Nanoparticle-mediated local depletion of tumour ...10.1038/s41551-017-0115... · Figure S7 MMP2...
Transcript of Nanoparticle-mediated local depletion of tumour ...10.1038/s41551-017-0115... · Figure S7 MMP2...
ArticlesDOI: 10.1038/s41551-017-0115-8
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Nanoparticle-mediated local depletion of tumour-associated platelets disrupts vascular barriers and augments drug accumulation in tumoursSuping Li1, Yinlong Zhang1,2, Jing Wang1, Ying Zhao1, Tianjiao Ji1, Xiao Zhao1, Yanping Ding1, Xiaozheng Zhao1, Ruifang Zhao1, Feng Li1, Xiao Yang1,2, Shaoli Liu1,2, Zhaofei Liu3 , Jianhao Lai3, Andrew K. Whittaker4, Gregory J. Anderson5, Jingyan Wei2 and Guangjun Nie1,6*
1 Chinese Academy of Sciences Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, Chinese Academy of Sciences Center for Excellence in Nanoscience, National Center for Nanoscience and Technology, Beijing 100190, China. 2 College of Pharmaceutical Science, Jilin University, Changchun 130021, China. 3 Medical Isotopes Research Center and Department of Radiation Medicine, School of Basic Medical Sciences, Peking University Health Science Center, Beijing 100191, China. 4 Australian Institute for Bioengineering and Nanotechnology, Centre for Magnetic Resonance, Australian Research Council Centre of Excellence in Convergent Bio–Nano Science and Technology, University of Queensland, St Lucia, QLD 4072, Australia. 5 QIMR Berghofer Medical Research Institute, 300 Herston Road, Brisbane, QLD 4006, Australia. 6 University of Chinese Academy of Sciences, Beijing 100049, China. Suping Li, Yinlong Zhang and Jing Wang contributed equally to this work. *e-mail: [email protected]
Corrected online: Author correction 2 August 2017
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Nanoparticle-mediated local depletion of tumour-associated platelets disrupts
vascular barriers and augments drug accumulation in tumours
Suping Li1#
, Yinlong Zhang1,2#
, Jing Wang1#
, Ying Zhao1, Tianjiao Ji
1, Xiao Zhao
1, Yanping Ding
1,
Xiaozheng Zhao1, Ruifang Zhao
1, Feng Li
1 , Xiao Yang
1,2, Shaoli Liu
1,2, Zhaofei Liu
3, Jianhao Lai
3,
Andrew K. Whittaker4, Gregory J Anderson
5, Jingyan Wei
2, Guangjun Nie
1,6*
1CAS Key Laboratory for Biomedical Effects of Nanomaterials & Nanosafety, CAS Center for
Excellence in Nanoscience, National Center for Nanoscience and Technology, Beijing 100190, China
2College of Pharmaceutical Science, Jilin University, Changchun, China
3Medical Isotopes Research Center and Department of Radiation Medicine, School of Basic
Medical Sciences, Peking University Health Science Center, Beijing 100191, China
4Australian Institute for Bioengineering and Nanotechnology, Centre for Magnetic Resonance,
ARC Centre of Excellence in Convergent Bio-Nano Science and Technology, The University of
Queensland, QLD 4072, Australia
5QIMR Berghofer Medical Research Institute, 300 Herston Road, Brisbane QLD 4006, Australia
6University of Chinese Academy of Sciences, Beijing 100049, China
#These authors contributed equally to this work.
*Address correspondence to:
Guangjun Nie, Ph.D
National Center for Nanoscience and Technology (NCNST), China
Tel: +86-10-82545529
Fax:+86-10-62656765
Email: [email protected]
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TABLE OF CONTENTS
Table S1-S3 Drug loading, morphology and pharmacokinetic parameters Page 3
Figure S1-S6 In vitro characteristics Page 4-6
Figure S7 MMP2 expression in tumor cell lines and in tissues of mice Page 7
Figure S8-S11 In vivo tumor accumulation, biodistribution and drug delivery Page 8-11
Figure S12-S16 In vivo biological effects Page 12-14
Figure S17 Anti-metastasis activity Page 15
Figure S18 Safety assessment Page 16
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Table S1. Encapsulation efficiency and loading efficiency of Dox in the core nanoparticles at
varying weight ratios of copolymer/Dox
Copolymer:Dox Ratio 10:1 20:1a 30:1
Encapsulation
efficiency of Dox (%) 80.13 ± 0.31 83.05 ± 1.25 83.19 ± 2.05
Loading efficiency
of Dox (%) 7.28 ± 0.11 3.82 ± 0.09 2.70 ± 0.06
a When the copolymer/Dox ratio was 20:1, the core nanoparticles displayed a high Dox encapsulation
efficacy as well as a satisfactory loading efficiency. This ratio was used in subsequent preparations of
the nanoparticles.
Table S2. Characterization of copolymer nanoparticles containing different components
Groups Particle size (nm)a Polydispersity (PDI)
a Zeta potential (mV)
a
P-D 130.6 ± 11.8
P-D-R 145.1 ± 4.5
LP-D-R 157.6 ± 3.5
PLP-D-R 156.3 ± 10.3
0.19
0.15
0.25
0.21
39.5 ± 2.2
17.1 ± 1.1
-17.1 ± 1.5
- 19.4 ± 0.9
a Determined by dynamic light scattering (DLS).
Table S3. Plasma pharmacokinetic parameters of Dox and R300 delivered in PLP-D-R and as
free drugs after intravenous injection
Parametersa
Dox (PLP-D-R) free Dox R300 (PLP-D-R) free R300
t1/2, dist (h) 2.05 0.89 1.49 1.20
t1/2, elim (h) 58.36 19.40 69.32 1.20
Vd (L kg-1
) 2.48 3.70 1.49 1.81
Cl (L h-1
kg-1
) 0.21 0.90 0.33 0.47
AUC0-∞
(mg h L-1
) 19.11 4.45 0.75 0.53
kel (h-1
) 0.085 0.24 0.22 0.26
a Mice were treated with 4 mg kg
-1 Dox or 0.25 mg kg
-1 R300 (free drug or equivalent doses in
PLP-D-R). Plasma concentrations were fitted to a two-compartment model. t1/2, dist , distribution
half-life; t1/2, elim, elimination half-life; Vd , initial volume of distribution; Cl, plasma clearance; AUC0-∞
,
total area under curve; kel, elimination constant.
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Figure S1. Effects of weight ratios of copolymer/antibody on core nanoparticle size distribution.
Representative DLS data are consistent with an increase in the absorption of R300 onto the core
nanoparticle surface as the antibody amount increases. When the amount of antibody reached a high
level (a ratio of antibody to copolymer of over 2.5:200), nanoparticle aggregation was observed.
Therefore, the weight ratio of copolymer/antibody of 200:2.5 was used in the current study.
Figure S2. Absorption efficiency of the R300 antibody onto the core nanoparticles. The antibody
absorption efficiency was determined by evaluating the amount of protein associated with the core
nanoparticles using SDS-PAGE on a gel stained with Coomassie blue (a) and quantified by optical
density analysis (b). Data are presented as the mean ± s.d. of three replicates.
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Figure S3. Effects of weight ratios of core copolymer/shell layer on nanoparticle surface charge.
When the copolymer/lipid-polypeptide shell layer weight ratio was 8:3, the zeta potential of the
nanoparticles was ~-20 eV, ensuring their high stability in the circulation. Data are presented as the
mean ± s.d. of three independent experiments.
Figure S4. Stability of PLP after drug loading (PLP-D-R). (a) Fluorescence spectra of
Cy5.5-labeled PLP-D-R after different periods of incubation in PBS containing 10% FBS, 37ºC, pH 7.4.
Cy5.5 was encapsulated into the core compartment. (b) Fluorescence spectra of PLP-D-R containing
the R300 antibody (covalently labeled with iFluor 700) after different periods of incubation in PBS
containing 10% FBS, 37ºC, pH 7.4.
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Figure S5. Mass spectra of free peptides before and after proteolysis by MMP2. Representative
MALDI-TOF-MS results demonstrate that, compared to the mass spectrum before cleavage (a), MMP2
treatment generated two new peaks at 998 and 1059 (b), indicating successful cleavage into two
smaller peptides. The peaks obtained were of the m/z expected for MMP2 cleavage products of the
target peptide.
Figure S6. Cellular internalization of PLP-D-R. (a) Flow cytometric analysis of HUVECs incubated
with PLP-D-R in the absence or presence of MMP2 inhibitor ARP100. PLP-D-R was more easily
internalized by HUVECs in the absence of the inhibitor (black) after incubation for 8 h, compared with
the presence of MMP2 inhibitor (green), (b) The mean fluorescence intensities of intracellular Dox
were quantified. The data represent the mean ± s.d. from three independent experiments; ***p<0.001.
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Figure S7. MMP2 expression in cell liness and in tissues from MCF7 tumor-bearing nude mice.
(a) MCF7, A549 and HUVEC cell lines were maintained at 37ºC and 5% CO2 in DMEM supplemented
with 10% FBS and 1% penicillin and streptomycin. Cells were trypsinized and treated with lysis buffer.
MMP2 expression was analyzed by immunoblotting (upper panel). The activity of MMP2 expressed in
the cells was determined by gelatin zymography assay (lower panel). Purified MMP2 was used as a
positive control. (b) Mice bearing ~200 mm3 MCF7 tumors were euthanized, and the tumors and major
normal organs were removed and immunostained with an antibody specific to human MMP2 (yellow).
Nuclei were stained with hematoxylin (blue). (c) Tissue samples were ground in lysis buffer, and
MMP2 expression was assessed by immunoblotting. (d) Results in (c) were normalized and quantified
by optical density analysis.
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Figure S8. In vivo tumor targeting efficiency. (a) In vivo fluorescence signals of MCF7
tumor-bearing mice before and after treatment with PLP-D-R or LP-D-R (without the responsive
peptide component). Nude mice bearing ~250 mm3
MCF7 tumors received 100 µL of Cy5.5-labeled
hybrid nanoparticles via a single tail vein injection. Images were collected for up to 24 hours
post-injection. A high-intensity fluorescence signal was detected only in the tumor region of
PLP-D-R-treated mice 24 h post-injection. Background fluorescence was subtracted. The white ovals
indicate the tumors, n = 3. (b) In vivo photoacoustic images of the tumor area (white ovals) 24 h after
intravenous injection of indocyanine green (ICG) labeled nanoparticles. Nude mice bearing ~250 mm3
MCF7 tumors received 100 µL of either PLP-D-ICG or PLP-D-R-ICG. Mice treated with saline served
as controls. Images showed a significantly higher photoacoustic signal in the tumor of mice treated
with PLP-D-R-ICG compared to PLP-D-ICG treatment.
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Figure S9. Enhancement of tumor accumulation of nanoparticles by PLP-D-R. (a) Mice bearing
~70 mm3 MCF7 tumors were pre-treated with LP-D-R or PLP-D-R (4 mg Dox equiv kg
-1 BW; 0.25 mg
R300 equiv kg-1
BW) for 6 h, then intravenously injected with 64
Cu-labeled LP-D-R
(64
Cu-NOTA-LP-D-R). At different time points post-injection, the mice were imaged by positron
emission tomography. Representative images are shown. The PLP-D-R pre-treated mice exhibit a
notably greater signal in the tumor region (white circles) at 6 h post-injection. (b) Tissue/organ uptake
of 64
Cu 6 h after injection of radiolabeled nanoparticles. Data are presented as percentage of injected
dose per g tissue (% ID/g), mean ± s.d. (n = 3); *** p<0.001.
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Figure S10. Biodistribution profiles of PLP-D-R in tumor bearing and normal mice. (a) Ex vivo
fluorescence imaging of the tumor and major organs from mice at different time points after
PLP-D-R-Cy5.5 administration. Nude mice bearing ~250 mm3 MCF7 tumors each received 100 µL of
Cy5.5-labeled hybrid nanoparticles via a single tail vein injection. Mice treated with saline were used
as controls. T, tumor; H, heart; Li, liver; S, spleen; Lu, lung; K, kidney. (b) Ex vivo fluorescence
imaging of major organs from normal BALB/c mice at different time points after intravenous injection
of 100 µL PLP-D-R-Cy5.5. Mice treated with saline were used as controls. (c,d) Time course of
changes in Cy5.5 fluorescence intensity in the liver (c) and kidney (d) based on the imaging
represented in (b). Three different mice were used for each time point. Fluorescence intensities were
quantified and shown as mean ± s.d. and the curves were made continuous using B-spline fitting.
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Figure S11. Enhanced intratumoral Dox delivery using PLP-D-R. Mice bearing ~250 mm3 MCF7
tumors were injected with 100 μL PLP-D-R (4 mg Dox equiv kg-1
BW; 0.25 mg R300 equiv kg-1
BW)
and after 24 h tumor tissues were removed and homogenized. The Dox concentration in the tumor
homogenates was measured using fluorescence spectrometry. Results are presented as mean ± s.d. (n =
3). *** p<0.001.
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Figure S12. Platelet depletion induced by PLP-D-R causes tumor vascular damage. (a) Tumors
harvested 24 h after treatment with saline or various drug formulations were immunostained for CD31
(a marker of vascular endothelial cells). CD31-positive blood vessels are shown in brown. (b) The
vessel-positive area in the tumors was quantified using Image-Pro Plus 6.0 software. The mean ± s.d.
from three independent replicates is shown; *** p<0.001.
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Figure S13. Representative H&E staining of the major organs of PLP-D-R-treated
tumor-bearing mice. The heart, liver, spleen, lung, and kidney of tumor-bearing mice showed no
visible red cell extravasation or hemorrhagic areas 24 h post-administration of PLP-D-R.
Figure S14. Impact of PLP-D-R treatment on circulating platelets. (a) Plasma platelet counts and
(b) tail bleeding time for normal BALB/c mice 12 h after intravenous injection with PLP-D-R, PLP-D,
free R300 or saline. For the PLP-D-R and PLP-D groups, the equivalent dose used for R300 was 0.25
mg kg-1
, which is the dose used for therapeutic studies. Free R300 at 3 mg kg-1
was used as a positive
control. Plasma platelet numbers were determined by phase contrast microscopy. Data are presented as
the mean ± s.d. (n = 5); *p < 0.05; *** p < 0.001.
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Figure S15. Representative H&E stains of some major organs from saline- and R300-treated
tumor-bearing mice. Tumor-bearing mice were injected intravenously with saline, R300, PLP-D or
PLP-D-R. Twenty-four hours after injection, the major organs were harvested, and tissue sections were
stained with H&E. Bleeding was only observed in the R300-treated mice. Black arrows indicate
hemorrhagic sites.
Figure S16. Evaluation of neutrophil recruitment within tumors treated with different drug
formulations. Total Ly6G (neutrophil marker) protein in tumor tissues was determined by
immunoblotting with an antibody specific for mouse Ly6G 24 h after the administration of saline, Dox,
PLP-D or PLP-D-R.
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Figure S17. Decreased lung metastasis in mice treated with PLP-D-R. (a) Lung sections of mice
bearing 4T1 mammary tumors were treated with saline, R300, PLP or PLP-D-R. Treatments
commenced when the tumors reached ~300 mm3 and were administered once every two days for five
injections in total. Two serial sections were then cut from each lung and examined by H&E staining
(upper panels) and immunostaining with an anti-PCNA antibody (lower panels) respectively, to assess
the occurrence of metastasis. Arrows indicate lung metastases. Both R300 and PLP-D-R showed
notable ability to inhibit lung metastasis. (b) Quantification of PCNA staining positive areas in the
tumors. Data are presented as the mean ± s.d. (n = 3); *** p < 0.001.
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Figure S18. Assessment of the safety of PLP-D-R treatment in normal mice. (a) Changes in the
body weight of non-tumor bearing BALB/c mice after three intravenous administrations of saline, Dox,
PLP or PLP-D-R. Data are presented as the mean ± s.d. (n = 5); ***p<0.001. (b) Heart sections from
mice treated with saline or various drug formulations were stained with H&E. Note the disruption of
myofilaments (black arrows) in the Dox treated group. (c-e) Serum levels of biochemical indicators of
heart (c), liver (d) and kidney (e) damage in mice after three treatments of saline or various drug
formulations. Serum samples were pooled from three mice in the same group and a single measurement
was carried out.