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Supporting Information
Au2Pt-PEG-Ce6 Nanoformulation with Dual Nanozyme Activities for
Synergistic Chemodynamic Therapy/Phototherapy
Man Wang a,c#, Mengyu Chang b#, Qing Chen c, Dongmei Wang c, Chunxia Li a,c*, Zhiyao Hou d*, Jun Lin b*, Dayong Jin e, Bengang Xing f
a Institute of Frontier and Interdisciplinarity Science and Institute of Molecular
Sciences and Engineering, Shandong University, Qingdao 266237, P. R. China
b State Key Laboratory of Rare Earth Resource Utilization, Changchun Institute of
Applied Chemistry, Chinese Academy of Sciences, Changchun, 130022, P. R. China
c Key Laboratory of the Ministry of Education for Advanced Catalysis Materials,
Zhejiang Normal University, Jinhua 321004, Zhejiang, P. R. China.
d Laboratory of Protein Modification and Degradation, School of Basic Medical
Sciences, Guangzhou Medical University, Xinzao Town, Panyu District, Guangzhou,
Guangdong 511436, P. R. China.
e Institute for Biomedical Materials and Devices, Faculty of Science, University of
Technology Sydney, NSW 2007, Australia
f School of Physical & Mathematical Sciences, Nanyang Technological University,
Singapore
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Experiment Section
Chemical
L-proline, chloroauric acid (HAuCl4), chloroplatinic acid (H2PtCl6) and ascorbic
acid (AA) were purchased from Shanghai Macklin Biochemical Co., Ltd. SH-
PEG3000-NH2 was purchased from PegBio Co., Ltd (Jilin, China). Chlorin e6, N-
hydroxysuccinimide (NHS) and 1-ethyl-3-[3-(dimethylamino) propyl] carbodiimide
hydrochloride (EDC) were obtained from J&K Scientific Ltd. DPBF, MTT, N,N-
Dimethylformamide (DMF), TMB and [Ru(dpp)3]Cl2 (RDPP) were purchased from
Aladdin. All chemicals were used directly without further purification.
Characterization
The X-ray diffraction (XRD) patterns were tested with a D8 Focus diffractometer
(Bruker) with the use of Cu Kα radiation (λ = 0.15405 nm). Transmission electron
microscopy (TEM) was recorded using a FEI Tecnai G2 S-Twin with a field emission
gun operating at 200 kV. The UV-Vis absorption spectra were recorded from UH5300
UV-Vis spectrophotometer. Thermal images were obtained by a FLIR T420 thermal
camera.
Photothermal properties of Au2Pt-PEG-Ce6 NPs
To measure the photothermal effect of Au2Pt-PEG-Ce6, Au2Pt-PEG-Ce6 with
different concentration (0, 100, 200, 300, and 400 µg mL-1) was exposed to NIR light
for 300 s (808 nm, 1 W cm-2). The real-time temperature was recorded every 15
seconds by infrared thermal camera. Then, the photothermal effect of Au2Pt-PEG-Ce6
(400 µg mL-1) was studied by irradiation with different power (0.5, 0.75, and 1 W cm -
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2).
Calculation of Photothermal Conversion Efficiency (η): The photothermal
conversion efficiency is calculated by formula (1):
ŋ=hS (Tmax , NP−T surr )−Qdis
I (1−10−A808 ) (1)
where h is the heat transfer coefficient, S is the surface area of the container, Tmax is
the maximum temperature of the solution, Tsurr is the surrounding temperature, I is the
laser power density, and A808 is the absorption value of the material at 808 nm. Qdis is
the heat generated after water and container absorbs light. To calculate hS, equation
(2) (3) was introduced:
Qdis=hS (T max, H 2 O−T surr) (2)
τ s=mD CD
hS (3)
mD is the mass of water, CD is the heat capacity of water (4.2 J·g-1·°C-1), τs is the
sample system time constant, which was calculated by formula (4) (5):
t=−τ s lnθ (4)
θ=T surr−T
T surr−T max
(5)
Cell compatibility
L929 and Hela cells were cultured in DMEM supplemented with 1% (v/v)
penicillin/streptomycin, and 10% (v/v) fetal bovine serum (FBS) at 37 °C under 5%
CO2. In total, 8000 L929 or Hela cells were seeded into 96 well plates and incubated
with different concentrations (0, 100, 200, 300, 400 and 500 µg mL-1) of Au2Pt or
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Au2Pt-PEG-Ce6 dispersed in DMEM for 24 h. Relative cell viabilities were detected
by the standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
assay.
Cellular uptake
Hela cells were seeded in 6-well plate at a density of 1×105 cells per well and
cultured overnight. Then the medium was replaced with fresh culture medium
containing Au2Pt-PEG-Ce6-RhB (200 µg mL-1), respectively. After incubation for 4 h,
the cells were washed with PBS several times and fixed with 4% paraformaldehyde
for 10 min. For nucleus labeling, the cells were incubated with DAPI solution for 10
min. Then the medium was removal and rinsed with PBS several times. The cellular
uptake was examined using a fluorescence microscope.
In vitro cytotoxicity evaluation
The cytotoxicity of Au2Pt-PEG-Ce6 to Hela cells was investigated. Briefly, Hela
cells (8000 cells per well) were seeded into 96-well plates and allowed to settle for 24
h. Subsequently, the medium was replaced with fresh culture medium containing
Au2Pt-PEG-Ce6 (0, 100, 200, 300 and 400 µg mL-1) with or without H2O2 (100 μM).
The pH value of medium was adjusted to 7.35 and 6.75 by the addition of HCl (1 M),
respectively. Then, the cells were further incubated for 24 h at 37 °C under 5% CO2.
At last, the cell viability was evaluated using MTT assay.
Enhancement of PDT in cell levels by Au2Pt-PEG-Ce6
Hela cells were inoculated into 96-well plates and incubated for 24 h in a 5% CO2
incubator at 37 °C followed by the addition of Au2Pt-PEG-Ce6 (0, 100, 200, 300 and
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400 µg mL-1) or free Ce6 (0, 6, 12, 18 and 24 µg mL -1) for 4 h. After washing three
times with PBS to remove excess nanoparticles or Ce6, the cells were irradiated (650
nm laser, 0.494 W cm-2) for 5 min in an air atmosphere. Then, the cells were further
incubated for 24 h at 37 °C under 5% CO2. At last, the cell viability was evaluated
using MTT assay. In order to prove that nanomaterials can decompose hydrogen
peroxide to produce oxygen for enhanced PDT, Hela cells were incubated with
various concentrations of Au2Pt-PEG-Ce6 or free Ce6 for 4 h and placed in a clear
box filled with nitrogen atmosphere. After washing three times with PBS to remove
excess nanoparticles or Ce6, the cells were irradiated (650 nm laser, 0.494 W cm-2) for
5 min in a nitrogen atmosphere. The cells continued to be incubated for 24 h and then
cell viability was determined by standard MTT method to evaluate the PDT effect of
Au2Pt-PEG-Ce6 in different atmospheres.
Toxicity evaluation
Healthy Balb/c mice were intravenously injected with saline (control group) and
Au2Pt-PEG-Ce6 at a dose of 20 mg kg-1 (test group), respectively. After injection at
day 14, the mice were euthanized and then the blood was collected for biochemistry
analysis. In addition, the major organs (heart, liver, spleen, lung and kidney) were
harvested and dissected to make paraffin sections for further haematoxylin and eosin
(H&E) staining.
Bio-distribution of Au2Pt-PEG-Ce6 in mice
Healthy Balb/c mice were injected with Au2Pt-PEG-Ce6 (100 μL, 20 mg kg-1)
intravenously. Then the mice (n = 3) were euthanized at different time points (1 h, 4
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h, 12 h, 1 day, 3 days, and 7 days). The major organs (heart, liver, spleen, lung and
kidney) and tumors were collected in a beaker to be weighed. Then all of the organs
and tumors were heated in concentrated nitric acid and H2O2 (v/v = 1:2) at 70 °C until
the solution became clear. The concentrations of Au in the solutions were measured
by ICP-MS, and the concentrations in each organ and tumor were calculated.
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Fig. S1. EDS spectrum of Au2Pt NPs.
Fig. S2. The line scanning profiles of Au and Pt in Au2Pt NPs.
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Fig. S3. (a) Hydrodynamic diameter of Au2Pt NPs and (b) Au2Pt-PEG-Ce6 NPs
measured in water, respectively. (c) The size distribution and (d) photographs of
Au2Pt-PEG-Ce6 NPs in water (1), PBS (2) and DMEM containing 10% fetal bovine
serum (FBS).
Fig. S4. The zeta potential of Au2Pt (1), Au2Pt-PEG-NH2 (2) and Au2Pt-PEG-Ce6 (3).
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Fig. S5. UV-Vis-NIR absorption spectra of Au2Pt-PEG-Ce6 after different treatments.
(a) 650 nm (0.25 W cm-2, 5 min) and 808 nm (1 W cm-2, 5 min) laser irradiation and
(b) 650 nm laser irradiation for different time.
Fig. S6. (a) UV-Vis-NIR absorption spectra of the Au2Pt-PEG-Ce6 NPs at varied
concentrations and (b) Mass extinction coefficient of Au2Pt-PEG-Ce6 NPs at 808 nm.
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Fig. S7. The thermal images of H2O and Au2Pt-PEG-Ce6 NPs exposed to 808 nm
laser irradiation for 5 min.
Fig. S8. (a) Images of systems at pH 6.75 in the presence of different concentrations
of Au2Pt. (1) 0 μg mL-1, (2) 3.25 μg mL-1, (3) 6.5 μg mL-1, (4) 9.75 μg mL-1, (5) 13 μg
mL-1, (6) 19.5 μg mL-1. (b) The influence of pH on CAT-like activity of Au2Pt. (c)
Kinetics for CAT-like activity of Au2Pt (pH 7.0).
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Fig. S9. The amount of oxygen produced in different temperature.
Fig. S10. (a) The influence of pH on POD-like activity of Au2Pt. (b) The influence of
temperature on POD-like activity of Au2Pt. (c) Kinetics for CAT-like activity of
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Au2Pt to H2O2 substrate or (d) TMB substrate (pH 4.5).
Fig. S11. The possible mechanism of Au2Pt nanozymes. (1) The process of slow
decomposition of H2O2 at room temperature to produce O2 and H2O. (2) H2O2 reacts
with the surface of Au2Pt nanozymes to form Au2Pt (O), releasing one molecule of
H2O. Next, a second molecule of H2O2 reduces Au2Pt (O) to metallic Au2Pt, releasing
a second molecule of H2O and O2 [1]. (Au2Pt (O) is used to mark chemisorbed oxygen
on Au2Pt.) (3) For the acidic condition with H pre-adsorbed onto the surface of Au2Pt,
H2O2 could still be adsorbed and produce H2O and Au2Pt (OH), followed by
conversion of Au2Pt (OH) into H2O and Au2Pt (O) on the surface of Au2Pt. When the
Au2Pt (O) attacked the substrates to abstract H atoms, a peroxidase-mimicking
process was completed [2]. (Au2Pt (OH/O) is used to mark species adsorbed on
Au2Pt.)
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Fig. S12. (a) The peroxidase activity of Au2Pt under 2 mM of H2O2 (pH 6.75) after 30
min incubation. (b) The changes of dissolved O2 of H2O2 (2 mM) catalyzed by Au2Pt
in pH 6.75 after 30 min incubation. According to the Beer-Lambert Law (A = εbc),
the concentration of oxidized TMB was calculated to be 10.38 μM, indicating 10.38
μM of H2O2 was involved in the peroxidase reaction of Au2Pt. Meanwhile, the change
of O2 content was measured by a portable dissolved oxygen meter, the concentration
of O2 was determined to increase by 0.4422 mM, suggesting 0.8844 mM of H2O2 was
involved in the catalase reaction of Au2Pt.
Fig. S13. (a) Time-dependent absorption changes at 652 nm of TMB (0.04 mM) in
the presence of Au2Pt (0.0166 mM) and (b) Au2Pt-PEG-Ce6 (0.0166 mM) and H2O2
(2 mM). (c) Oxygen generation in H2O2 solution (3.2 mM) in the presence of Au2Pt
(0.028 mM) and (b) Au2Pt-PEG-Ce6 (0.028 mM).
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Fig. S14. L929 and Hela cells viability incubated with Au2Pt or Au2Pt-PEG-Ce6 for
24 h at different concentrations.
Fig. S15. Hela cells viability after treatment with Au2Pt at various concentrations (0,
100, 200, 300 and 400 μg mL-1) for 24 h under different pH conditions with or
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without H2O2.
Fig. S16. Bio-distribution of Au in major organ and tumor of mice after injection of
Au2Pt-PEG-Ce6 (20 mg kg-1, 100 μL) at different time points.
Fig. S17. Blood biochemical and haematological analysis of the healthy mice
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intravenously injected with normal saline or Au2Pt-PEG-Ce6 (20 mg kg-1, 100 μL) at
14 days post-injection.
Fig. S18. The H&E stained histological slices from mice receiving intravenous
injection of normal saline (control group) or Au2Pt-PEG-Ce6 (20 mg kg-1, 100 μL)
after 14 days. Scale bar: 20 μm.
Fig. S19. The H&E stained images of major organs after the different treatments.
Scale bar: 20 μm.
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Table S1. Comparison of the Km and Vmax between Au2Pt and HRP.
catalyst Substrate Km (mM) Vmax (10-8 M s-1)
Au2Pt H2O2 5.045 14.11
Au2Pt TMB 0.05378 18.405
HRP H2O2 6.36 18.8
HRP TMB 0.041 4.3
References
[1] S. Rui, B. Marion, C. Stephen, T. Kevin, S. Andrew, J. Rimstidt, Pavel M. Usov,
M. Amanda, and F. Marc Michel, Mechanism and Kinetics of Hydrogen Peroxide
Decomposition on Platinum Nanocatalysts, ACS Appl. Mater. Interfaces 10 (2018)
21224−21234.
[2] J. Li, W. Liu, X. Wu, X. Gao, Mechanism of pH-switchable peroxidase and
catalase-like activities of gold, silver, platinum and palladium, Biomaterials 48 (2015)
37-44.
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