Reasonably retard O2 consumption through a photoactivity conversion nanocomposite for oxygenated photodynamic therapy

Article abstract of DOI:10.1016/j.biomaterials.2019.119312

Photodynamic therapy (PDT) brings excellent treatment outcome while also causing poor tumor microenvironment and prognosis due to the uncontrolled oxygen consumption. To solve this issue, a novel PDT strategy, oxygenated PDT (maintain the tumor oxygenation before and after PDT) was carried out by a tumor and apoptosis responsive photoactivity conversion nanocomposite (MPPa-DP). Under physiological conditions, this nanocomposite has a low photoactivity. While at H₂O₂-rich tumor microenvironment, the nanocomposite could react with overexpressed H₂O₂ to produce O₂ and release high photoactivity chimeric peptide PPa-DP for oxygenated tumor and PDT. Importantly, when the PDT mediates cell apoptosis, the photoactivity of PPa-DP be effectively quenched and the O₂ consumption appeared retard, which avoided further consumption of residual O₂ on apoptotic cells. In vitro and vivo studies revealed that this nanocomposite could efficiently change photoactivity, reasonable control O₂ consumption and increase residual O₂ content of tumor after PDT.

Full text of DOI:10.1016/j.biomaterials.2019.119312

Accepted Manuscript
Reasonably retard O consumption through a photoactivity conversion
2
nanocomposite for oxygenated photodynamic therapy
Jin Zhang, Mengqing Xu, Yongli Mu, Jinjie Li, Mohamed F. Foda, Weiyun Zhang, Kai
Han, Heyou Han
PII:
S0142-9612(19)30411-9
DOI:
https://doi.org/10.1016/j.biomaterials.2019.119312
Article Number: 119312
Reference:
JBMT 119312
To appear in: Biomaterials
Received Date: 24 February 2019
Revised Date: 16 June 2019
Accepted Date: 26 June 2019
Please cite this article as: Zhang J, Xu M, Mu Y, Li J, Foda MF, Zhang W, Han K, Han H, Reasonably
retard O consumption through a photoactivity conversion nanocomposite for oxygenated photodynamic
2
therapy, Biomaterials (2019), doi: https://doi.org/10.1016/j.biomaterials.2019.119312.
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ACCEPTED MANUSCRIPT
Reasonably retard O2 consumption through a
photoactivity conversion nanocomposite for
oxygenated photodynamic therapy
Jin Zhanga, Mengqing Xub, Yongli Mub, Jinjie Lic, Mohamed F. Fodab,d, Weiyun
Zhanga, Kai Hanb and Heyou Hana,b,*
a State Key Laboratory of Agricultural Microbiology, College of Life Science and
Technology, College of Science, Huazhong Agricultural University, Wuhan 430070,
Hubei, P. R. China
b State Key Laboratory of Agricultural Microbiology, College of Science, Huazhong
Agricultural University, Wuhan 430070, Hubei, P. R. China
c State Key Laboratory of Agricultural Microbiology, College of Food Science and
Technology, College of Science, Huazhong Agricultural University, Wuhan, Hubei
430070, People’s Republic of China
d
Department of Biochemistry, Faculty of Agriculture, Benha University, Moshtohor,
Toukh 13736, Egypt
*Corresponding author.
E-mail address: hyhan@mail.hzau.edu.
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Abstract
Photodynamic therapy (PDT) brings excellent treatment outcome while also
causing poor tumor microenvironment and prognosis due to the uncontrolled oxygen
consumption. To solve this issue, a novel PDT strategy, oxygenated PDT (maintain
the tumor oxygenation before and after PDT) was carried out by a tumor and
apoptosis responsive photoactivity conversion nanocomposite (MPPa-DP). Under
physiological conditions, this nanocomposite has a low photoactivity. While at
H2O2-rich tumor microenvironment, the nanocomposite could react with
overexpressed H2O2 to produce O2 and release high photoactivity chimeric peptide
PPa-DP for oxygenated tumor and PDT. Importantly, when the PDT mediates cell
apoptosis, the photoactivity of PPa-DP be effectively quenched and the O2
consumption appeared retard, which avoided further consumption of residual O2 on
apoptotic cells. In vitro and vivo studies revealed that this nanocomposite could
efficiently change photoactivity, reasonable control O2 consumption and increase
residual O2 content of tumor after PDT.
Keywords: tumor and apoptosis responsive, photoactivity conversion, reasonable O2
consumption, oxygenated photodynamic therapy
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1. Introduction
Photodynamic therapy (PDT) as a potent therapy modality can efficiently convert
dissolved oxygen (O2) into reactive oxygen species (ROS) to elicit cell death.[1, 2]
However, the inherent hypoxia in tumor arising from the uncontrolled proliferation of
cancer cells and distorted tumor blood vessels severely impairs ROS production and
PDT treatment. Besides, the photoactivity of most photosensitizers (PS) during PDT
are always ON. In order to maximize therapeutic effect, the persistent irradiation time
1
and constantly activated PS would continuously convert O2 into O2 to oxidize the
bio-substances of tumor cells containing apoptotic cells, which will further aggravate
the extreme hypoxia of tumor[3-6] and induced the adverse Domino effects such as
compromised therapy and poor prognosis.[7, 8]. To this end, many pioneering
strategies have been developed to improve the intratumoral O2 level by delivered or in
situ generation of O2.[7, 9-14] However, they mainly highlight how to supply O2 to
enhance the therapeutic effect. The side effect caused by O2 consumption in PDT is
rarely considered. Although the tumor can be oxygenated to some extent, these O2 are
still limited compared to the large amount of O2 loss in PDT, e. g. the low O2 loading
efficiency and low H2O2 content (< 100 ꢀM).[15, 16] Finally, the generated O2 in
tumor would be rapidly depleted again. Therefore, how to prevent immoderate O2
consumption to improve the hypoxic tumor after PDT is of considerable importance.
On the other hand, insufficient attention to controlling O2 consumption may be
attributed to the challenge of fabricating an appropriate O2 consumption conversion
system. Currently, there are many activateable PDT systems that realized
photoactivity conversion to precisely treat tumor without off-target PS
phototoxicity.[17, 18] These approaches mainly were “OFF-ON” mode that quenched
PS by sophisticated synthetic method in vitro and activated based on various triggers
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such as acidic,[19] redox,[20] protease,[21] H2S,[22] and hypoxia[23] in tumor site.
However, the photoactivity “ON-OFF” system was hard to build due to the difficulty
of performing complex chemical synthesis in vivo and rare triggers during PDT. More
importantly, untimely intervention of O2 consumption will severely impair the
efficiency of PDT, resulting in the severe compromise of treatment. It can be
imagined that if the tumor can be pre-oxygenated before PDT and a suitable trigger
can inhibit the O2 consumption reasonably to keep tumor oxygenation after PDT, the
oxygenated PDT could be expected.
Keeping all these issues in mind, we prepared a caspase-3-triggered photoactivity
conversion chimeric peptide to control the O2 consumption in PDT based on
aggregation-caused quenching (ACQ) theory[24, 25] due to the fact that PDT mainly
mediates cell apoptosis by caspase-3/7 pathway.[26] The chimeric peptide was
fabricated using i) a specified photosensitizer, pheophorbide-a (PPa), which has
significant difference in photoactivity between monomeric and aggregated states[27,
28] for PDT and ii) a hydrophilic PEGylated Asp-Glu-Val-Asp (DEVD) peptide
sequence which can be specifically cleaved by caspase-3 for changing the
hydrophile-lipophilic-balance of PPa molecules. Under physiological conditions, the
hydrophilic PEGylated DEVD polypeptide sequence could significantly increase the
photoactivity of PPa molecules for facilitated the ROS generation.[29, 30] However,
under enzyme caspase-3, the hydrophobic PPa molecules in PPa-DP underwent sharp
aggregation due to detachment of the PEGylated DEVD fragment, resulting in a
significant decrease in photoactivity. The change in photoactivity also changes the
rate of O2 consumption and realize control of O2 consumption. To pre-oxygenated the
tumor, we further adsorbed the chimeric peptide on high biocompatible MnO2
nanosheets due to the high catalytic efficiency of MnO2 with H2O2. As shown in
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Scheme 1, the MnO2 sheets could highly deliver PPa-DP into tumor cells and react
quickly with intracellular H2O2 to release O2, Mn2+ and high photoactivity PPa-DP for
PDT. Upon the apoptosis of cells, the activated caspase-3 in apoptotic cells would
trigger quenching of photoactivity of PPa-DP and prevent the O2 consumption. This
apoptosis-triggered photoactivity conversion strategy was quite rational because i)
there was no impair to treatment outcome, and ii) no additional oxygen is
inappropriately acted on apoptotic tumor cells, which should maximize the O2 content
of the tumor after PDT.
2. Materials and methods
2.1.Reagents and materials
Rink Amide resin, N-fluorenyl-9-methoxycarbonyl (Fmoc)-protected L-amino
acids,
diisopropylethylamine
(DIEA),
o-benzotriazole-N,N,N′,N′-tetramethyluroniumhexa fluorophosphates (HBTU) and
piperidine were purchased from GL Biochem Ltd. (Shanghai, China). The
Pheophorbide-a, PPa, was provided by Frontier Scientific (Logan, UT, USA). The
caspase-3 was purchased from the R&D systems. Dulbecco’s modified Eagle’s
medium (DMEM), trypsin, fetal bovine serum (FBS), MTT, and
penicillinstreptomycin were purchased from GIBCO Invitrogen Corp.
Tetramethylammonium hydroxide (TMA·OH), Manganese (II) chloride tetrahydrate
(MnCl2·4H2O), and trifluoroacetic acid (TFA) were obtained from Shanghai Reagent
Chemical Co. (China). 2′,7′-dichlorofluorescin diacetate (DCFH-DA) were bought
from Sigma-Aldrich. The hypoxia probe HP2-100Kit was acquired from
Hypoxyprobe, Inc. The All other reagents and solvents were of analytical grade and
used directly without further purification.
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2.2.Preparation and characterization of MnO2 nanosheets and PPa-DP
The MnO2 nanosheets were prepared as previously reported.[31] Typically, 20 mL
of a mixed aqueous solution of 0.6 M tetramethylammonium hydroxide and 3 wt %
H2O2 was added to 10 mL 0.3 M MnCl2 solution within 15 s. When the two solutions
were mixed, the mixture became dark brown immediately, indicating that Mn2+ was
oxidized to Mn4+. The resulting dark brown suspension was stirred vigorously
overnight in the open air at room temperature. The as-prepared bulk manganese
dioxide was centrifuged at 2000 rpm for 10 minutes and washed with copious
o
amounts of distilled water and methanol, followed drying at 60 C and storage for
further use. The MnO2 nanosheets were prepared by dispersing 10 mg bulk MnO2 in
10 mL water, followed by ultrasonication for 10 h, centrifugation at the speed of 2000
rpm, and collecting the supernatant for further use. The concentration of as-prepared
MnO2 nanosheets was quantified through inductively coupled plasma optical emission
spectrometry (Optima 8000 ICP-OES spectrometer, Perkin-Elmer) and diluted with
ultrapure water to 1 g L-1 for further use. The UV−vis absorption and XPS was
conducted to characterize the successful formation of MnO2 nanosheets.
PPa-DP was manually synthesized on Rink Amide resin (0.596 mmol g-1) via the
conventional solid phase peptide synthesis procedure. After the resin was swollen in
DMF for 1 h in nitrogen atmosphere, Fmoc group was deprotected using 20%
piperidine in DMF at room temperature for 20 min. HBTU/DIPEA was used as
coupling agents to sequentially couple amino acids to the N-terminus of peptide on
resin. The PPa was introduced to peptide chain by coupling to ε-NH2 of Lys using
FMOC-Lys(Mtt)-OH as a linker. The PPa-DP was cleaved from the resin by a
cleavage solution (TFA : TIS : H2O = 95 : 2.5 : 2.5) for 1.5 h. Then the crude product
was prepared by precipitating the mixed solution in cold anhydrous diethyl ether and
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drying under vacuum overnight. The crude was dissolved in distilled water and
freeze-dried to obtain PPa-DP. The PPa-MP was prepared as a control using a similar
method. The molecular weight was verified by electrospray ionization mass
spectrometry (ESI-MS).
The UV−vis absorption of PPa-DP (50 mg L-1) was measured using a UV-2450
UV-vis spectrophotometer. The hydrodynamic size was determined by Nano-ZS
ZEN3600 (Malvern Instruments). The morphology of PPa-DP (50 mg L-1) was
observed with a transmission electron microscope (TEM, JEM-2100 microscope).
2.3.Preparation and characterization of MPPa-DP
The MPPa-DP were fabricated by mixing the PPa-DP with MnO2 under vigorous
stirring for 2 h and further incubation for 6 h at room temperature. Then, the mixture
was centrifuged through 30 kDa MWCO Amicon filters to remove the excessive
PPa-DP. The MPPa-DP was obtained via re-dispersed the residue in water. The
morphology, size and zeta potential of MnO2 (140 mg L-1), MPPa-DP (240 mg L-1)
and MPPa-DP with H2O2 was observed by TEM and Nano-ZS ZEN3600. The
concentration of PPa-DP is 100 mg L-1. Besides, the morphologys of MnO2 and
MPPa-DP were further studied by scanning electron microscope (SEM, JSM-6390LV)
and atomic force microscope (AFM, DIMultiMode8). The concentration of MnO2 is
10 mg L-1. The fluorescence analysis of PPa-DP (30 mg L-1), MPPa-DP (75 mg L-1)
and MPPa-DP with H2O2 was performed with an RF-5301PC fluorospectro
photometer.
The degradation of MnO2 in H2O2 was studied by optical imaging and ultraviolet
spectrophotometer. 100 ꢀL MnO2 and MPPa-DP (The concentration of MnO2 is 300
mg L-1, pH 6.5) was added to ELISA plate strips with different concentrations of H2O2.
After 15 min, the optical imaging and UV-Vis spectrum were obtained. The O2
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generation by the reaction between MPPa-DP (200 mg L-1 MnO2) and H2O2/H+ (0.1
or 1 mM) was measured with an oxygen probe (JPBJ-608 portable Dissolved Oxygen
Meters, Shanghai REX Instrument Factory).
2.4.ROS detection
The ROS generation was measured using DCFH-DA as the sensor. Briefly,
PPa-DP (30 mg L-1) and DCFH-DA (250 ꢀL, 20 mg L-1) were mixed with 730 ꢀL
PBS in a quartz cuvette, followed by light irradiation with 660 nm LED at preset time.
The fluorescence spectrum from 500-550 nm was recorded at an excitation
wavelength of 488 nm. PBS, Free PPa in 0.05% DMSO was used as control. The
ROS generation ability was calculated with the formula: Ft/F0. F0 is the initial
fluorescence of samples without irradiation. Ft represents the fluorescence of samples
with irradiation for a certain period of time.
2.5.The effect of Mn2+ on characterization of PPa-DP
Specifically, 30 mg L-1 PPa-DP solution (30 ꢀL, 1 g L-1) was added into 1 mL of
MnCl2·4H2O solution with different concentrations of Mn2+. After stirring for 1 h at
room temperature, the effect of Mn2+ on PPa-DP was evaluated by UV-Vis \
pectrophotometer and fluorospectro photometer. The ROS generation of
MnPPa-DP was measured by mixing PPa-DP (30 mg L-1) with 30 equiv of Mn2+ via
DCFH method.
2.6.Caspase-3 mediated aggregation of PPa-DP
The caspase-3 induced aggregation of PPa molecules was investigated using TEM,
UV-vis and fluorospectro spectrophotometer. For the UV-vis measurement, caspase-3
(20 ꢀL, 10 mg L-1) was added to PPa-DP solution (200 ꢀL, 0.5 g L-1), followed by
immediate recording of the UV-vis absorption spectrum from 300 to 750 nm at preset
time. For the fluorescence analysis, after mixing the caspase-3 solution and PPa-DP
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solution, 10 ꢀL mixture was withdrawn at different time points and then diluted to
500 ꢀL for fluorescence analysis (Ex = 410 nm, Em = band-pass 600-700 nm). The
aggregation behavior was further observed by TEM after incubation with caspase-3
over night. For comparison, the control group PPa-MP was tested under the same
condition.
2.7.Caspase-3 mediated photoactive quenching of MPPa-DP and reduction of O2
consumption
1
To investigate the photoactivity change of PPa-DP, the generation of O2 was
detected by the UV-vis absorbance spectrum using RNO plus histidine as the sensor.
1
Briefly, RNO (50 ꢀL, 500 × 10−6 M) and histidine (150 ꢀL, 60 × 10−3 M, as O2
trapping agent) were mixed to a total volume of 1 mL PBS, MPPa-DP, MnPPa-DP
and PPa-DP with caspase-3 (pre-incubated for 1.5 h at 37 °C). The concentration of
PPa-DP is 30 mg L-1. Then, these samples were exposed to 660 nm LED and the
UV-vis absorption change at 440 nm was evaluated at different time points. For O2
consumption assay, the sample, l-histidine (60 × 10−3 M) and dissolved oxygen meter
(JPBJ-608) were packed in a sealed glass bottle without residual air. When the
dissolved O2 was stable, the samples were exposed to 660 nm LED, and the dissolved
O2 was recorded at preset time. Finally, the ꢁDO was calculated as the initial value
minus the measured value.
2.8.Cellular uptake and intracellular ROS generation study
The cellular uptake of MPPa-DP nanoparticles was examined by confocal laser
scanning microscopy (CLSM) and flow cytometry. For CLSM observation, the cells
were seeded in a 35-mm confocal dish and incubated for 24 h. After washing three
times with PBS, the cells were incubated with PPa-DP or MPPa-DP (30 mg L-1) in a 1
mL DMEM for 0.5 and 2 h. After incubation, the cells were washed repeatedly with
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PBS and the cell nuclei were stained with Hoechst 33342 for 15 min, followed by
another wash with PBS for CLSM (Leica SP8, Germany). For flow cytometry, 4T1
cells were seeded in a 6-well plate and incubated for 24 h. Then the medium was
replaced with new DMEM containing PPa-DP or MPPa-DP. After 0.5 and 2 h
incubation, cells were washed, collected and resuspended in PBS. Finally, the samples
were quantified via a FACS Calibur flow cytometer (BD Biosciences, USA).
The intracellular ROS generation was tested by CLSM, the 4T1 cells were seeded
in confocal imaging chambers and incubated for 24 h, followed by addition of
PPa-DP and MPPa-DP in DMEM at a concentration of 10 mg L-1 to the respective
chambers. After 2 h incubation, the cells were washed three times and incubated with
the ROS probe (DCFH-DA, 10 × 10−6 M). After 20 min incubation, the cells were
washed again and exposed to 45 s light irradiation for CLSM. For quantification of
intracellular ROS generation, the cells were digested by trypsin, collected, and
resuspended in PBS for flow cytometry. Datas were analyzed using the Flowjo
software.
2.9.Cell cytotoxicity assay
The cell cytotoxicity of PPa-DP or MPPa-DP was determined by MTT assay
under N2 or O2 condition. Typically, 4T1 cells were seeded in 96-well plate and
incubated for 24 h, followed by culture in the fresh medium with PPa-DP or
MPPa-DP at various concentrations. After 2 h culture, the 96-well plates were placed
in a transparent box ventilated with either N2 or O2 in advance and further incubated
for 1 h, followed by irradiating the cells with 660 nm LED (10 mW cm-2) for 45 s.
After irradiation, the cells were incubated in fresh culture medium for another 24 h.
The relative cell viabilities were measured by the standard MTT assay.
The dark cytotoxicity was analyzed by incubating 4T1 cells with different
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concentrations of PPa-DP or MPPa-DP in 96-well plates. After incubation for 24 h,
the standard MTT assay was conducted to measure the cell viabilities.
2.10.
Caspase-3 mediated aggregation of MPPa-DP in cell
First, the intracellular caspase-3 expression during PDT was examined by Western
blotting analysis. After irradiation with a 660 nm laser (10 mW cm-2) for a different
period of time, the MPPa-DP-pretreated cells were lysed and collected immediately
for WB analysis. The intracellular caspase-3 mediated aggregation of MPPa-DP was
investigated via CLSM. Briefly, 4T1 cells were incubated with the MPPa-DP in a
confocal dish for 2 h, followed by washing repeatedly in PBS and staining with
Hoechst 33342 for 15 min. Next, cells were washed with PBS again and irradiated
with 660 nm laser for 45 s. Finally, the cells were observed by CLSM at 0, 0.5 and 2 h
post-irradiation.
2.11.
Optical imaging, tissue distribution in vivo
The optical imaging study was performed using a small animal imaging system.
Typically, MPPa-DP (200 ꢀg in 100 ꢀL PBS, 4 mg/kg PPa per mouse) was injected
into mice via tail vein. At preset time, the mice were anesthetized and imaged directly
using the Maestro small animal imaging system (Cri inc.). At 24 h post-injection, the
mice were sacrificed and the heart, liver, spleen, lung, kidney, and tumor were
exfoliated and imaged for tissue distribution analysis.
2.12.
Evaluation of tumor hypoxia, HIF-1α and VEGF during PDT
Female BALB/c mice bearing 4T1 tumor (≈ 200 mm3) were administered
intravenously with 100 ꢀL of MPPa-MP and MPPa-DP at the PPa-equivalent
concentration of 4 mg kg−1. At 24 h post-injection, the mice were irradiated with a
660 nm laser (10 mW cm-2) for 0, 0.5, 1, 1.5 and 2 h, followed by the intraperitoneal
injection of hypoxia-specific probe pimonidazole (hypoxyprobe-1 plus kit,
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Hypoxyprobe Inc.) at 60 mg kg−1 for covalent conjugation to thiolcontaining proteins
in hypoxic tumor tissues. After 90 min, the tumors were excised and sectioned into 10
ꢀm thick slices with a cryostat, followed by incubation first with mouse
anti-pimonidazole antibody and then with Alexa Fluor 488-conjugated goat
anti-mouse secondary antibody according to the manufacturer’s protocols. The
hypoxia was assessed with a fluorescence microscope and the hypoxia area was
analyzed with ImageJ software (Threshold value: 21). Then, we collected the tumors
from MPPa-DP with 0 and 2 h irradiation, MPPa-MP with 2 h irradiation and PBS to
further evaluate the expression of HIF-1α and VEGF using the immunofluorescence
and immunohistochemistry analysis. The slices were observed by a digital microscope
(Leica QWin).
2.13.
Antitumor therapy in vivo and histological analysis
Animal experiments were conducted in compliance with the criteria of The
National Regulation of China. 107 of 4T1 tumor cells (100 ꢀL) was injected
subcutaneously into the Female BALB/c mice. When the volume of 4T1 tumor
xenograft growth was around 100 mm3, the mice were randomly divided into 4 groups.
PPa-DP, and MPPa-DP (dose of PPa = 4 mg kg−1, MnO2 = 6 mg kg−1) were
intravenously injected into mice every two days. PBS and MPPa-DP without light
irradiation were used as controls. At 24 h post-injection, the mice received 1 h light
irradiation (660 nm, 10 mW cm-2). The tumor volume was measured per day and was
calculated as follows: volume = (tumor length) × (tumor width)2/2. At the end of
treatment, the tumors in various groups were exfoliated and weighted for further
evaluation of the antitumor effect. Relative tumor volumes were calculated as V/V0
(V0 is the tumor volume before treatment).
After treatment, the mice were sacrificed and the main organs and tumor were
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exfoliated and fixed in 4% formalin, followed by embedding in paraffin for H&E
staining. For the serum analysis, blood samples extracted from different groups were
solidified and centrifuged at 3000 rpm for 15 min to obtain serum. Then, the TNF-a
level in serum was detected using ELISA kits (Pub. No. MAN0017423).
2.14.
The studies of MPP-DP mediated CDT
The MPPa-DP was decomposed into Mn2+ under H2O2/H+ and further caused
Fenton-like reaction with H2O2 to generate ·OH for CDT was investigated by the
degradation of methylene blue (MB) and cytotoxicity analysis. For the degradation
of MB, the MnO2 (200 mg L-1, pretreated with H2O2/H+) was added to 50 mM
NaHCO3 solution with different pHs of 6.5 and 7.4 containing 10 ꢀg mL-1 MB and 5
mM H2O2. Meanwhile, the group without H2O2 and with pure Mn2+ (MnCl2) were
o
used as negative and positive control, respectively. After 1 h at 37 C, the optical
imaging of ELISA strips and UV-Vis adsorption spectra were recorded. For
cytotoxicity assay, the 4T1 cells were seeded in 96-well plate and incubated for 24 h,
and then the medium was replaced with different concentrations of MPPa-DP. After
incubation for 24 or 48 h, the culture was extracted and 200 ꢀL new culture medium
was added. After other 24 h, cell viability was evaluated by the MTT method. The
PPa-DP and MPP-DP with Vitamin C (VC) was use as control.
2.15.
Statistical analysis
Statistical significance was analyzed by a three-sample Student’s test. Statistical
significance was inferred at a value of P < 0.05.
3. Results and discussion
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3.1. Synthesis and characterization of MPPa-DP
The PPa-DP was synthesized through the tandard Fmoc solid phase peptide
synthesis (SPPS) method (Fig. S1) and the electrospray ionization mass spectrometry
(ESI-MS) confirmed the validity of PPa-DP (Fig. S2). The MnO2 sheets were
prepared as previously reported[32]. The UV-vis adsorption spectrum (Fig. S3A),
X-ray photoelectron spectrometer (XPS, Fig. S3B), SEM (Fig. S4) and AFM (Fig.
S5A) indicated that the typical 2D MnO2 sheet with a thickness of 2 nm was
successfully prepared. Before loading the PPa-DP on MnO2 sheets, we firstly verified
that the pegylated DEVD peptide sequence could improve the water solubility and
photochemical property of PPa (Fig. 1). Transmission electron microscopy (TEM)
result showed that the amphiphilic PPa-DP could form well-dispersed and uniform
spherical nanoparticles in PBS buffer (Fig. 1A). Dynamic light scattering (DLS)
results further revealed that the corresponding hydrodynamic size was 96.15 ± 7.4 nm
(Fig. 1 B). There was discrepancy between DLS and TEM results due to the facts that
i) the nanoparticles suffered from the shrinkage in vacuum state during TEM
observation and ii) only high contrast part of PPa-DP could be extracted from TEM
picture.[31, 33] The intermolecular interaction of PPa molecules in PPa-DP in various
environmental conditions was also examined by the UV−vis spectroscopy. As shown
in Fig. 1C, the Qy-band of free PPa in DMSO was a single peak at 677 nm, which is
characteristic of the PPa monomer. However, the peak was redshifted to 685 nm in
PBS, which is characteristic of the PPa dimer.[34] This difference was due to the π-π
stacking of PPa molecules in aqueous solution, and it can transform into monomers in
DMSO. Note that the PPa-DP also displayed a sharp Qy-band at 677 nm in PBS,
suggesting that the hydrophilic PEG and carboxyl groups in PPa-DP dramatically
decreased the π-π stacking among PPa molecules. The negligible π–π stacking in this
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nanoparticle would remarkably improve the bioactivity of PPa, especially in the
generation efficacy of ROS.[27] Herein, we examined the ROS generation of PPa-DP
using 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) as a ROS sensor. In Fig.
1D, it can be seen that the free PPa and PBS displayed a low fluorescence increment
with irradiation time prolonging. However, the fluorescence intensity increased
quickly in PPa-DP under light irradiation, indicating that the pegylated DEVD peptide
sequence can effectively improve the water solubility of the PPa and facilitate ROS
generation.
The multifunctional nanocomposites (MPPa-DP) were fabricated as previously
reported.[35] After mixing the PPa-DP with MnO2 under vigorous stirring for 2 h and
further incubation for 6 h, the AFM images (Fig. S5 A and B) showed a significant
increase in the height of the MnO2 after loading PPa-DP, indicating that the chimeric
peptide was effectively adsorbed on MnO2 sheet (the drug loading efficiency is 45%).
Meanwhile, the appearance of LMCT absorption (Fig. S6) band peak at 415 nm in the
optical absorption spectrum revealed the formation of Mn-N coordinate bonds
between PPa-DP and MnO2.[36] which was consistent with previous reports.[35, 37]
The optical images and DLS results (Fig. 2A and B) showed that the MnO2 sheets and
MPPa-DP could be uniformly dispersed in aqueous solution with an average
hydrodynamic size of 137.9 and 212.5 nm, respectively. The zeta potential also
changed from -26.9 mV to -21.2 mV (Fig. 2C). These dates implied the successful
preparation of chimeric peptide -MnO2 nanocomposites.
3.2. H2O2-triggered photoactivity enhancement and oxygen generation
Because MnO2 can react with acid H2O2 solution and be decomposed into
water-soluble Mn2+.[37] Next, we demonstrated the reaction of MPPa-DP with H2O2.
As the concentration of H2O2 increases, the color and UV-vis absorption of MnO2 at
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pH 6.5 gradually disappeared (Fig. S7). The TEM results (Fig. 2A) also revealed that
the MnO2 with sheet-like morphology disappeared under acidic H2O2 and existed as a
well-dispersed and uniform spherical nanoparticles. The corresponding size and zeta
potential was 85.8 nm and -10.7 mV, which was similar to pure PPa-DP, indicating
that the MnO2 was decomposed and the PPa-DP was released from MPPa-DP. Owing
to MnO2 nanosheets have an intense and broad optical absorption spectrum, it can be
used as an efficient fluorescence quencher, the fluorescence changes of MPPa-DP
were also investigated. As shown in Fig. 2D, the fluorescence of PPa-DP decreased
obviously after loading on MnO2 and recovered as expected under acidic H2O2
solution, which further confirmed the decomposition of MnO2. Meanwhile, we have
further studied the effect of Mn2+ on PPa-DP by mixing Mn2+ with PPa-DP at
different ratios. Only negligible change was observed in UV-vis adsorption spectrum
of PPa-DP and the Qy-band still remained as a single peak (Fig. S8A). Interestingly,
although the molecular structure of PPa-DP unchanged, the fluorescence intensity
appeared obvious decreased (Fig. S8B) after addition of Mn2+, which was consistent
with incomplete fluorescence recovery in Fig. 2D. A possible explanation is that the
partial PPa molecules in PPa-DP might be coordinated with Mn2+, giving a
considerable enhancement to the deactivation and intersystem crossing of the excited
singlet state.[35, 38, 39] The enhancement of intersystem crossing further increases the
quantum yield of the triplet state and the ROS generation. Then, we evaluated the
ROS generation of PPa-DP at same molar amount of Mn2+ as MnO2 (designed as
MnPPa-DP). As expected, the Mn2+ obviously improved the ROS generation of
PPa-DP, as evidenced by faster increment of fluorescence (Fig. 2E). In addition to
MnO2, H2O2 can also be decomposed into O2. We also monitored the O2 generation
using a dissolved oxygen (DO) meter. As shown in Fig. 2F, the DO in MPPa-DP with
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H2O2 increased remarkably within minutes while no change occurred in MPPa-DP or
H2O2 solution, suggesting that MPPa-DP could trigger rapid decomposition of H2O2
to generate O2.
MnO2 has been reported to facilitate drug internalization. The delivery efficacy of
MPPa-DP was examined by confocal laser scanning microscopy (CLSM) and flow
cytometry. Compared to the pure PPa-DP, a stronger red fluorescence was found in
MPPa-DP at 0.5 h and 2 h (Fig. 3A). Flow cytometry (Fig. 3C) also showed a similar
result, demonstrating the excellent delivery efficacy of MnO2 sheets. The intracellular
ROS levels were also examined using ROS probe DCFH-DA. A brighter green
fluorescence in MPPa-DP (Fig. 3B and C) demonstrated that MPPa-DP could
generate more intracellular ROS. Subsequently, we investigated the PDT efficiency of
MPPa-DP against hypoxia by MTT assay under N2 and O2 conditions. Without light
irradiation, both PPa-DP and MPPa-DP presented negligible cell dark-cytotoxicity
(Fig. S9). Under light irradiation (10 mW cm-2) and O2 conditions, both PPa-DP and
MPPa-DP exhibited excellent phototoxicity to 4T1 cells (Fig. 3D). Meanwhile, the
MPPa-DP had higher cell toxicity than PPa-DP, partly due to the efficient delivery
efficacy and Mn2+-mediated enhancement of photoactivity. However, under N2
atmosphere, the cell cytotoxicity of MPPa-DP and PPa-DP displayed great difference.
MPPa-DP still maintained the excellent PDT efficacy as proved by the death of more
than 50% cells at the PPa concentration of 15 ꢀM. In sharp contrast, more than 85%
cells in PPa-DP group survived under the same condition. All these results indicated
that MPPa-DP could effectively improve the hypoxic environment of tumor and
facilitate the PDT.
3.3. Caspase-3 induced reduction of photoactivity and oxygen consumption
Thereafter, we studied the aggregation behavior of PPa molecular mediated by
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caspase-3 in vitro (Fig. 4A). TEM date showed that the size of nanocomposites
significant increase in TEM after treatment with caspase-3 (Fig. 4B). The Qy-band
also became a broadened double peak at 667 nm and 683 nm as depicted in Fig. 4C.
The time-dependent UV-vis spectrum from 600 nm to 700 nm (Fig. 4D) further
revealed that the Qy-band gradually shifted from 667 nm to 683 nm, clearly indicating
the aggregation of PPa molecules. Fluorescence analysis was conducted to further
confirm this aggregation behavior due to the ACQ effect. After incubation with
caspase-3, the fluorescence intensity (Fig. 4E) quickly dropped and was less than 50%
within 30 min. The caspase-3 could effectively target the cleavage of the carboxylic
terminal of PEGylated DEVD peptide sequence and induce the aggregation of
hydrophobic PPa via π–π stacking, which caused the red-shift of Qy-band and
fluorescence quenching. The aggregation behavior would greatly impair the
bioactivity of PPa, and the ROS production was detected again by using the
well-established N, N-dimethyl-4-nitrosoaniline (RNO) photobleaching method. The
1O2 generation capacity of PBS, MPPa-DP, MnPPa-DP and MnPPa-DP with caspase-3
at the same PPa concentration was assessed by calculating the change value (-ꢁA) of
the UV–vis absorption at 440 nm for RNO. As shown in Fig. 4F, a gradual increase of
-ꢁA implied that the MPPa-DP could generate 1O2 to oxidize RNO under irradiation.
However, a faster increment of -ꢁA in MnPPa-DP suggested the high photoactivity
after decomposition of MnO2 as demonstrated above. In sharp contrast, the increment
of -ꢁA was significantly retarded after incubation with caspase-3, implying the low
photoactivity of PPa. The low photoactivity would reduce the conversion efficiency of
3O2 to 1O2, and decrease O2 consumption correspondingly. To confirm this, we tested
the O2 consumption of various samples by using a simple method. The sample,
1
l-histidine (60 × 10−3 M, as O2 trapping agent) and a dissolved oxygen meter were
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packed in a sealed glass bottle without residual air in the dark. When the DO value
reached a constant value, the glass bottles were exposed to laser irradiation and the
DO value was recorded at preset time. As expected, after treatment with caspase-3,
only a slight O2 consumption was observed in Fig. 4G which agreed fairly well with
the curve of 1O2 generation, suggesting the low O2 consumption.
The caspase-3-mediated aggregation of MPPa-DP was also investigated in cells. As
PDT mainly mediates cell apoptosis by caspase-3/7 pathway, we examined the
expression of caspase-3 in 4T1 cells during PDT by Western Blot (WB) analysis. The
4T1 cells were seeded in a 10 cm2 culture dish and incubated with MPPa-DP for 2 h.
After removing the excess reagents, the cells were treated with laser irradiation or not
and collecting the cells immediately for WB analysis. A identifiable black band
representing the expression of caspase-3 was found in MPPa-DP with irradiation (Fig.
4H), whereas no or little caspase-3 expression was observed in control group and
MPPa-DP without irradiation, suggesting that the PDT could effectively and rapidly
induce the expression of caspase-3 in cells. Then, the intracellular aggregation was
observed via CLSM analysis. Meanwhile, we synthesized a caspase-3-insensitive
chimeric peptide ((PPa)KMGEDD-PEG8), designated as PPa-MP) as the control
group. No difference occurred in UV-vis spectrum (Fig. S10A) and fluorescence
intensity (Fig. S10B) before and after incubation with caspase-3 confirmed that the
PPa-MP could not aggregate under caspase-3. For CLSM observation, the MPPa-DP
and MPPa-MP was incubated with cells for 2 h, the cells were washed with PBS to
remove the excess reagents and received 45 s light irradiation. At 0, 0.5 and 2 h
post-irradiation, the intracellular fluorescence was observed by CLSM. As shown in
Fig. 4I, a stronger intracellular red fluorescence representing MPPa-DP is clustered
near the nuclear before light irradiation. However, the fluorescence was distributed
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throughout the cell at 0.5 h post-irradiation, which might be due to the escape of
MPPa-DP from endosome into the cytoplasm via photochemical internalization
(PCI).[40] Interestingly, the red intracellular fluorescence became very weak in
MPPa-DP at 2 h post-irradiation, whereas the fluorescence signal in MPPa-MP still
remained strong. The CLSM images of MPPa-MP (Fig. S11) also showed that there
was no obvious difference in the fluorescence intensity at 0.5 and 2 h post-irradiation,
demonstrating that the aggregation could occur in cells. The caspase-3-mediated the
aggregation behavior can prevent the transformation of 3O2 to 1O2 to oxidize apoptotic
cells, thereby precisely converting the rate of oxygen consumption after apoptosis and
only targeting ROS to living cancer cells. Compared to the PDT systems with
constantly active photosensitizer, the present system may ensure that the tumor
maintains a high oxygen concentration.
3.4. Elevation of oxygen and control of oxygen consumption in vivo
Based on the in vitro results, MPPa-DP was expected to boost O2 concentration in
tumor tissues. Prior to studying the O2 content of tumor, we evaluated the in vivo
bio-distribution of MPPa-DP using 4T1 tumor bearing nude mouse. Based on a small
animal imaging system, we found that the MPPa-DP was accumulated quickly in
tumor tissue within 2 h and reached its maximum at 4 h (Fig. 5A). The ex vivo
fluorescence images of major organs and tumor (Fig. 5B) dissected from mice
revealed that the tumor region still had high fluorescence signal at 24 h post-injection,
indicating the high accumulation of MPPa-DP in tumor. Next, we performed PDT on
mice with different treatment and evaluated tumor hypoxic by a hypoxia-specific
probe pimonidazole. MPPa-MP and MPPa-DP were separately injected to mice
intravenously. At 24 h post-injection, the tumors underwent different time light
irradiation by a 660 nm LED (10 mW cm-2). At the end of light irradiation,
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pimonidazole was injected intraperitoneally into mice for imaging tumor hypoxia.
Without laser irradiation, both MPPa-MP and MPPa-DP had lower expression of
green fluorescence (Fig. 5C), evidencing that MnO2 could ameliorate tumor hypoxia
due to the ability of O2 supply. As the extension of irradiation time, an increasing
green fluorescence signal in all groups was observed. The quantitative results (Fig. 5D)
also showed that the hypoxia positive area gradually increased. Obviously, the
photosensitizers accumulated in tumor could transfer energy from light irradiation to
the surrounding dissolved O2 and generate therapeutical ROS to oxidize the adjacent
bio-substances, leading to the rapid elimination of O2. Notably, the increment of
hypoxia positive area in MPPa-MP was remarkably faster than that of MPPa-DP, as
seen by the brighter green fluorescence, clearly demonstrating that the MPPa-DP with
photoactivity conversion could improve the hypoxia microenvironment before and
after PDT.
After 2 h irradiation, we further evaluated the HIF-1α and VEGF expression using
the immunofluorescence and immunohistochemistry analysis. The HIF-1α, a key
marker of hypoxia, and its downstream target, VEGF, is often associated with tumor
growth and metastasis, which play a key role in progression of tumor. Without light
irradiation, the PBS group exhibited strong red immunofluorescence as depicted in
Fig. 5E due to the intrinsic tumor hypoxia. However, only an extremely weak red
fluorescence was found in MPPa-DP without irradiation due to the production of O2.
After light irradiation, the MPPa-MP group had a strong red fluorescence indicated
high expression of HIF-1α. In contrast, the MPPa-dP had only a small amount of
HIF-1α expression. The VEGF expression also displayed similar result that only a
little brown staining was found in MPPa-DP groups while a large number of brown
staining appeared in the PBS and MPPa-MP group. The down-regulation of HIF-1α
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and VEGF may be able to reduce the risk of tumor invasion and metastasis for better
prognosis. [7, 41, 42]
3.5. Antitumor study in vivo
For antitumor effect, the 4T1 tumor-bearing mice were intravenously injected with
PBS, MPPa-DP without irradiation, PPa-MP and MPPa-DP, respectively. At 24 h
postinjection, the mice in treatment groups received irradiation (660 nm, 10 mW
cm−2 ) for 1 h. The tumor volume of mice was recorded daily over treatment and the
therapeutic effect was expressed by the relative tumor volume. As shown in Fig. 6A,
the PBS and MPPa-DP without light irradiation afforded no antitumor performance.
Besides, PPa-DP with light irradiation could inhibit tumor growth to some extent. By
contrast, MPPa-DP exhibited a significant tumor inhibition effect. Meanwhile, the the
changes of body weight were monitored daily. Unobvious change of body weight had
been observed in Fig. 6B. Additionally, the physiological morphology (Fig. S12) of
various organs (heart, liver, spleen, lung, and kidney) in various groups was identified
to be normal by H&E staining, with no pathological changes observed in the images
of stained organ slices, suggesting the low systemic toxicity of MPPa-DP. At the end
of treatment, the average tumor weight (Fig. 6C) and images of tumor tissues (Fig. 6D)
were recorded. Significant difference that was similar to that of average tumor volume
between MPPa-DP and other groups in tumor image and weight was found. The
expression of TNF-α in murine serum and Hematoxylin and Eosin (H&E) staining of
tumor slices further confirmed the therapeutical effect. A higher level of TNF-α and a
more severe cell damage (the absence of blue dots) could be found in MPPa-DP with
irradiation (Fig. S13 and Fig. 6E). All results suggested that MPPa-DP with
self-supplied O2 and the photoactivity conversion not only greatly enhanced
photodynamic efficiency, but also improved the poor tumor microenvironment in
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PDT.
Besides, it is noteworthy that the MPPa-DP group also showed a discernible
antitumor effect. This result might be attributed to Mn2+-mediate chemodynamic
therapy (CDT) described in previous literature.[43, 44] The decomposed Mn2+ in
tumor acidic microenvironment could further react with H2O2 to produce ·OH with
-
the help of physiological HCO3 and lead to a discernible antitumor effect. In order to
confirm it, the Mn2+-mediate CDT was studied by the degradation of MB and
cytotoxicity analysis. The Fig. S14A and B revealed that the pure and decomposed
Mn2+ could rapidly produce hydroxyl radicals and completely degrade MB, a dye that
can be degraded by hydroxyl radicals. The cell cytotoxic results (Fig. S14 C)
indicated that the MPPa-DP could effectively cause the loss of cell viability after 48 h
of incubation. Moreover, the cell damage could be alleviated by the addition of
Vitamin C (VC, a ROS scavenger). Therefore, the excellent antitumor effect in
MPPa-DP with light could also be attributed to the coexistence of CDT and PDT.
4. Conclusions
In this study, we fabricated a photoactivity conversion MnO2-chimeric peptide
nanocomposite (MPPa-DP) to improve the O2 content of tumor before and after PDT.
Firstly, low photoactive MPPa-DP could react with the endogenous H2O2 in tumor to
generate O2, Mn2+ and chimeric peptide PPa-DP for highly effective PDT. Secondly,
upon the apoptosis of cells, the caspase-3 could specifically cleave the PEGylated
DEVD peptide sequence, resulting in aggregation of PPa molecules. The aggregated
PS with low photoactivity significantly reduced O2 consumption in subsequent PDT.
Compared with the control group whose photoactivity was always on, MPPa-DP
improved O2 content in tumor and downregulated the expression of HIF-1α and
VEGF after PDT, which should be a novel strategy to decrease the side effect caused
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by uncontrollable elimination of O2 during PDT.
Acknowledgements
This work was supported by the Natural Science Foundation of China (21778020
and 31750110464) and Sci-tech Innovation Foundation of Huazhong Agriculture
University (2662017PY042 and 2662018PY024). We thank Wuhan Institute of
Virology, Chinese Academy of Sciences, for our in vivo fluorescence imaging work.
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