Neutrophil-mediated anticancer drug delivery for …...I o ed. S1 Neutrophil-mediated anticancer...
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Neutrophil-mediated anticancer drug delivery for suppression of
postoperative malignant glioma recurrence
Jingwei Xue, Zekai Zhao, Lei Zhang, Lingjing Xue, Shiyang Shen, Yajing Wen, Zhuoyuan
Wei, Lu Wang, Lingyi Kong, Hongbin Sun, Qineng Ping, Ran Mo*, Can Zhang*
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Materials. Soy phosphatidylcholine (SPC) was purchased from Taiwei Pharmaceutical Co.,
Ltd. Cholesterol (Chol) was provided by Huixing Biochemistry Reagent Co., Ltd. Paclitaxel
(PTX) was purchased from Yew Pharmaceutical Co., Ltd. Coumarin 6 (Cou6) was purchased
from Sigma-Aldrich Co. Antibodies: FITC-conjugated Ly-6G/Ly-6C (Gr-1) antibody
(BioLegend, RB6-8C5, 108406), PE-conjugated MAIR-IV (CLM-5) antibody (BioLegend,
TX69, 139605), PE-conjugated CD11b antibody (BioLegend, M1/70, 101208), Alexa Fluor
488-conjugated TNF-α antibody (Bioss, Polyclonal, bs-2081R-A488), Alexa Fluor 647-
conjugated CXCL1 antibody (Bioss, Polyclonal, bs-10234R-A647). ELISA kits: mouse IL-10
ELISA kit (Elabscience, E-EL-M0046c), mouse TNF-α ELISA kit (Elabscience, E-EL-
M0049c), mouse CXCL1/KC ELISA kit (Xinle, xl-Em0420).
Synthesis of cationic lipid, 1,5-dioctadecyl-N-histidyl-L-glutamate (HG2C18). HG2C18 was
synthesized as illustrated in Supplementary Fig. 1.1 Briefly, glutamic acid (11.8 g, 80.2 mmol)
and p-Tos (18.3 g, 96.2 mmol) were dissolved in toluene (350 mL) with stirring for 1 h.
Octadecanol (47.8 g, 176.7 mol) was then added to the reaction mixture with stirring for 12 h.
After removing toluene by the rotary evaporation, the reactant was dissolved in
dichloromethane (DCM), followed by washing with 5% NaHCO3 (100 mL × 2) and distilled
water (100 mL × 1). The organic layer was dried with anhydrous sodium sulphate. 1,5-
Dioctadecyl-L-glutamate (G2C18, 29 g) was recrystallized from methanol (600 mL) with a
yield of 55.4%.
Boc-L-His(Tos)-OH (6.9 g, 16.8 mmol), DCC (10.4 g, 50.4 mmol) and NHS (2.9 g, 25.2
mmol) were dissolved in N,N’-dimethylformamide (DMF) (100 mL) with stirring for 3 h at
room temperature. The synthesized G2C18 (11 g, 16.9 mmol) and TEA were dissolved in
DMF (300 mL) with stirring for 1 h at room temperature. Afterward, two solutions mentioned
above were mixed together with further stirring for 12 h at room temperature. The reaction
solution was then suspended in water (2.4 L), and the solid precipitate was collected by
vacuum filtration. Upon recrystallization from methanol, a white powder ((Boc)H(Tos)G2C18)
was obtained with a yield of 50.8%. The obtained (Boc)H(Tos)G2C18 (8.9 g, 8.5 mmol) was
dissolved in 50% solution of trifluoroacetic acid (TFA) (30 mL) with stirring for 4 h at room
temperature. The pH of the reaction mixture was then adjusted to 8-9 with 5% sodium
bicarbonate solution and the organic layer was collected. After dried with anhydrous sodium
sulfate and concentrated, 1,5-dioctadecyl N-(N-g-tosyl) histidyl-L-glutamate (H(Tos)G2C18)
was obtained with a yield of 93.2%.
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H(Tos)G2C18 (2.06 g, 2.18 mmol) and HOBt (3.54 g, 26.0 mmol) were dissolved in
tetrahydrofuran (100 mL) with stirring for 5 h at room temperature. 1,5-dioctadecyl-N-
histidyl-L-glutamate (HG2C18) was obtained with a yield of 69.8% from the resulting solution
above by column chromatography separation (dichloromethane:methanol, 30:1, v:v), followed
by the rotary evaporation.
HG2C18: 1H NMR (CDCl3+CD3OD, 500 MHz, δ ppm): 7.16 (d, 1H, N=CH), 6.94 (d, 1H,
CH2C=CH), 4.50 (q, 1H, NHCH), 4.08 (t, 4H, COOCH2), 3.88 (m, 1H, NH2CH), 2.93-3.10 (q,
2H, NHCHCH2), 2.37(t, 2H, CH2CO), 1.98-2.18 (m, 2H, NH2CHCH2), 1.25-1.62 (m, 60H,
CH2(octadecyl)), 0.88 (t, 6H, CH3); HRMS (ESI): m/z ([M+H]+) calcd for C47H88N4O5 (788.68),
found 788.6827.
Preparation and characterization of PTX-loaded liposomes. PTX-loaded cationic
liposomes (PTX-CL) composed of SPC, HG2C18 and Chol were prepared using the thin-film
hydration method.2,3 PTX, SPC, HG2C18 and Chol (1:24:6:3, w:w:w) were dissolved in the
mixture of chloroform and methanol (2:1, v:v), followed by removal of organic solvents via
the vacuum rotary evaporation at 40°C. After vacuum dry overnight, the lipid film containing
PTX was hydrated with the distilled water at 37°C. The liposome solution was ultrasonicated
under an ice water bath and repeatedly extruded through the membrane filters with a pore size
of 0.45 and 0.22 μm. PTX-loaded conventional liposomes (PTX-L) without HG2C18 were
prepared as a control using the same method above except that the mass ratio of PTX, SPC
and Chol was adjusted to be 1:30:3. For fluorescence tagging, Cou6 (0.02% of total lipid, w:w)
was added into the liposome compositions.
The encapsulation efficiency (W/W0 × 100%) and drug-loading efficiency (W/(W+Wlipid)
× 100%) of PTX were calculated, where W and W0 are the amount of PTX in the liposomes
after and before passing over Sephadex G-50 column, respectively, and Wlipid is the amount of
total lipids in the liposomes. The encapsulation efficiency and drug-loading capacity of PTX
in PTX-CL were about 93.2% and 2.6%, respectively. The PTX concentration in PTX-CL
was about 1 mg/mL.
The particle size and zeta potential of the liposomes were measured using the Zetasizer
(Nano ZS, Malvern). For the transmission electron microscope (TEM) characterization, the
liposomes were dropped onto a copper grid (300 mesh) and stained by phosphotungstic acid
(1%, v:v). After air-dry, the sample was observed by TEM (H-7650, Hitachi) at the
accelerating voltage of 80 kV.
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The release of PTX from liposomes was investigated using a dialysis tube.2 1 mL of the
liposomes was added into a dialysis tube (10 K MWCO) against 50 mL of PBS (pH 7.4 or 4.5)
containing Tween 80 (1%, w:v), and gently shaken at 50 rpm and 37°C. At predetermined
time intervals, 1 mL of sample was withdrawn and filtered through a 0.22 pore-sized
polycarbonate membrane filter, followed by replacing with 1 mL of fresh buffer solution with
the same pH value. The amount of PTX released was determined by HPLC (LC-2010AHT,
SHIMADZU).
Cytotoxicity of PTX-CL towards NEs. The in vitro cytotoxicity of PTX-CL against NEs
was estimated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
assay. The blank NEs (1 × 104 cells/well) were seeded in 96-well plates and cultured in the
FBS free medium for 1 h. Afterward, NEs were incubated with different concentrations of
PTX-CL or Taxol for 12 h, followed by adding the MTT solution to a final concentration of
0.5 mg/mL. After 4 h of incubation, the medium was removed and the cells were mixed with
150 μL of dimethyl sulfoxide (DMSO). The absorbance was measured at a test wavelength of
570 nm by a microplate reader (Multiskan MK3, Thermo Scientific). The cell viability was
calculated as Asample/Acontrol × 100%, where Asample and Acontrol were the absorbance at 570 nm
after the cells were treated with or without the testing samples.
Tumour penetration in 3D tumour spheroids. The 3D tumour spheroids of G422 cells were
obtained using a liquid overlay method.4 Each well of 96-well plates was pre-coated with 100
μL of the FBS free medium containing sterile agarose (2%, w:v). Subsequently, G422 cells
(5000 cells/well) were seeded into each well and cultured in the medium containing FBS
(10%, v:v). The tumour spheroids were allowed to grow up to attain the diameter about 300
μm for 14 days at 37°C. The formation of 3D tumour spheroids was monitored using an
optical microscope. The uniform and compact tumour spheroids were selected for the
subsequent studies.
The cytokines such as TNF-α and CXCL1/KC in the culture medium (extratumoural)
and tumour spheroid (intratumoural) were detected within 14 days of culture using an
enzyme-linked immunosorbent assay (ELISA) method (extratumoural: in the culture medium;
intratumoural: inside the tumour spheroid). Briefly, at predetermined time intervals, the
culture medium was sampled, and the G422 spheroids were gently dispersed to single cells,
followed by centrifugation at 400 g for 3 min. The levels of TNF-α and CXCL1/KC in the
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supernatant and culture medium were assayed using the corresponding ELISA kits,
respectively.
To assess the tumour penetration capability of the NE formulation, the cell membranes
of Cou6-CL/NEs were first stained with 1,1’-dioctadecyl-3,3,3’,3’-tetramethylindotricarbo
cyanine iodide (DiR) (Life Technologies) by incubating with DiR (2 μM) for 30 min upon the
firm anchoring of the hydrophobic alkyl chain of DiR into the lipid bilayer of the NE
membrane. After washing with ice-cold PBS thrice, the double-stained Cou6-CL/DiR-NEs
was obtained. The distribution of Cou6 and DiR signals in Cou6-CL/DiR-NEs was observed
using the confocal laser scanning microscope (CLSM) (LSM 700, Zeiss). The tumour
spheroid transferred in a confocal dish was incubated with 1 mL of Cou6-CL/DiR-NEs (2 ×
105 cells equivalent to 33 ng/mL Cou6) for 8 h. Afterward, the tumour spheroids were washed
with ice-cold PBS twice. The images of the tumour spheroid were acquired from the top to the
middle of the spheroid using Z-stack tomoscanning (LSM 700, Zeiss). By comparison, the
tumour spheroids were incubated with 1 mL of the Cou6 solution (33 ng/mL Cou6) and
Cou6-CL (33 ng/mL Cou6), respectively. In addition, the images of the tumour spheroids
after incubation with Cou6-CL/DiR-NEs were obtained in real time at the fixed depth of 120
μm from the surface to the middle of the tumour spheroid.
Intercellular transport of liposomes from NEs to tumour cells. G422 cells (1 × 105
cells/well) were seeded in confocal dishes. After 12 h of culture, G422 cells were incubated
with 1 mL of Cou6-CL/DiR-NEs (1 × 105 cells equivalent to 16.5 ng/mL Cou6) or PMA
treated Cou6-CL/DiR-NEs. At predetermined time intervals (4 and 8 h), the cells were gently
washed with ice-cold PBS thrice, followed by staining with PI (5 μg/mL) for 30 min. Hoechst
33342 was used for nuclear counterstaining. The cells were observed using CLSM (TCS SP5,
Leica).
Cytotoxicity of PMA-treated PTX-CL/NEs against G422 cells. The in vitro cytotoxicity of
PTX-CL/NEs after PMA treatment against G422 cells was determined using the MTT assay.
G422 cells (1 × 104 cells/well) was seeded in 96-well plates and cultured for 24 h. PTX-
CL/NEs were pretreated with PMA (100 nM) for 4 h. Afterward, both the untreated and
PMA-treated PTX-CL/NEs were centrifuged at 2000 rpm for 5 min. The supernatant was
incubated with G422 cells for different times (24, 48, 72, 96 h), followed by adding the MTT
solution to a final concentration of 0.5 mg/mL. After 4 h of incubation, the medium was
removed and the cells were mixed with 150 μL of DMSO. The absorbance was measured at a
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test wavelength of 570 nm by a microplate reader (Multiskan MK3, Thermo Scientific). The
cell viability was calculated.
Determination of the presence of TANs in brain tumour. The mice were intracranially
implanted with G422 cells (1 × 105 cells/mouse), and the brain was harvested at different
times (2, 8, 16 days) after implantation, followed by cryotomy. TANs in the frozen brain
section was stained with FITC-conjugated Ly-6G/Ly-6C (Gr-1) antibody (0.5 μg/mL)
(BioLegend, RB6-8C5), and observed using CLSM.
Quantification of PTX in cells and organs by HPLC. The HPLC system consisted of a
SHIMADZU LC-2010AHT system. The mobile phase contained methanol and water (75:25,
v:v). A C18 column (250 mm × 4.6 mm × 5 μm, Inertsil ODS-SP) was used to separate the
samples at a flow rate of 1 mL/min. The detection wavelength was set at 227 nm and the
column temperature was 35°C. The amount of PTX was determined using the HPLC system
above. Briefly, the cells were disrupted by the cell lysis buffer. The cell lysate was
centrifuged at 10000 g for 5 min. 50 μL of supernatant was mixed with 200 μL of methanol,
vortexed for 5 min, and centrifuged at 10000 g for 10 min. 20 μL of the supernatant was
injected into the HPLC system for the PTX quantification. The concentration of PTX in
different organs was also determined by HPLC described above. The weighed organs were
added with 800 μL of saline, followed by homogenization. 200 μL of the homogenate was
mixed with 200 μL of acetonitrile, vortexed for 5 min, and centrifuged at 10000 g for 10 min.
20 μL of the supernatant was injected into the HPLC system for the PTX quantification.
References
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amino acid based lipids for plasmid DNA delivery. Bioconjugate Chem. 19, 1055-1063
(2008).
2. Assanhou, A.G. et al. Reversal of multidrug resistance by co-delivery of paclitaxel and
lonidamine using a TPGS and hyaluronic acid dual-functionalized liposome for cancer
treatment. Biomaterials 73, 284-295 (2015).
3. Mo, R. et al. Multistage pH-responsive liposomes for mitochondrial-targeted anticancer
drug delivery. Adv. Mater. 24, 3659-3665 (2012).
4. Ju, C. et al. Sequential intra-intercellular nanoparticle delivery system for deep tumor
penetration. Angew. Chem. Int. Ed. 53, 6253-6258 (2014).
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Supplementary Figure 1. Synthesis of the cationic lipid, HG2C18.
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Supplementary Figure 2. a, Histogram of particle size distribution of PTX-L obtained by the
DLS measurement. PTX-L was prepared by SPC and Chol. The encapsulation efficiency and
drug-loading capacity of PTX in PTX-L was determined to be 97.5% and 2.8%, respectively.
b, Zeta potential of PTX-L and PTX-CL at different pH values. Data are shown as mean ± s.d.
(n = 3 independent experiments). c, In vitro release profiles of PTX from PTX-L and PTX-CL
at pH 7.4 and 4.5. Data are shown as mean ± s.d. (n = 2 independent experiments).
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Supplementary Figure 3. Cytotoxicity of Taxol and PTX-CL towards NEs for 12 h. Data are
shown as mean ± s.d. (n = 3 independent experiments). *P < 0.05, **P < 0.01 (two-tailed
Student’s t-test).
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Supplementary Figure 4. Cellular uptake of PTX-CL by NEs. a, Quantity of PTX in NEs
after NEs (1 × 105 cells/mL) were incubated with PTX-CL at different concentrations of PTX
for 2 h. Data are shown as mean ± s.d. (n = 2 independent experiments). b, Quantity of PTX
in NEs after NEs (1 × 105 cells/mL) were incubated with PTX-CL (50 μg/mL) for different
times. Data are shown as mean ± s.d. (n = 3 independent experiments).
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Supplementary Figure 5. Images of NEs transported in the lower chamber of the transwell
system in the presence of different concentrations of fMLP. Scale bar, 50 μm.
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Supplementary Figure 6. Determination of the quantity of PTX released from and retained
in PTX-CL/NEs in the presence of sulfasalazine (SSZ) (300 μM) over time. Data are shown
as mean ± s.d. (n = 3 independent experiments).
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Supplementary Figure 7. CLSM images of Cou6-CL/NEs before and after treatment with
fMLP or PMA. Scale bar, 20 μm. In sharp contrast with both untreated and fMLP-treated
Cou6-CL/NEs, PMA-treated Cou6-CL/NEs showed a nearly complete release of Cou6 and an
evident formation of NETs as the extracellular NE-derived DNA webs that were readily
stained with PI displaying red fluorescence.
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Supplementary Figure 8. TEM images of PTX-CL/NEs (a) and the blank NEs without PTX-
CL (b) after treatment with PMA for 4 h. Scale bars, 100 nm (left) and 1 μm (right). The
intact PTX-CL, which were secreted from NEs after the PMA treatment, were collected in the
medium.
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Supplementary Figure 9. Change in the TEER value of the bEnd.3 cell monolayer during
culture. Data are shown as mean ± s.d. (n = 3 independent experiments). The well-established
bEnd.3 monolayers with the TEER value higher than 300 Ω cm2 were used for the BBB
penetration study.
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Supplementary Figure 10. a, Migration of the blank NEs and PTX-CL/NEs (2 × 105 cells)
across the bEnd.3 cell monolayer after 3 h of incubation in the absence and presence of fMLP
(10 nM). Migration (%) is the ratio of the number of NEs in the lower chamber of the
transwell system to the number of NEs that were added. Data are shown as mean ± s.d. (n = 5
independent experiments). ***P < 0.001, untreated compared with fMLP-treated (two-tailed
Student’s t-test). b, Change in the TEER value of the bEnd.3 cell monolayer after incubation
with PTX-CL/NEs (2 × 105 cells equivalent to 18 μg/mL PTX) over time. Data are shown as
mean ± s.d. (n = 3 independent experiments). *P < 0.05, **P < 0.01, PTX-CL/NEs compared
with control (two-tailed Student’s t-test).
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Supplementary Figure 11. Expression of TNF-α (a) and CXCL-1/KC (b) in the culture
medium (extratumoural) and in the G422 tumour spheroid (intratumoural) during 14 days of
culture (extratumoural: in the culture medium; intratumoural: inside the tumour spheroid).
Data are shown as mean ± s.d. (n = 3 independent experiments).
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Supplementary Figure 12. CLSM image of Cou6-CL/DiR-NEs. Scale bar, 4 μm. The cell
membranes of Cou6-CL/NEs were stained with DiR to achieve the double-stained Cou6-
CL/DiR-NEs, in which the Cou6 signal was observed within NEs, and the DiR signal was
visualized on the plasma membrane of NEs. The nucleus of NEs was stained with Hoechst
33342.
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Supplementary Figure 13. Penetration of Cou6 into the 3D G422 tumour spheroids after
incubation with Cou6-CL/DiR-NEs over time. CLSM images were obtained using the Z-stack
scanning from the surface to the middle of the tumour spheroid at the fixed depth of 120 μm.
Scale bar, 100 μm.
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Supplementary Figure 14. a, CLSM images of G422 cells after incubation with Cou6-
CL/DiR-NEs or PMA-treated Cou6-CL/DiR-NEs over time. b, The enlarged overlaid CLSM
images. The nuclei of G422 cells were stained with Hoechst 33342. Scale bar, 50 μm.
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Supplementary Figure 15. Cytotoxicity of PMA-treated PTX-CL/NEs against G422 cells for
72 (a) and 96 h (b). In the PMA-treated PTX-CL/NE group, PTX-CL/NEs were pretreated
with PMA (100 nM) for 4 h. Afterward, the PMA-treated or untreated PTX-CL/NEs were
collected, followed by centrifugation. The supernatant was incubated with G422 cells for 72
or 96 h. Data are shown as mean ± s.d. (n = 3 independent experiments).
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Supplementary Figure 16. Histological observation of the brain collected from the mice 8,
12 and 16 days after implantation. The brain sections were stained with hematoxylin and
eosin (H&E). Scale bar, 100 μm. G422 tumours were formed 8 days after implantation, which
were round in shape with an average diameter of 0.5 mm and displayed no focal local
infiltration. After 12 days, the tumours became larger with an average diameter of 1.3 mm.
Initial focal peripheral infiltration was observed, in which short projections of tumour cells
invaded the normal brain parenchyma and a few islets of tumour cells were found inside. At
16 days post-implantation, the tumours with an average diameter of 2.1 mm presented a
prominently greater infiltration pattern, which showed apparently wider projections of
invading tumour cells together with much more islands of tumour cells both around the
primary tumour mass and inside the surrounding normal brain parenchyma.
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Supplementary Figure 17. CLSM images of the brain tumour section collected from the
mice different days after intracranial implantation. TANs were stained with FITC-conjugated
Ly-6G/Ly-6C (Gr-1) antibody, and the nuclei were stained with Hoechst 33342. Scale bar, 50
μm.
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Supplementary Figure 18. a, In vivo fluorescence imaging of the Luc-G422 tumour growth
in the brain of the mice over time (n = 4 mice per group). b, Change in the luminescence
intensity of Luc-G422 cells during the tumour growth and after surgical tumour removal.
After tumour removal at 16 days post-implantation of Luc-G422 cells, the mice were treated
with the blank NEs (5 × 106 cells/mouse), Taxol (10 mg/kg PTX), PTX-CL (10 mg/kg PTX)
and PTX-CL/NEs (5 × 106 cells/mouse equivalent to 5 mg/kg PTX), respectively. Data are
shown as mean ± s.d. (n = 4 independent experiments). c, Time of death during treatment with
different formulations. Arrows indicate the time of the surgery. All of 4 studied mice treated
with the blank NEs, Taxol and PTX-CL died within 43 days. The 50% survival rate of the
mice treated with the blank NEs, Taxol and PTX-CL was 29, 27 and 40 days, respectively. By
comparison, the 50% survival rate of the mice treated with PTX-CL/NEs was 65 days, and
one of them survived during 4 month of monitoring. Data are shown as mean ± s.d. (n = 4
mice per group). d, Histological observation of the brain collected from the surgically treated
Luc-G422-bearing mice after treatment with different formulations. The mice were sacrificed
at the onset of neurological deficits, and the brains were harvested for histological analysis
using the H&E staining (from left to right: NEs, Taxol, PTX-CL, PTX-CL/NEs (I)). The brain
section marked as PTX-CL/NEs (II) was harvested from the surviving mouse treated with
PTX-CL/NEs. Scale bar, 50 μm.
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Supplementary Fig. 19. a, Ex vivo fluorescence imaging of the brain harvested from the
GFP-G422-bearing mice before and after surgical tumour removal. The efficiency of the
surgery was determined to be about 96% by comparing the fluorescence intensity of GFP-
G422 cells before and after surgery obtained using the quantitative ROI analysis. b, CLSM
images of the frozen brain section of the GFP-G422-bearing mice after surgical tumour
removal. The nuclei were stained with Hoechst 33342. SC indicates the surgical cavity. Scale
bar, 100 μm. The remaining infiltrating GFP-G422 cells were observed in the normal brain
parenchyma distal to the surgical cavity.
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Supplementary Figure 20. Histological observation of the brain collected from the G422-
bearing mice 2, 6 and 12 days after surgical tumour removal. The brain sections were stained
with H&E. SC indicates the surgical cavity. Scale bar, 100 μm. At 2 days post-surgery,
several small islands of tumour cells obviously remained in the normal brain parenchyma
around the surgical cavity, although the primary tumour mass was completely removed. After
6 days, the islets became larger, and small tumours formed visibly. At 12 days after surgery,
an apparent recurrence was found in the surgical cavity in the mice.
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Supplementary Figure 21. CLSM images of TNF-α and CXCL1/KC present in the frozen
brain sections harvested from the G422-bearing mice at 6 h, 2 days and 10 days post surgical
tumour removal. TNF-α and CXCL1/KC were stained with Alexa Fluor 488-conjugated TNF-
α antibody and Alexa Fluor 647-conjugated CXCL1/KC antibody, respectively. SC indicates
the surgical cavity. Scale bar, 100 μm.
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Supplementary Figure 22. a-c, Expression of IL-10 (a), TNF-α (b) and CXCL1/KC (c) in
the brain of the C6-bearing mice before and after surgical tumour removal for 10 days. d-f,
Expression of IL-10 (d), TNF-α (e) and CXCL1/KC (f) in the serum of the C6-bearing mice
before and after surgical tumour removal for 3 days. Data are shown as mean ± s.d. (n = 3
independent experiments).
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Supplementary Figure 23. a-c, Expression of IL-10 (a), TNF-α (b) and CXCL1/KC (c) in
the brain of the U-87 MG-bearing mice before and after surgical tumour removal for 10 days.
d-f, Expression of IL-10 (d), TNF-α (e) and CXCL1/KC (f) in the serum of the U-87 MG-
bearing mice before and after surgical tumour removal for 3 days. Data are shown as mean ±
s.d. (n = 3 independent experiments). The U-87 MG model has been reported as a
controversial model that does not reflect the behaviour of human glioma completely.
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Supplementary Figure 24. Construction of mouse C6 glioma surgical resection model.
Histological observation of the brain collected from the mice 3, 5 and 7 days after
implantation (a), and from the glioma-bearing mice 3, 5 and 7 days after surgical tumour
removal (b). The brain sections were stained with H&E. SC indicates the surgical cavity.
Scale bar, 100 μm. The C6 tumours were formed at 3 days post-implantation in the brain, and
presented a prominently greater infiltration pattern at 7 days post-implantation, which showed
apparently wider projections of invading tumour cells together with much more islands of
tumour cells both around the primary tumour mass and inside the surrounding normal brain
parenchyma. The time of the surgery was therefore selected as 7 days post-implantation of C6
cells. After surgery for 3 days, the primary tumour mass was completely removed, and
whereas several small islands of tumour cells obviously remained in the normal brain
parenchyma around the surgical cavity. At 7 days after surgery, an apparent recurrence was
found in the surgical cavity in the mice.
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Supplementary Figure 25. In vivo fluorescence imaging of the normal mice (a), the G422-
bearing mice (b), the surgically treated G422-bearing mice (c) and the sham-operated mice (d)
after intravenous administration of PTX-CL/DiR-NEs at a dosage of 5 × 106 cells/mouse over
time (n = 4 mice per group).
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Supplementary Figure 26. In vivo fluorescence imaging of the normal mice (a), the C6-
bearing mice (b), the surgically treated C6-bearing mice (c) and the sham-operated mice (d)
after intravenous administration of PTX-CL/DiR-NEs at a dosage of 5 × 106 cells/mouse over
time (n = 4 mice per group).
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Supplementary Figure 27. ROI analysis of the DiR signal from the brains of the normal
mice (I), the glioma-bearing mice (II), the surgically treated glioma-bearing mice (III) and the
sham-operated mice (IV) after intravenous administration of PTX-CL/DiR-NEs at a dosage of
5 × 106 cells/mouse over time. Left: the G422 model. Right: the C6 model. Data are shown as
mean ± s.d. (n = 4 independent experiments). The area under the concentration versus time
curve in the brain (AUCbrain) for the DiR signal of PTX-CL/DiR-NEs in the surgically treated
glioma-bearing mice was significantly higher than that in three other models. *P < 0.05, **P <
0.01, ***P < 0.001 (two-tailed Student’s t-test).
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Supplementary Figure 28. a,b, Ex vivo fluorescence imaging of the brain harvested from the
normal mice (I), the glioma-bearing mice (II), the surgically treated glioma-bearing mice (III)
and the sham-operated mice (IV) at 48 h post-injection of PTX-CL/DiR-NEs (n = 4 mice per
group). a: G422 model; b: C6 model. c,d, ROI analysis of the DiR signal from the brain
harvested from the normal mice (I), the glioma-bearing mice (II), the surgically treated
glioma-bearing mice (III) and the sham-operated mice (IV) at 48 h post-injection of PTX-
CL/DiR-NEs. c: G422 model; d: C6 model. Data are shown as mean ± s.d. (n = 4 independent
experiments). *P < 0.05, **P < 0.01, ***P < 0.001 compared with III (two-tailed Student’s t-
test).
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Supplementary Figure 29. CLSM images of the frozen brain sections harvested from the
surgically treated GFP-G422-bearing mice after intravenous injection of PTX-CL/DiR-NEs
for different times. SC indicates the surgical cavity. Scale bar, 50 μm.
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Supplementary Figure 30. Biodistribution profiles of PTX accumulation in the lung (a) and
kidney (b) of the surgically treated G422-bearing mice after intravenous injection of different
PTX formulations at a PTX dosage of 5 mg/kg. PTX/organ is the ratio of the quantity of PTX
in the organ (μg) to the weight of the organ (g). Data are shown as mean ± s.d. (n = 3
independent experiments).
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Supplementary Figure 31. Brain targeting efficiency of different PTX formulations after
intravenous administration into the surgically treated G422-bearing mice. Brain targeting
efficiency was indicated by AUCbrain/AUCother organs, where AUCbrain and AUCother organs are
AUC of brain and other organs after administration of Taxol, PTX-CL or PTX-CL/NEs,
respectively. Data are shown as mean ± s.d. (n = 3 independent experiments).
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Supplementary Figure 32. a-e, Quantification of the DiR distribution in the liver (a), spleen
(b), lung (c), kidney (d) and brain (e) of the normal mice (I), the G422-bearing mice (II), the
surgically treated G422-bearing mice (III), the sham-operated mice (IV) after intravenous
administration of PTX-CL/DiR-NEs (5 × 106 cells/mouse). DiR/organ is the ratio of the
quantity of DiR in the organ (ng) to the weight of the organ (g). Data are shown as mean ± s.d.
(n = 3 independent experiments). f, Brain targeting efficiency of PTX-CL/DiR-NEs on
different mouse models, including the normal mice (I), the G422-bearing mice (II), the
surgically treated G422-bearing mice (III), the sham-operated mice (IV). Brain targeting
efficiency was indicated by AUCbrain/AUCother organs, where AUCbrain and AUCother organs are
AUC of brain and other organs after administration of PTX-CL/DiR-NEs, respectively. Data
are shown as mean ± s.d. (n = 3 independent experiments).
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Supplementary Figure 33. Change in the body weight of the surgically treated G422-bearing
mice after treatment with different formulations. Arrow indicates the time of the surgery. Data
are shown as mean ± s.d. (n = 12 mice per group).
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Supplementary Figure 34. Change in the activities of alanine transaminase (ALT) (a),
aspartate transaminase (AST) (b) and alkaline phosphatase (ALP) (c) in the serum of the mice
over time after treatment with different formulations. Data are shown as mean ± s.d. (n = 3
independent experiments). *P < 0.05, **P < 0.01, ***P < 0.001 (two-tailed Student’s t-test).
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Supplementary Figure 35. Histological observation of the brain collected from three
surgically treated G422-bearing mice that were survived during 4 months of monitoring after
treatment with PTX-CL/NEs. Scale bar, 100 μm. Several islands of G422 cells were observed
in the normal brain parenchyma, indicating that treatment with PTX-CL/NEs did not cure the
mice completely, but slow the recurrent growth efficiently.
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Supplementary Figure 36. Histological observation of different main organs collected from
the surgically treated G422-bearing mice after treatment with different formulations. Scale bar,
100 μm. No pathological variation after treatment with PTX-CL/NEs was observed in the
main organs.
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Supplementary Figure 37. a,b, Survival curve (a) and change in the body weight (b) of the
non-surgically treated G422-bearing mice after treatment with saline, the blank NEs (5 × 106
cells/mouse), CL/NEs without PTX (5 × 106 cells/mouse), Taxol (10 mg/kg PTX), PTX-CL
(10 mg/kg PTX) and PTX-CL/NEs (5 × 106 cells/mouse equivalent to 5 mg/kg PTX) (n = 10
mice per group). Treatment with different formulations started at 16 days post-implantation.
Error bars represent s.d. (n = 10). c,d, Survival curve (c) and change in the body weight (d) of
the non-surgically treated G422-bearing mice that were pre-injected with different dosages of
IL-8 in the brain tumour after treatment with or without PTX-CL/NEs (5 × 106 cells/mouse
equivalent to 5 mg/kg PTX) (n = 10 mice per group). Treatment with PTX-CL/NEs started at
16 days post-implantation. The low (L), medium (M) and high (H) dosages of IL-8 were set
as about 16, 32 and 48 pg/kg, respectively. In b and d, data are shown as mean ± s.d.
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Supplementary Figure 38. a,b, Survival curve (a) and change in the body weight (b) of the
surgically treated C6-bearing mice after treatment with saline, the blank NEs (5 × 106
cells/mouse), CL/NEs without PTX (5 × 106 cells/mouse), Taxol (10 mg/kg PTX), PTX-CL
(10 mg/kg PTX) and PTX-CL/NEs (5 × 106 cells/mouse equivalent to 5 mg/kg PTX) (n = 12
mice per group). Arrow indicates the time of the surgery. In b, data are shown as mean ± s.d. **P < 0.01, ***P < 0.001 (log-rank (Mantel-Cox) test). c, Histological observation of the brain
collected from the surgically treated C6-bearing mice after treatment with different
formulations. Scale bar, 100 μm.
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Supplementary Figure 39. a,b, Survival curve (a) and change in the body weight (b) of the
non-surgically treated C6-bearing mice after treatment with saline, the blank NEs (5 × 106
cells/mouse), CL/NEs without PTX (5 × 106 cells/mouse), Taxol (10 mg/kg PTX), PTX-CL
(10 mg/kg PTX) and PTX-CL/NEs (5 × 106 cells/mouse equivalent to 5 mg/kg PTX) (n = 10
mice per group). Treatment with different formulations started at 7 days post-implantation.
c,d, Survival curve (c) and change in the body weight (d) of the non-surgically treated C6-
bearing mice that were pre-injected with different dosages of IL-8 in the brain tumour after
treatment with or without PTX-CL/NEs (5 × 106 cells/mouse equivalent to 5 mg/kg PTX) (n =
8 mice per group). Treatment with PTX-CL/NEs started at 7 days post-implantation. The low
(L), medium (M) and high (H) dosages of IL-8 were set as about 16, 32 and 48 pg/kg,
respectively. In b and d, data are shown as mean ± s.d.
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Supplementary Table 1. AUC of PTX in different organs after intravenous administration of
different PTX formulations into the surgically treated G422-bearing mice at a PTX dosage of
5 mg/kg. Data are shown as mean ± s.d. (n = 4 independent experiments).
AUC (μg × h/g) Taxol PTX-CL PTX-CL/NEs Liver 34 ± 6 43 ± 10 152 ± 40 Spleen 15 ± 1 32 ± 3 285 ± 7 Lung 11 ± 2 16 ± 3 41 ± 13 Kidney 12 ± 3 6 ± 2 36 ± 7 Brain 0.3 ± 0.1 4 ± 0.4 349 ± 115
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