via dark/light relay catalysis remote-controlled multi ... · via dark/light relay catalysis ying...
Post on 26-Mar-2021
3 Views
Preview:
TRANSCRIPT
S1
Electronic Supplementary Material (ESI) for Nanoscale Horizons. This journal is © The Royal
Society of Chemistry 2019
Remote-controlled multi-enzyme system for enhanced tumor therapy
via dark/light relay catalysis
Ying Chen,‡ Zi-Hao Li,‡ Jing-Jing Hu, Si-Yuan Peng, Lei Rong,* Yunxia Sun* and Xian-Zheng
Zhang*
Key Laboratory of Biomedical Polymers of Ministry of Education & Department of Chemistry,
Wuhan University, Wuhan 430072, P. R. China
* Corresponding authors. E-mail addresses: rong-lei@outlook.com (L. Rong), yx-
sun@whu.edu.cn (Y. X. Sun), xz-zhang@whu.edu.cn (X. Z. Zhang)
‡ These authors contributed equally to this work.
Electronic Supplementary Material (ESI) for Nanoscale Horizons.This journal is © The Royal Society of Chemistry 2019
S2
Experimental Section
Materials: Potassium ferricyanide, polyvinylpyrrolidone (PVP K30), poly
(dimethyldiallylammonium chloride) (PDDA) and hydrogen peroxide was obtained from
Sinopharm Chemical Reagent Co. 3,3',5,5'-Tetramethylbenzidine and purified terephthalic acid
were purchased from Sigma Aldrich. 5,5-dimethyl-1-pyrroline-1-oxide and 2,2,6,6-
Tetramethylpiperidine were purchased from Adamas Reagent. The BCA reagents and the
Superoxide Dismutase (SOD) assay kit were purchased from Beyotime. Cy5-NHS and glucose
oxidase were purchased from Aladdin Reagent Co. Ltd. (Shanghai, China). Aminophenyl
fluorescein (APF), Singlet oxygen sensor green (SOSG), 2',7'-Dichlorodihydrofluorescein
diacetate (DCFH-DA), Calcein-AM, and propidium iodide (PI) were obtained from Sigma-
Aldrich. All the reagents used were obtained from commercial suppliers without further
purification.
Preparation of PB, HMPB, GPB and mGPB: PB particles were synthesized according to a
previous report. Briefly, 131.7 mg of K3[Fe(CN)6]·3H2O and 3 g of PVP were dissolved into
40 mL of HCl solution (0.01 M) under strring conditions for 30 min, then heated to 80 °C for
20 h. And the synthetic products were acquired by centrifugation and washed with ethanol and
deionized water for three times.
HMPB was obtain by an acid etching method. 20 mg of PB and 100 mg of PVP were
dissolved into 20 mL of HCl solution (1.0 M). Then the mixture solution was added into a
Teflon vessel and heated at 140 °C for 4 h. After cooling and centrifuging, PDDA was added
to gain HMPB nanocube with positive potential. The obtained HMPB nanoparticles (20 mg)
were dissolved into 10 mL of deionized water, then 2 mg of GOx was added. After 12 h stirring,
the GPB was collected by centrifugation and washed with deionized water for three times. 1
mg of GPB and 0.5 mL of 4T1 cell membrane (2 mg/mL) were dissolved into deionized water.
Then, the mixture was extruded to obtain mGPB.
S3
Peroxidase-like activity of GPB: RhB and TMB were chosen as indicators to evaluate the
peroxidase-like activity of GPB. 20 μL of GPB (1 mg/mL) and 10 μL of RhB (9 mg/mL) were
applied to the reaction system. 10 μL of H2O2 (30% w/w) or D-glucose (1 mM) was added as
substrates. DI water was used to fill volume of 1 ml. When excited at 350 nm, the emission at
550 nm of RhB was recorded after 15 min treatment (NIR 660 nm: 30 mW cm−2). TMB, which
can be oxidized to ox-TMB with an absorption of 652 nm was used to further evaluate the
Michaelis–Menten kinetics of POD-like activity for GPB. 0.8 mM of TMB was applied to 20
μL/mL of GPB solution, followed addition of a series H2O2 or D-glucose concentration. All
reactions were monitored by measuring the absorbance of 652 nm at different times. The
Michaelis–Menten constant (KM) and maximal velocity Vmax were calculated according to the
Michaelis–Menten saturation curve.
Oxidase-like activity of GPB: The oxidase-like activity of GPB was evaluated by monitoring
the absorption spectra of ox-TMB at 652 nm. First, O2 was purged in HAC-NaAC buffer
solution (20 mM pH = 5.2) to saturate dissolved oxygen. 1 mL of TMB (3.2 mM) was applied
to 20 μL/mL of GPB solution and HAc-NaAc buffer solution (20 mM pH = 5.2) was used to
fill volume of 3 mL. All reactions were monitored by measuring the absorbance of 652 nm after
15 min treatment (NIR 660 nm: 30 mW cm−2).
Detection of •OH and 1O2: The detection of •OH was first detected based on the reaction of
between terephthalic acid (PTA) and •OH. Upon capturing •OH, negligibly fluorescent PTA
would generate 2-hydroxy terephthalic acid with unique fluorescence around 435 nm. DMPO,
as a trapping probe, were further employed to demonstrate the existence of •OH by ESR. GPB
(1 mg/mL, 5 μL), PBS (pH 7.4, 80 μL), H2O2 (1%, w/w, 5 μL) and DMPO (0.8 M, 10 μL) were
mixed to prepare the test solution. After NIR 660 nm treatment (30 mW cm−2, 15 min), ESR
spectra were recorded. The following instrument settings were used for collecting ESR spectra:
1 G field modulation, 150 G scan range.
S4
The detection of 1O2 was first identified by Singlet Oxygen Sensor Green (SOSG) (Ex = 504
nm, EM = 525 nm). TEMP, as a trapping probe of 1O2, were further employed to demonstrate
the existence of 1O2 by ESR. GPB (1 mg/mL, 5 μL), PBS (pH = 7.4, 80 μL), H2O2 (1%, w/w,
5 μL) and TEMP (0.8 M, 10 μL) were mixed to prepare the test solution. After NIR 660 nm
treatment, ESR spectra were recorded. The following instrument settings were used for
collecting ESR spectra: 1 G field modulation, 150 G scan range.
Study the immune evasive and tumor-targeted capabilities of mGPB: RAW 264.7 cells were
seeded in the glass bottom dishes at a density of 1×105 cells per well for 24 h. Then the cells
were incubated with Cy5-GPB or Cy5-mGPB for 4 h. After being washed by PBS, the cells
were incubated with Hoechst 33342 at 37 ℃ for 15 min to stain the cellular nuclei. To assess
the tumor-targeting capability of Cy5-mGPB, COS7, CT26, HeLa and 4T1 cells were seeded
onto in the glass bottom dishes at a density of 1×105 cells respectively. After cultured for 24 h,
the cells were incubated with mGPB for 4 h. Afterward, the cells were washed with PBS and
digested by trypsin and incubated with Hoechst 33342 at 37 ℃ for 15 min to stain the cellular
nuclei. Finally, all the cells were visualized by CLSM (Leica TCS SP8).
In vitro cytotoxicity study: The cytotoxicity of mPB and mGPB under different
concentrations was estimated in 4T1 cells by MTT assay. 4T1 cells (6000/well) were seeded in
the 96-well plates for 24 h. The cells were co-incubated with mPB or mGPB for 4 h and
irradiated by 660 nm laser(30 mW cm−2, 6 min). After co-incubation for 24 h, 20 μL of MTT
(5 mg/mL) was added and incubated for another 4 h. Next, the medium was replaced with 150
μL DMSO in each well. Finally, the optical density at 570 nm was measured by a microplate
reader (Bio-Rad, Model 550, USA). The relative cell viability was calculated according to the
following equation: cell viability = (OD570(samples)/OD570(control))×100%, where OD570(samples) was
obtained in the presence of nanoparticles and OD570(control) was obtained without treatment.
Animal tumor xenograft models: All animal experiments were performed according to the
guidelines for laboratory animals established by the Wuhan University Center for Animal
S5
Center Experiment/A3-Lab. All study protocols were approved by the Institutional Animal
Care and Use Committee (IACUC) of the Animal Experiment Center of Wuhan University
(Wuhan, China). Injecting 4T1 cells or CT26 cells (1 × 106) subcutaneously on the right back
the BALB/C mice (4-5 weeks old) to establish tumor model.
In vivo fluorescence imaging and biodistribution: When tumors reached a size of
approximately 150 mm3 in volume, 200 μL of DiR-mGPB (containing 200 μg/mL mGPB) was
injected into the 4T1 or CT26 bearing mice via the tail vein. Then the mice were anesthetized
and imaged by the IVIS Spectrum (PerkinElmer) at 0 h, 12 h, 24 h, 36 h, 48 h, 60 h, 72 h, 84 h
and 96 h after injection. The mice were sacrificed at 96 h after systemic injection and the major
organs (heart, liver, spleen, lung and kidney) and tumor tissues were harvested for imaging.
In vivo anti-tumor study and histochemistry analysis: When the tumor volume reached 100
mm3, 4T1 tumor-bearing mice were randomly divided into 8 groups (n = 6) and injected via the
tail vein with (1) PBS, (2) HMPB, (3) GPB, (4) mGPB and (5) PBS + Light, 6) HMPB + Light,
7) GPB + Light, 8) mGPB + Light. The light groups were irradiated by 660 nm laser (150 mW
cm−2) for 6 min after injection for 24 h. And tumor volumes and body weights of mice in eight
groups were measured every day. And tumor volume was calculated as tumor length×tumor
width2/2. All the mice were excised after 14 days treatment. Simultaneously, the main organs
(heart, liver, spleen, lung and kidney) and tumors were harvested and used for H&E staining,
immunofluorescence staining, and immunohistochemical staining.
Statistical Analysis: Without specifically mention, all data were averaged from four
independent experiments. Statistical analysis between two groups was performed using a
Student’s t-test, while more than 2 groups were performed using the Two-way ANOVA. The
differences were considered to be statistically significant for a P value < 0.05 (*P < 0.05, **P
< 0.01, ***P < 0.001).
S6
Table S1. Fitting Results of Fe Element on the Surface of GPB.
Element (%) C1s O1s N1s Fe2p
GPB+H2O2 49.64 11.8 30.95 7.61
GPB+H2O2+NIR 57.87 12.76 24.4 4.97
S7
Table S2. Room temperature 57Fe Mössbauer parameters of GPB.
System Component IS (mm/s) QS (mm/s) Г/2 (mm/s) Area (%)
LS FeII/III -0.13 0.19 56.9GPB+H2O2
HS FeIII 0.44 0.36 0.21 43.1
LS FeII/III -0.15 0.17 44.6GPB+H2O2+NIR
HS FeIII 0.39 0.42 0.24 55.4
S8
Table S3. Blood Routine Examination of mice after different treatments with PBS, HMPB,
GPB and mGPB.
Indicator PBS HMPB GPB mGPB
WBC (109/L) 6.24±0.30 6.17±0.37 6.27±0.27 5.67±0.34
RBC (1012/L) 9.32±0.75 9.55±0.52 8.64±0.41 9.19±1.13
HGB (g/L) 125±6.68 127±8.48 116±2.49 125.33±9.81
MCV(fL) 52.56±1.10 52.77±0.65 51.83±0.74 51.40±0.91
MCH (pg) 13.47±0.45 13.33±0.20 13.37±0.32 13.80±0.75
MCHC (g/L) 256.00±2.94 252.33±2.05 258.33±4.49 268.33±1.69
HCT (%) 48.93±3.14 50.47±3.43 44.77±1.82 48.76±5.14
PLT (109/L) 524.33±9.23 511.00±10.82 488.33±15.19 517.33±6.55
MPV (fL) 6.83±0.47 7.26±0.28 6.79±0.22 7.13±0.21
PDW (%CV) 8.03±0.53 9.13±0.87 8.03±0.48 9.27±0.54
PCT (%) 0.23±0.063 0.37±0.024 0.27±0.015 0.26±0.040
S9
Fig. S1 SEM images of HMPB. Scale bar = 200 nm.
S10
Fig. S2 SEM images of HMPB. Scale bar = 200 nm.
S11
Fig. S3 (A) Brunauer-Emmett-Teller (BET) nitrogen adsorption/desorption isotherms and (B)
pore size distribution for GPB calculated by Barrett Joyner-Halenda (BJH) method.
S12
Fig.S4 Fourier transform diffraction pattern of GPB nanoparticles.
S13
Fig. S5 The standard curve of BSA for BCA Protein Assay Kit.
S14
Fig. S6 (A) Fluorescence spectra of RhB degradation. (B) Relative quantification of
absorption intensity.
S15
0 2 4 6 8 10 12 14 1630
35
40
45
50
55
Tem
pera
ture
(℃)
Time (min)
PBS 2.5 mM 5 mM 10 mM 20 mM
Fig. S7 Temperature curve of GPB under 660 nm (150 mW cm−2).
S16
Fig. S8 OXD-like activity of GPB.
S17
Fig. S9 PXRD patterns of GPB with and without irradiation.
S18
Fig. S10 XPS survey of GPB, GPB +H2O2 and GPB +H2O2+Light.
S19
Fig. S11 Fourier transform infrared spectroscopy of HMPB, GPB and mGPB.
S20
400 500 600 700 800 900 1000
0.04
0.06
0.08
0.10
0.12
0.14
0.16
0.18
0.20
Abs
orpt
ion
Wavelength (nm)
Fig. S12 UV-vis spectrum of mGPB.
S21
10 20 30 40 50 60 70 80
Inte
nsity
(a.u
.)
2 (degree)
Fig. S13 PXRD pattern of mGPB.
S22
PBSmPB
mGPB
PBS+Ligh
t
mPB+Ligh
t
mGPB+Ligh
t0.0
5.0x104
1.0x105
1.5x105
2.0x105
MFI
Fig. S14 Flow quantitative analysis 4T1 cells after stain with DCFH-DA.
S23
Contro
l
DMSO VC0.0
5.0x104
1.0x105
1.5x105
2.0x105
2.5x105
3.0x105
MFI
NaN 3
Fig. S15 Flow quantitative analysis after treated with different inhibitors.
S24
Fig. S16 Live/dead cell staining after mPB and mGPB treatment with/without irradiation.
Scale bar = 100 μm.
S25
Fig. S17 Apoptosis assay of 4T1 cells after different treatments.
S26
Fig. S18 TUNEL staining of tumor tissues after different treatment with PBS, HMPB, GPB
and mGPB for 14 days.
S27
Fig. S19 Blood biochemistry analysis of mice (A) liver and (B) kidney.
S28
Fig. S20 H&E staining against the major organs (heart, liver, spleen, lung, and kidney) after
different treatment with PBS, PBS+Light, HMPB, HMPB+Light, GPB, GPB+Light, mGPB
and mGPB+Light for 14 days. H&E staining: 200× magnification.
top related