric analysis was performed to study the effect of
various formulations of drug on the cell cycle. A549 cells were
seeded in 24-well plates at a density of 1×107 /well. After
incubated in incubator for 12 h, after exposure to various drug
formulations DMEM solution (10% FBS), Cu2+, ZPCN, and ZnPc-
(COOH)8, at equivalent concentrations (ZnPc-(COOH)8: 7.5 μM;
Cu2+: 60 μM) for 24 hours, the cells were irradiated by light for
5 min. Then, the cells were re-incubated in incubator for 24 h.
After that, cells were washed with PBS and their cell cycle
were detected using cell cycle analysis Kit.
ZPCN formation and ATP triggered decomposition
The carboxylate group has strong affinity with Cu2+. Therefore,
we proposed that in ZPCN, each ZnPc-(COOH)8 linker is
coordinated to eight different Cu2(COO)4 paddlewheel units
through the carboxylate groups and interconnects these
27
binuclear paddlewheel units into 2D infinite layers.
All the
parallel layers are slipped with each other and further stacked
into a 3D supramolecular architecture through π-π stacking
41
interactions.
To verify this hypothesis, the complexation
processes between ZnPc-(COOH)8 and Cu2+ was monitored
clearly by UV-visible and fluorescence spectra by molar ratio
methods. For the molar ratio method, a series of solutions
containing different concentrations of Cu2+ and a constant
concentration of ZnPc-(COOH)8 (10 μM) were prepared, and
the absorbance at a wavelength at 680 nm was plotted against
the molar ratio of [Cu2+] / [ ZnPc-(COOH)8]. Two straight lines
of different slopes can be derived from the plot, and the molar
Mitochondria dysfunction detection
Methods for quantifying mitochondrial transmembrane
potential based on rhodamine 123 fluorescence were similar
to those reported previously. 40 Briefly, following experimental
treatment, cultures were incubated for 30 min in DMEM
containing 5 μM rhodamine 123. Cultures were washed with
PBS and fluorescence was quantified using a flow cytometer
(488 nm excitation).
ratio where the two lines cross reflects the composition of the
42
complex.
And the results indicated that Cu2+ form 8:1
In vivo biocompatibility and anti-tumour growth efficiency
complexes with ZnPc-(COOH)8 (Fig. 1). Besides, the two
absorbance peaks of ZnPc-(COOH)8 at 640 nm and 680 nm
were ascribed to its aggregate and monomer form, separately.
Female BALB/c nude mice (3-4 weeks old) were purchased
from Comparative Medicine Centre of Yangzhou University. All
4 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Please do not adjust margins
Journal of Materials Chemistry B
Page 5 of 10
View Article Online
DOI: 10.1039/C8TB00334C
Journal Name
ARTICLE
Interaction between Cu2+ and ZnPc-(COOH)8 induced ZnPc-
(COOH)8 aggregation. The PDT activity of aggregate was much
lower than the monomer form. Therefore, ZPCN could
distribution (2) of ZPCN; (D) TEM image (1) and size distribution (2) of ZPCN+ATP.
Bar=200 nm.
Upon the introduction of ATP, the strong affinity between
Cu2+ and ATP can promote the disassembly of the ZPCN and
the process can be observed by monitoring the monomer form
and fluorescence recovery of ZnPc-(COOH)8. The
disassemblyprocess was also verified by fluorescence life time
and quantum yield regain results (Table S1). And TEM image
also indicated that after ATP adding for 30 min, the structure
of ZPCN was damaged and their size was decreased
accordingly (Fig. 2).
1.2
ZnPc-(COOH)8:Cu2+=1:0
1.2
ZnPc-(COOH)8:Cu2+=1:1
ZnPc-(COOH)8:Cu2+=1:2
1.0
1.0
0.8
0.6
0.4
0.2
0.0
B
A
ZnPc-(COOH)8:Cu2+=1:3
ZnPc-(COOH)8:Cu2+=1:4
ZnPc-(COOH)8:Cu2+=1:5
ZnPc-(COOH)8:Cu2+=1:6
ZnPc-(COOH)8:Cu2+=1:7
ZnPc-(COOH)8:Cu2+=1:8
ZnPc-(COOH)8:Cu2+=1:9
ZnPc-(COOH)8:Cu2+=1:10
0.8
0.6
0.4
0.2
0
2
4
6
8
10
500
550
600
650
700
750
800
Wavelength /nm
Mol ratio between Cu2+ and ZnPc-(COOH)8
1O2 generation ability and photobleaching property
800
700
600
500
400
300
200
100
0
800
600
400
200
0
ZnPc-(COOH)8:Cu2+=1:0
ZnPc-(COOH)8:Cu2+=1:1
ZnPc-(COOH)8:Cu2+=1:2
ZnPc-(COOH)8:Cu2+=1:3
ZnPc-(COOH)8:Cu2+=1:4
ZnPc-(COOH)8:Cu2+=1:5
ZnPc-(COOH)8:Cu2+=1:6
ZnPc-(COOH)8:Cu2+=1:7
ZnPc-(COOH)8:Cu2+=1:8
ZnPc-(COOH)8:Cu2+=1:9
C
D
As we mentioned above, forming ZPCN greatly induce ZnPc-
(COOH)8 aggregation, which could obviously minimize its 1O2
generation capability. ATP adding could induce monomer
recovering, which could cause 1O2 generation capability
ZnPc-(COOH)8:Cu2+=1:10
1
0
2
4
6
8
10
620 640 660 680 700 720 740 760 780 800 820
Wavelength /nm
recovering. To verify this hypothesis. O2 generation ability of
Mol ratio between Cu2+ and ZnPc-(COOH)8
ZnPc-(COOH)8, ZPCN and ZPCN+ATP were studied and
compared. ADPA can react with 1O2 to produce its
endoperoxide, which would cause its absorption intensity
decreasing. Fig. 3 shows the decrease in absorption intensity in
different samples, including ZnPc-(COOH)8, ZPCN and
ZPCN+ATP, as a function of the time of light exposure. The
absorption intensity of ZnPc-(COOH)8 and ZPCN+ATP show a
sharp decrease with the time of light exposure, indicating the
Fig. 1 (A) UV-Vis spectrum changes of ZnPc-(COOH)8 in aqueous solution upon addition
of Cu2+. [ZnPc-(COOH)8] = 10 μM, [Cu2+] = 0, 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100
μM; (B) Molar ratio plots for Cu2+-ZnPc-(COOH)8 obtained by plotting the absorbance at
680 nm as a function of the molar ratio of Cu2+ to ZnPc, (C) Fluorescence spectrum
changes of ZnPc-(COOH)8 in aqueous solution upon addition of Cu2+. [ZnPc-(COOH)8] =
10 μM, [Cu2+] = 0, 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 μM; (D) Molar ratio plots for
Cu2+-ZnPc-(COOH)8 obtained by plotting the fluorescence intensity at 695 nm as a
function of the molar ratio of Cu2+ to ZnPc-(COOH)8.
1
generation of O2. On the contrary, ZPCN produced almost no
significantly minimized the ROS production efficiency of ZnPc-
(COOH)8. Similar results were detected in fluorescence results.
The fluorescence of ZnPc-(COOH)8 was sharply decreased after
interacting with Cu2+ and also proved Cu2+ form 8:1 complexes
with ZnPc-(COOH)8. The formation of ZPCN was also proved by
IR and TGA spectra (Fig. S2). As showing in Fig. 2, the 3D
supramolecular ZPCN were well dispersed in aqueous solution
with the average diameter of 50 nm.
change in the absorption intensity of ADPA with the same
irradiation time. The relative rate constant k of ZnPc-(COOH)8,
ZPCN and ZPCN+ATP were -1.26×10-2, -0.03×10-2 and -0.93×10-
2. The 1O2 generation ability of ZPCN and ZPCN+ATP were much
higher than that of commercial photosensitizer porphyrin (k=-
0.04×10-2) under the same experiment condition. Above
results confirmed that forming ZPCN could significantly
1
minimized the O2 production efficiency of ZnPc-(COOH)8 and
1
ATP could deprive Cu2+ in ZPCN and induce its O2 production
1.0
0.8
0.6
0.4
0.2
0.0
800
600
400
200
0
A
B
ZnPc-(COOH)8
efficiency recovering.
ZnPc-(COOH)8
ZPCN
ZPCN+ATP
ZPCN
ZPCN+ATP
All the Ps used in PDT are known to be degraded or when
exposed to light. Such photobleaching of Ps can be either a
disadvantage or an advantage. If the sensitizer bleaches too
rapidly during illumination, tumor destruction may be
incomplete. On the other hand, the photobleaching could be
used to decrease the concentration of sensitizer in the body
550
600
650
Wavelength / nm
700
750
640
680
720
760
800
Wavelength / nm
and thus shorten the period of photosensitivity in patients
43
following PDT.
Thus, ideal PS should have efficient ROS
generation capability to ensure its PDT efficacy and high
photobleaching rates to avoid side effect after PDT treatment.
Above results indicated that our PS have satisfied 1O2
1
generation ability. Therefore, their photobleaching during O2
generation process were detected and compared. As shown in
Fig. 3, upon exposure to the 665 nm light, the color of ZnPc-
(COOH)8 (or ZPCN+ATP) in aqueous solution changed from
blue to colorless and a distinct decrease in absorbance was
observed, indicating their photobleaching process during 1O2
generation process. On the contrary, no photobleaching was
detected in ZPCN because ZnPc-(COOH)8 existing in aggregate
Fig. 2 (A) Absorbance spectra of ZnPc-(COOH)8, ZPCN and ZPCN+ATP; (B) fluorescence
emission spectra of ZnPc-(COOH)8, ZPCN and ZPCN+ATP; (C) TEM image (1) and size
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 5
Please do not adjust margins

Please do not adjust margins
Journal of Materials Chemistry B
Page 6 of 10
View Article Online
DOI: 10.1039/C8TB00334C
ARTICLE
Journal Name
form, which has low 1O2 generation ability but strong
photostability. These results indicated that ZPCN has efficient
1.0
0.8
0.6
0.4
0.2
0.0
1.0
0.8
0.6
0.4
0.2
0.0
A
B
300
320
340
360
380
400
420
300
320
340
360
380
400
420
Wavelength /nm
Wavelength /nm
Fig. 4 Localization fluorescence image and in situ fluorescence analysis of ZnPc-(COOH)8
and ZPCN, with different treatment, with lysotracker blue and mitotracker green in
cancer cells (Bar = 25 nm).
1.0
0.8
0.6
0.4
0.2
0.0
0.0
ZPCN
C
-0.1
-0.2
-0.3
-0.4
-0.5
-0.6
ZPCN+ATP
In vitro anticancer activity
D
The in vitro anticancer activity of the different drugs against
A549 cells were evaluated by MTT assay. As shown in Fig. 5A,
cell survival percent of dark incubation cycle for ZPCN was
about 85.9%, which is possibly caused by Cu2+ mediated
anticancer pathway. No obvious dark toxicity was detected in
ZnPc-(COOH)8 treated cells. After irradiation (Fig. 5B), cell
survival percent of ZnPc-(COOH)8 treated cells was about
55.4%. Almost same cell death percent were detected in Cu2+
treated cell before (85.9%) and after irradiation (84.5%).
Interestingly, the cell survival decreasing degree of ZPCN
(23.7%) was much bigger than the sum effect of ZnPc-(COOH)8
and Cu2+, which indicated that there are that there are additive
and synergistic anticancer mechanisms in the anticancer
process of ZPCN (synergistic index=1.97) (Table 1).
ZnPc-(COOH)8
30 40
300
320
340
360
380
400
420
0
10
20
Time /s
Wavelength /nm
1.0
0.8
0.6
0.4
0.2
0.0
0.30
0.25
0.20
0.15
0.10
0.05
0.00
E
F
550
600
650
700
750
550
600
650
700
750
Wavelength /nm
Wavelength /nm
1.0
0.8
0.6
0.4
0.2
0.0
120
100
80
60
40
20
0
ZnPc-(COOH)8
G
H
ZPCN+ATP
ZPCN
Defective apoptosis (programmed cell death) represents a
major causative factor in the development and progression of
cancer. The ability of tumor cells to evade engagement of
0
10
20
30
40
550
600
650
700
750
Irradiation time /
s
Wavelength /nm
apoptosis can play a significant role in their resistance to
47
Fig. 3 Absorbance spectra of the drugs, including ZnPc-(COOH)8 (A), ZPCN (B) and (C)
ZPCN+ATP, with ADPA, after irradiation at 665 nm for 0, 20, 30 and 40 s. (D) The best fit conventional therapeutic regimens. And, failure to undergo
to the experimental points of the same sample, where the value of k obtained; the
48
apoptosis may result in treatment resistance.
Therefore,
absorption spectra of ZnPc-(COOH)8 (E), ZPCN (F) and (G) ZPCN+ATP after irradiating
with 665 nm LED; (H) photobleaching percent of ZnPc-(COOH)8, ZPCN and ZPCN+ATP in
40 s irradiation; insert panel: colour of all drugs after irradiation for 40 s.
finding effective ways to induce cancer cells apoptosis could
uncover new and effective strategies to tackle the complexity
of tumor treatment resistance. The apoptosis-inducing effect
of various drugs on A549 cells were detected using Annexin V-
FITC/PI apoptosis detection kit. Annexin V-FITC labels the
phosphatidylserine sites that translocate to the extracellular
membrane upon initiation of apoptosis. PI labels the
intracellular DNA in the late apoptotic cells where the plasma
membrane has been compromised. Above combination results
could differentiate the apoptotic and viable cells by the
1O2 generation capability to ensure its PDT efficacy and high
photobleaching rates to avoid side effect after PDT treatment.
Comparative Intracellular Localization.
The subcellular localization of PSs could also determine the
outcome of PDT. 44 Although lysosomes were considered to be
mere “waste bags” used by the cell to dispose of unwanted
biomolecules, they are now known to partake in numerous key
physiological processes in cancer, such as endocytosis,
autophagy, exocytosis, plasma membrane repair, homeostatic
maintenance, apoptosis and multidrug resistance. Therefore,
in light of this correlation between lysosomes and cancer,
Table 1. Dark (ATP depletion process) and light (ATP depletion process + PDT process, a
synergistic process) cell toxicity comparison of various drugs. Synergistic index was
calculated by dividing the expected growth inhibition rate by the observed growth
inhibition rate. An index more than 1 indicates synergistic effect and less than 1
indicates no synergistic effect. 49
lysosomes have been recently considered as a novel target for
45, 46
Dark
83.5%
97.0%
85.9%
---
Light
84.5%
55.4%
23.7%
46.8%
23.7%
1.97
anticancer therapy.
The localization of ZnPc-(COOH)8 and
Cu2+
ZnPc-(COOH)8
ZPCN
ZPCN were investigated by co-incubating photosensitized cells
with lysotracker blue and mitotracker green. As shown in Fig.
4, ZnPc-(COOH)8 and ZPCN localize in the same subcellular
region as the lysosome marker, suggesting their affinity to
lysosome. These data implied that ZPCN would decompose
and release ZnPc-(COOH)8 inside cancer cells.
Drugs
Expected
Observed
---
Synergistic index
---
6 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Please do not adjust margins
Journal of Materials Chemistry B
Page 7 of 10
View Article Online
DOI: 10.1039/C8TB00334C
Journal Name
ARTICLE
flow cytometry. As shown in Fig. 5C, ZnPc-(COOH)8 and Cu2+
To verify this hypothesis, ATP concentration influence by
showed the apoptosis-inducing characteristics, and the various drugs were studied. In vitro ATP deprivation and
apoptosis ratios were 71.10% and 28.22%, separately. Of note, hydrolysis of various drugs were carried out using ATP assay kit.
ZPCN had a prominent apoptosis ratio of 89.41%.
Results indicated that the cells incubated with Cu2+ and ZPCN
could cause obviously ATP concentration decrease (Fig. 6A),
and thus lead to energy shortages within the cells under
investigation. On the contrary, no obvious changing of ATP
decreasing was detected on ZnPc-(COOH)8 treated cell
comparing with control cells.
Anticancer mechanism of dark and light cycle
ATP is classified as a nucleoside triphosphate, which indicates
that it consists of three components, a nitrogenous base
(adenine), the sugar ribose, and the triphosphate. ATP is one
of four "monomers" required in the synthesis of RNA.
Therefore, DNA replication and DNA transcription also
consumes ATP. Besides, ATP is often referred to as the energy
Then, variation of cell cycle arrest induced by various
drugs were studied and compared. Almost no cell cycle
changing was detected in ZnPc-(COOH)8 treated cell after 48 h
dark incubation comparing with the untreated cells. The Cu2+
and ZPCN treated cells incubated for 48 h dark incubation
displayed arrest in the S phase (Fig. 6C). Fig. 6B shows the
statistical data for all of the treatment groups to enable
comparison of the proportion of A549 cells arrested in the S
phase. DNA replication occurs during this phase. S phase arrest
indicated that cell DNA replication inhibition, which is possibly
because of the insufficient ATP supplying. Such a notable
enhancement in cell cycle arrest of ZPCN shows its potential in
combination therapies.
supplier for
a lot of biological process, such as GSH
biosynthesis. Tumor cells need large amount of ATP as energy
because of their rapid proliferation and glycolysis. Intracellular
ATP concentration sharply decreases could affect ATP needed
physiological processes, such as DNA replication and GSH
50, 51
biosynthesis.
reports indicated that although tumor cells
have selected many mechanisms to escape and resist
programmed cell death, they cannot avoid ATP depletion-
induced death, which indicated that ATP is very crucial for cell
23
surviving.
Cu2+ has very strong affinity with phosphate.
Therefore, the bond between Cu and COO could be destroyed
and replaced by the bond between Cu2+ and phosphate in the
present of ATP. The Fenton-type reaction, in which copper ion
reacts with H2O2 (malignant cancerous cells would produce
excessive amounts of H2O2 and cause significant increase of
H2O2 level in the tumor microenvironment 34) to form hydroxyl
radical (·OH), a reactive oxygen species that can damage ATP
and induce ATP depletion inside cells. 35
Besides, ATP depletion and ·OH generation could also
inhibit GSH formation or directly destroy GSH (The GSH
depletion is not due to the direct interaction between ZPCN
and GSH because spectral results indicated no direct
interaction between them, Fig. S3). GSH is an important
16
120
A
B
14
12
10
8
S
phase
G2/M phase
G1 phase
100
80
60
40
20
0
#
#
#
# #
**
**
120
100
80
60
40
20
0
120
100
80
60
40
20
0
B
6
A
#
# #
4
**
**
**
2
#
# #
0
***
Control
ZnPc-(COOH)8
Cu2+
ZPCN
Control
ZnPc-(COOH)8
Cu2+
ZPCN
***
Cu2+
ZPCN
Control
ZnPc-(COOH)8
Cu2+
ZPCN
Control
ZnPc-(COOH)8
1.0
0.8
0.6
0.4
0.2
0.0
14
12
10
8
E
D
**
*
**
**
*
6
4
2
0
ZnPc-(COOH)8 Cu2+
ZPCN
Control
ZnPc-(COOH)8
Cu2+
ZPCN
Control
Fig. 6 (A) ATP concentration comparison of various drugs after 48 h dark incubation;
(Data are expressed as means ± SD; **P < 0.01, ***P < 0.001 drugs versus control; ##P <
0.01, ###P < 0.001 Cu2+ or ZPCN versus ZnPc-(COOH)8) (B) The statistical data for the
impact of drugs on the distribution of A549 cell cycle populations after 48 h dark
incubation; (C) The impact of drugs on the distribution of A549 cell cycle populations
after 48 h dark incubation; (D) GSH level inside cells comparison of various drugs after
48 h dark incubation(*P < 0.05,**P < 0.01, drugs versus control); (E) GSH/GSSG ratio
inside cells comparison of various drugs after 48 h dark incubation(*P < 0.05,**P <
0.01, drugs versus control).
Fig. 5 (A) Dark cell toxicity comparison of various drugs (data are expressed as means ±
SD; **P < 0.01 drugs versus control). (B) Light toxicity comparison of various drugs.
(Data are expressed as means ± SD; **P < 0.01, ***P < 0.001 drugs versus control; ###P
< 0.001 ZnPc-(COOH)8 or Cu2+ versus ZPCN). (C) Flow cytometric analysis of A549 cell
apoptosis induced by different drugs using Annexin V-FITC/PI staining. In each panel,
the lower-left (Q4, Annexin V-FITC-, PI-), lower-right (Q3, Annexin V-FITC+, PI-) and
upper-right (Q2, Annexin V-FITC+, PI+) quadrants represent the populations of the
viable, early apoptotic and late apoptotic cells, respectively.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 7
Please do not adjust margins

Please do not adjust margins
Journal of Materials Chemistry B
Page 8 of 10
View Article Online
DOI: 10.1039/C8TB00334C
ARTICLE
Journal Name
antioxidant defence of cells because it could prevent the
damage by ROSs. Therefore, GSH in cancer cells could
consume ROS and sharply reduce PDT activity. In other words,
reducing the GSH concentration inside cells might greatly
improve PDT activity. Reports indicated that an increase in
GSH or GSH/GSSG is indicative of augmented antioxidant
capacity, whereas a decrease is suggestive of diminished
antioxidant defences. 52 53As shown in Fig. 6D and E, Cu2+ and
ZPCN treated cells all showed GSH and GSH/GSSG decreasing,
which indicated that they could diminished the antioxidant
defences of cancer cells and helpful for the light cycle PDT
process. Based on above results, we could get a conclusion
that during the dark cycle of the treatment of ZPCN, ATP in
cells was depletion, which induce DNA replication inhibition
and antioxidant defence destruction.
Fig. 7 Fluorescence images and their Normalized average intracellular fluorescence
intensity of various ROS generation various drugs and light irradiation (Bar=100 nm, Fd
was the fluorescence intensity of drug treated cells, Fc was the fluorescence intensity of
control cells, * P<0.05, ** P<0.01, *** P<0.001 drug treated cells vs. control).
We hypothesis that during the light cycle, ROS, including
·
1
H2O2, ·OH, O2 - and O2, was generated inside cancer cells and
induced cell death. DCFH-DA is oxidized by total ROS to a
fluorescent product, dichlorofluorescein (DCF) 54. DHE, BES-
As shown in Fig. 8, ATP depletion could induce
mitochondrial hyperpolarization. But the effect of ROS burst
plus ATP depletion of ZPCN could induce 68.72% cell
mitochondria transmembrane potential depolarization, which
in much higher that ZnPc-(COOH)8 treated one (49.69%). These
results indicated that the anticancer mechanism of ZPCN is a
synergistic process rather than being simply aggregated.
Mitochondria transmembrane potential depolarization can
directly induce cell apoptosis. Therefore, the synergistic of
above cycles could cause mitochondrial dysfunction and
induce effective cell apoptosis under hypoxic condition.
1
H2O2-AC and SOSG are specific dye for O2·-, H2O2 and O2. 55-57
·
As shown in Fig. 7, no obvious O2 - were detected in all groups,
which indicated that it is not the main ROS during this PDT
1
process. No O2 generation was detected in Cu2+ treated cell.
But, Cu2+ could increase DCF fluorescence intensity but
degrease BES-H2O2-AC fluorescence intensity, which indicated
that DCF fluorescence intensity increasing was caused by the
generated ·OH from the Fenton reaction of Cu (consuming
H2O2 simultaneously). ZnPc-(COOH)8 generated H2O2 during
PDT process. Theoretically, H2O2 generation by ZPCN should be
higher than that of ZnPc-(COOH)8 because of the decreased
antioxidant defence of cells. However, the H2O2 generation by
ZPCN was not higher than that of ZnPc-(COOH)8, which is
possibly because the Fenton reaction of released Cu2+
consumed the extra H2O2. The total ROS generation ability of
ZPCN was obviously higher than that of ZnPc-(COOH)8. And
1
such ROS increasing was partly contributed by O2 generation
capability improving. Furthermore, the decreased antioxidant
defence of cells also very helpful for the increasing efficacy.
Based on above results, we could get a conclusion that during
the light cycle of the treatment of ZPCN, abundant ROS was
generated to kill the cancer cells. The dark cycle could not only
induce cell death directly, but also provide a weak antioxidant
microenvironment for light cycle PDT process.
Mitochondria is a very important ATP and endogenous
ROS generate site, so, ATP depletion and extra ROS burst could
induce mitochondria dysfunction, which can activate apoptosis
pathways capable of inducing cell death. Based on above
conception, we proposed that the dark and light cycle could
have synergistic effect to induce mitochondria dysfunction and
activate apoptosis pathways. The level of mitochondrial
rhodamine 123 uptake is directly related to mitochondrial
transmembrane potential, an indirect indicator of the
mitochondria dysfunction and energetic status of
mitochondria.58 Mitochondrial damage induces quenching or
enhancing of rhodamine 123, which is called depolarization or
hyperpolarization, and finally induce cell death.
Fig. 8 Rhodamine 123 fluorencense changing of varies drug.
8 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Please do not adjust margins
Journal of Materials Chemistry B
Page 9 of 10
View Article Online
DOI: 10.1039/C8TB00334C
Journal Name
ARTICLE
In vivo biocompatibility and anticancer activity
ZPCN treated mice, which was obviously smaller than ZnPc-
(COOH)8 treated one and the control group.
The in vivo biocompatibility of ZnPc-(COOH)8 and ZPCN was
very important for their safety using guarantee. Healthy
female Kunming mice were intravenously injected with ZnPc-
(COOH)8 or ZPCN. The group intravenously injected with saline
was set as the control group. The body weights of the mice
were recorded every day and the results indicated that no
obvious body-weight losing during 20 days treating (Fig. 9A).
Besides, the tissue sections of heart, liver, spleen, lung, kidney
and brain stained by H&E showed no apparent pathological
changes (Fig. 9B). These preliminary data indicate the high
biocompatibility of ZnPc-(COOH)8 and ZPCN.
Conclusions
Solid tumors hypoxic microenvironment is believed to be one
of the major causes for the failure of PDT. Therefore, finding
new PDT system with high activity under hypoxia condition has
attracted great attention during past years. Classic solutions
were based on directly increasing in situ O2 concentration
combining PS with
a hypoxia-activated prodrug. In this
manuscript, a new strategy was exploited based on ATP
depletion. ATP generation route for hypoxia tumor cells was
an ineffective glycolysis processes, but ATP is very important
for rapid and uncontrolled growing and dividing. Therefore,
finding effective ways to deplete ATP could make cancer cell
weak and sensitive. Based on this concept, we prepared a
nanoscale metal-organic framework nanoparticle ZPCN, which
could deplete ATP after entrapped by cancer cells and induce
satisfied PDT activity. Our research demonstrated the potential
applicability of the new strategy to overcome hypoxic tumor
treatment by PDT.
The in vivo anticancer efficiency assessed on A549 tumor
xenograft in nude mice. The tumor bearing mice were divided
into 3 groups: control, ZnPc-(COOH)8 and ZPCN treated groups.
After the intravenous administration of drugs for 6 h, 665 nm
light was directly irradiated on the tumor position. As shown in
Fig. 9C, no obvious body-weight losing was detected after
treating by ZnPc-(COOH)8 or ZPCN comparing with control
group during 20 days treatment. On the contrary, the
tumorgrowth was significantly suppressed in the ZnPc-(COOH)8
and ZPCN treated group compared to the control groups (Fig.
9D). In the H&E stained tumor section, only a small portion of
purple blue (nuclei stained by hematoxylin) area in the
Conflicts of interest
40
There are no conflicts to declare.
35
A
30
25
20
15
Acknowledgements
Saline
ZnPc-(COOH)8
10
ZPCN
5
This research was financially supported by the National
Natural Science Foundation of China (NO. 21571105 and
21671105), the project BK20161554 supported by NSF of
Jiangsu Province of China. The Priority Academic Program
Development of Jiangsu Higher Education Institutions (PAPD),
the Foundation of Jiangsu Collaborative Innovation Centre of
Biomedical Functional Materials (161090H001).
0
0
2
4
6
8
10 12 14 16 18 20
Time / day
Notes and references
1.
2.
3.
C. Zhang, K. Zhao, W. Bu, D. Ni, Y. Liu, J. Feng and J. Shi, Angew.
Chem. Int. Edit., 2015, 54, 1770-1774.
M. Lismont, L. Dreesen and S. Wuttke, Adv. Fun. Mater., 2017,
27,1606314.
I. Roy, T. Y. Ohulchanskyy, H. E. Pudavar, E. J. Bergey, A. R.
Oseroff, J. Morgan, T. J. Dougherty and P. N. Prasad, J. Am.
Chem. Soc., 2003, 125, 7860-7865.
30
25
20
15
10
5
10
8
C
D
***
***
Saline
ZnPc-(COOH)8
ZPCN
6
4
Saline
ZnPc-(COOH)8
ZPCN
2
0
0
0
2
4
6
8
10 12 14 16 18 20
0
2
4
6
8
10 12 14 16 18 20 22
Time / day
4.
5.
H. Chen, J. Tian, W. He and Z. Guo, J. Am. Chem. Soc., 2015,
137, 1539-1547.
W. L. Lu, Y. Q. Lan, K. J. Xiao, Q. M. Xu, L. L. Qu, Q. Y. Chen, T.
Huang, J. Gao and Y. Zhao, J. Mater. Chem. B., 2017, 5, 1275-
1283.
E. Ju, K. Dong, Z. Liu, F. Pu, J. Ren and X. Qu, Adv. Fun. Mater.,
2015, 25, 1574-1580.
M. Wartenberg, F. C. Ling, M. Muschen, F. Klein, H. Acker, M.
Gassmann, K. Petrat, V. Putz, J. Hescheler and H. Sauer, FASEB
J., 2003, 17, 503.
Time / day
Fig. 9 (A) Kunming mice body weight change as a function of feeding time after ZnPc-
(COOH)8, ZPCN or saline treatment; (B) H&E stained histological sections of heart, liver,
spleen, lung, kidney and brain from the Kunming mice and H&E stained histological
sections of tumor from BALB/c nude mice the after 20 days treating (Bar =100 µm); (C)
BALB/c nude mice body weight change as a function of drugs and light treating time
after drugs treatment; (D) tumor-volume change as a function of drugs and light
treating time after treating with ZnPc-(COOH)8, ZPCN or saline and 665 nm irradiation
(*** P＜0.001 ZPCN treated cells vs. control or ZnPc-(COOH)8 treated cells).
6.
7.
8.
H. Tian, Z. Luo, L. Liu, M. Zheng, Z. Chen, A. Ma, R. Liang, Z.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 9
Please do not adjust margins

Please do not adjust margins
Journal of Materials Chemistry B
Page 10 of 10
View Article Online
DOI: 10.1039/C8TB00334C
ARTICLE
Han, C. Lu and L. Cai, Adv. Fun. Mater., 2017, 27, 1703197.
Journal Name
38. L. Zhou, E. Chen, W. Jin, Y. Wang, J. Zhou and S. Wei, Dalton T.,
2016, 45, 15170-15179.
9.
G. Song, C. Liang, X. Yi, Q. Zhao, L. Cheng, K. Yang and Z. Liu,
Adv. Mater., 2016, 28, 2716-2723.
10. J. Zhou, T. Schmid, R. Frank and B. Brune, J. Biol. Chem., 2004,
279, 13506-13513.
11. Z. Wang, S. Li, M. Zhang, Y. Ma, Y. Liu, W. Gao, J. Zhang and Y.
Gu, Adv. Sci., 2017, 4, 1600327.
39. B. Thienpont, J. Steinbacher, H. Zhao, F. D'Anna, A. Kuchnio, A.
Ploumakis, B. Ghesquiere, L. Van Dyck, B. Boeckx, L.
Schoonjans, E. Hermans, F. Amant, V. N. Kristensen, K. P. Koh,
M. Mazzone, M. L. Coleman, T. Carell, P. Carmeliet and D.
Lambrechts, Nature, 2016, 537, 63-68.
12. Y. Liu, Y. Liu, W. Bu, Q. Xiao, Y. Sun, K. Zhao, W. Fan, J. Liu and J. 40. J. Yang, J. Zhao, Q. Cao, L. Hao, D. Zhou, Z. Gan, L. Ji and Z.
Shi, Biomaterials, 2015, 49, 1-8. Mao, ACS appl. Mater. Inter., 2017, 9, 13900-13912.
13. Y. Zhang, F. Wang, C. Liu, Z. Wang, L. Kang, Y. Huang, K. Dong, 41. S. Dong, H. Tian, L. Huang, J. Zhang, D. Yan, Y. Geng and F.
J. Ren and X. Qu, Acs Nano, 2018, 651-661. Wang, Adv. Mater., 2011, 23, 2850–2854.
14. A. Srivatsan, J. R. Missert, S. K. Upadhyay and R. K. Pandey, J. 42. Z. Zeng, J. Zhou, Y. Zhang, R. Qiao, S. Xia, J. Chen, X. Wang and
Porphyr. Phthalocya., 2015, 19, 109-134. B. Zhang, J. Phys. Chem. B., 2007, 111, 2688-2696.
15. C. Qian, J. Yu, Y. Chen, Q. Hu, X. Xiao, W. Sun, C. Wang, P. Feng, 43. C. L. Yu, S. J. Xu, S. Chen, M. H. Zhang and T. Shen, J. Photoch.
Q. D. Shen and Z. Gu, Adv. Mater., 2016, 28, 3313-3320. Photobio.B., 2002, 68, 73-78.
16. D. Zhang, M. Wu, Y. Zeng, N. Liao, Z. Cai, G. Liu, X. Liu and J. 44. T. Jiang, R. Mo, A. Bellotti, J. Zhou and Z. Gu, Adv. Funct.
Liu, J. Mater. Chem. B, 2016, 4, 589-599. Mater., 2014, 24, 2295-2304.
17. Y. Cheng, H. Cheng, C. Jiang, X. Qiu, K. Wang, W. Huan, A. 45. B. Zhitomirsky and Y. G. Assaraf, Drug Resist. Update., 2016,
Yuan, J. Wu and Y. Hu, Nat. Commun., 2015, 6.8785. 24, 23-33.
18. W. Fan, W. Bu, B. Shen, Q. He, Z. Cui, Y. Liu, X. Zheng, K. Zhao 46. N. Seebacher, D. J. R. Lane, D. R. Richardson and P. J. Jansson,
and J. Shi, Adv. Mater., 2015, 27, 4155-4161. Free Radical Bio. Med., 2016, 96, 432-445.
19. M. Gao, C. Liang, X. Song, Q. Chen, Q. Jin, C. Wang and Z. Liu, 47. S. Kasibhatla and B. Tseng, Mol. Cancer Ther., 2003, 2, 573-
Adv. Mater., 2017, 29, 1701429. 580.
20. L. Feng, L. Cheng, Z. Dong, D. Tao, T. E. Barnhart, W. Cai, M. 48. S. Fulda and K. M. Debatin, Oncogene, 2006, 25, 4798-4811.
Chen and Z. Liu, Acs Nano., 2017, 11, 927-937.
49. D. H. Shin, H.-Y. Min, A. K. El-Naggar, S. M. Lippman, B. Glisson
21. Y. Liu, Y. Liu, W. Bu, C. Cheng, C. Zuo, Q. Xiao, Y. Sun, D. Ni, C.
and H.-Y. Lee, Mol. Cancer Ther., 2011, 10, 2437-2448.
Zhang, J. Liu and J. Shi, Angew. Chem. Int. Edit., 2015, 54, 50. E. L. Hansen, E. B. Sozer, S. Romeo, S. K. Frandsen, P. T. Vernier
8105-8109. and J. Gehl, PloS one, 2015, 10, e0122973.
22. S. Tuncel, A. Trivella, D. Atilla, K. Bennis, H. Savoie, F. Albrieux, 51. M. A. Moro, A. Almeida, J. P. Bolaños and I. Lizasoain, Free
L. Delort, H. Billard, V. Dubois, V. Ahsen, F. Caldefie-Chezet, C. Radical Bio. Med., 2005, 39, 1291-1304.
Richard, R. W. Boyle, S. Ducki and F. Dumoulin, Mol. 52. Y. Zhou, D. E. Harrison, K. Love-Myers, Y. Chen, A. Grider, K.
Pharmaceutics, 2013, 10, 3706-3716.
23. J. Pouyssegur, F. Dayan and N. M. Mazure, Nature, 2006, 441,
437-443.
24. A. M. Poveda, M. Le Clech and P. Pasero, Transcription, 2010,
1, 99-102.
Wickwire, J. R. Burgess, M. A. Stochelski and R. Pazdro, Free
Radical Bio. Med., 2014, 71, 157-164.
53. J. Yang, Y. Duan, X. Zhang, Y. Wang and A. Yu, J. Mater. Chem.
B., 2016, 4, 3868-3873.
54. Y. Zhao, C. Ye, W. Liu, R. Chen and X. Jiang, Angew. Chem. Int.
Edit., 2014, 53, 8127-8131.
25. H. Fan, G. Yan, Z. Zhao, X. Hu, W. Zhang, H. Liu, X. Fu, T. Fu, X.-
B. Zhang and W. Tan, Angew. Chem. Int. Edit., 2016, 55, 5477- 55. Z. Zhang, S. S. Leonard, C. S. Huang, V. Vallyathan, V.
5482.
Castranova and X. L. Shi, Free Radical Bio. Med., 2003, 34,
26. J. Marx, Science, 1994, 263, 319-321.
1333-1342.
27. Y. Ma, X. Li, A. Li, P. Yang, C. Zhang and B. Tang, Angew. Chem. 56. H. Kanzaki, F. Shinohara, M. Kajiya, S. Fukaya, Y. Miyamoto and
Int. Edit., 2017, 56, 13752-13756. Y. Nakamura, Free Radical Bio. Med., 2014, 77, 239-248.
28. Y. M. Zhang and J. F. Lovell, Wires. Nanomed. Nanobi, 2017, 9: 57. Z. Zhu, Z. Tang, J. A. Phillips, R. Yang, H. Wang and W. Tan, J.
e1420. Am. Chem. Soc., 2008, 130, 10856-10857.
29. L. Wang, Q. Song, Q. Liu, D. He and J. Ouyang, Adv. Fun. 58. R. J. Mark, J. N. Keller, I. Kruman and M. P. Mattson, Brain Res.,
Mater., 2015, 25, 7017-7027.
1997, 756, 205-214.
30. C. Walling and S. Kato, J. Am. Chem. Soc., 1971, 93, 4275-4281.
31. Y. Ohta, N. Shiraishi, T. Nishikawa and M. Nishikimi, BBA-Gen.
Subjects., 2000, 1474, 378-382.
32. B. Halliwell, The Lancet, 1994, 344, 721-724.
33. S. Karthik, A. Jana, M. Selvakumar, Y. Venkatesh, A. Paul, S. S.
Shah and N. D. P. Singh, J. Mater. Chem. B., 2017, 5, 1734-
1741.
34. G. Song, Y. Chen, C. Liang, X. Yi, J. Liu, X. Sun, S. Shen, K. Yang
and Z. Liu, Adv. Mater., 2016, 28, 7143-71487.
35. Y. Ohta, N. Shiraishi, T. Nishikawa and M. Nishikimi, Biochim.
Biophys. Acta, 2000, 1474, 378-382.
36. E. Ju, K. Dong, Z. Chen, Z. Liu, C. Liu, Y. Huang, Z. Wang, F. Pu, J.
Ren and X. Qu, Angew. Chem. Int. Edit., 2016, 55, 11639–
11643.
37. Q. Zhan, X. Shi, X. Yan, Q. Liu, J. Zhou, L. Zhou and S. Wei, J.
Mater. Chem. B., 2017, 5, 6752-6761
10 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins