Neutrophil-mediated anticancer drug delivery for …...I o ed. S1 Neutrophil-mediated anticancer...

47
In the format provided by the authors and unedited. 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* © 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved. SUPPLEMENTARY INFORMATION DOI: 10.1038/NNANO.2017.54 NATURE NANOTECHNOLOGY | www.nature.com/naturenanotechnology 1

Transcript of Neutrophil-mediated anticancer drug delivery for …...I o ed. S1 Neutrophil-mediated anticancer...

Page 1: Neutrophil-mediated anticancer drug delivery for …...I o ed. S1 Neutrophil-mediated anticancer drug delivery for suppression of postoperative malignant glioma recurrence Jingwei

In the format provided by the authors and unedited.

S1

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*

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

SUPPLEMENTARY INFORMATIONDOI: 10.1038/NNANO.2017.54

NATURE NANOTECHNOLOGY | www.nature.com/naturenanotechnology 1

Page 2: Neutrophil-mediated anticancer drug delivery for …...I o ed. S1 Neutrophil-mediated anticancer drug delivery for suppression of postoperative malignant glioma recurrence Jingwei

S2

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%.

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

NATURE NANOTECHNOLOGY | www.nature.com/naturenanotechnology 2

SUPPLEMENTARY INFORMATIONDOI: 10.1038/NNANO.2017.54

Page 3: Neutrophil-mediated anticancer drug delivery for …...I o ed. S1 Neutrophil-mediated anticancer drug delivery for suppression of postoperative malignant glioma recurrence Jingwei

S3

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.

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

NATURE NANOTECHNOLOGY | www.nature.com/naturenanotechnology 3

SUPPLEMENTARY INFORMATIONDOI: 10.1038/NNANO.2017.54

Page 4: Neutrophil-mediated anticancer drug delivery for …...I o ed. S1 Neutrophil-mediated anticancer drug delivery for suppression of postoperative malignant glioma recurrence Jingwei

S4

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

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

NATURE NANOTECHNOLOGY | www.nature.com/naturenanotechnology 4

SUPPLEMENTARY INFORMATIONDOI: 10.1038/NNANO.2017.54

Page 5: Neutrophil-mediated anticancer drug delivery for …...I o ed. S1 Neutrophil-mediated anticancer drug delivery for suppression of postoperative malignant glioma recurrence Jingwei

S5

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

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

NATURE NANOTECHNOLOGY | www.nature.com/naturenanotechnology 5

SUPPLEMENTARY INFORMATIONDOI: 10.1038/NNANO.2017.54

Page 6: Neutrophil-mediated anticancer drug delivery for …...I o ed. S1 Neutrophil-mediated anticancer drug delivery for suppression of postoperative malignant glioma recurrence Jingwei

S6

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

1. Obata, Y., Suzuki, D. & Takeoka, S. Evaluation of cationic assemblies constructed with

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).

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

NATURE NANOTECHNOLOGY | www.nature.com/naturenanotechnology 6

SUPPLEMENTARY INFORMATIONDOI: 10.1038/NNANO.2017.54

Page 7: Neutrophil-mediated anticancer drug delivery for …...I o ed. S1 Neutrophil-mediated anticancer drug delivery for suppression of postoperative malignant glioma recurrence Jingwei

S7

Supplementary Figure 1. Synthesis of the cationic lipid, HG2C18.

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

NATURE NANOTECHNOLOGY | www.nature.com/naturenanotechnology 7

SUPPLEMENTARY INFORMATIONDOI: 10.1038/NNANO.2017.54

Page 8: Neutrophil-mediated anticancer drug delivery for …...I o ed. S1 Neutrophil-mediated anticancer drug delivery for suppression of postoperative malignant glioma recurrence Jingwei

S8

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).

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

NATURE NANOTECHNOLOGY | www.nature.com/naturenanotechnology 8

SUPPLEMENTARY INFORMATIONDOI: 10.1038/NNANO.2017.54

Page 9: Neutrophil-mediated anticancer drug delivery for …...I o ed. S1 Neutrophil-mediated anticancer drug delivery for suppression of postoperative malignant glioma recurrence Jingwei

S9

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).

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

NATURE NANOTECHNOLOGY | www.nature.com/naturenanotechnology 9

SUPPLEMENTARY INFORMATIONDOI: 10.1038/NNANO.2017.54

Page 10: Neutrophil-mediated anticancer drug delivery for …...I o ed. S1 Neutrophil-mediated anticancer drug delivery for suppression of postoperative malignant glioma recurrence Jingwei

S10

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).

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

NATURE NANOTECHNOLOGY | www.nature.com/naturenanotechnology 10

SUPPLEMENTARY INFORMATIONDOI: 10.1038/NNANO.2017.54

Page 11: Neutrophil-mediated anticancer drug delivery for …...I o ed. S1 Neutrophil-mediated anticancer drug delivery for suppression of postoperative malignant glioma recurrence Jingwei

S11

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.

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

NATURE NANOTECHNOLOGY | www.nature.com/naturenanotechnology 11

SUPPLEMENTARY INFORMATIONDOI: 10.1038/NNANO.2017.54

Page 12: Neutrophil-mediated anticancer drug delivery for …...I o ed. S1 Neutrophil-mediated anticancer drug delivery for suppression of postoperative malignant glioma recurrence Jingwei

S12

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).

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

NATURE NANOTECHNOLOGY | www.nature.com/naturenanotechnology 12

SUPPLEMENTARY INFORMATIONDOI: 10.1038/NNANO.2017.54

Page 13: Neutrophil-mediated anticancer drug delivery for …...I o ed. S1 Neutrophil-mediated anticancer drug delivery for suppression of postoperative malignant glioma recurrence Jingwei

S13

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.

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

NATURE NANOTECHNOLOGY | www.nature.com/naturenanotechnology 13

SUPPLEMENTARY INFORMATIONDOI: 10.1038/NNANO.2017.54

Page 14: Neutrophil-mediated anticancer drug delivery for …...I o ed. S1 Neutrophil-mediated anticancer drug delivery for suppression of postoperative malignant glioma recurrence Jingwei

S14

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.

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

NATURE NANOTECHNOLOGY | www.nature.com/naturenanotechnology 14

SUPPLEMENTARY INFORMATIONDOI: 10.1038/NNANO.2017.54

Page 15: Neutrophil-mediated anticancer drug delivery for …...I o ed. S1 Neutrophil-mediated anticancer drug delivery for suppression of postoperative malignant glioma recurrence Jingwei

S15

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.

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

NATURE NANOTECHNOLOGY | www.nature.com/naturenanotechnology 15

SUPPLEMENTARY INFORMATIONDOI: 10.1038/NNANO.2017.54

Page 16: Neutrophil-mediated anticancer drug delivery for …...I o ed. S1 Neutrophil-mediated anticancer drug delivery for suppression of postoperative malignant glioma recurrence Jingwei

S16

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).

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

NATURE NANOTECHNOLOGY | www.nature.com/naturenanotechnology 16

SUPPLEMENTARY INFORMATIONDOI: 10.1038/NNANO.2017.54

Page 17: Neutrophil-mediated anticancer drug delivery for …...I o ed. S1 Neutrophil-mediated anticancer drug delivery for suppression of postoperative malignant glioma recurrence Jingwei

S17

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).

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

NATURE NANOTECHNOLOGY | www.nature.com/naturenanotechnology 17

SUPPLEMENTARY INFORMATIONDOI: 10.1038/NNANO.2017.54

Page 18: Neutrophil-mediated anticancer drug delivery for …...I o ed. S1 Neutrophil-mediated anticancer drug delivery for suppression of postoperative malignant glioma recurrence Jingwei

S18

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.

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

NATURE NANOTECHNOLOGY | www.nature.com/naturenanotechnology 18

SUPPLEMENTARY INFORMATIONDOI: 10.1038/NNANO.2017.54

Page 19: Neutrophil-mediated anticancer drug delivery for …...I o ed. S1 Neutrophil-mediated anticancer drug delivery for suppression of postoperative malignant glioma recurrence Jingwei

S19

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.

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

NATURE NANOTECHNOLOGY | www.nature.com/naturenanotechnology 19

SUPPLEMENTARY INFORMATIONDOI: 10.1038/NNANO.2017.54

Page 20: Neutrophil-mediated anticancer drug delivery for …...I o ed. S1 Neutrophil-mediated anticancer drug delivery for suppression of postoperative malignant glioma recurrence Jingwei

S20

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.

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

NATURE NANOTECHNOLOGY | www.nature.com/naturenanotechnology 20

SUPPLEMENTARY INFORMATIONDOI: 10.1038/NNANO.2017.54

Page 21: Neutrophil-mediated anticancer drug delivery for …...I o ed. S1 Neutrophil-mediated anticancer drug delivery for suppression of postoperative malignant glioma recurrence Jingwei

S21

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).

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

NATURE NANOTECHNOLOGY | www.nature.com/naturenanotechnology 21

SUPPLEMENTARY INFORMATIONDOI: 10.1038/NNANO.2017.54

Page 22: Neutrophil-mediated anticancer drug delivery for …...I o ed. S1 Neutrophil-mediated anticancer drug delivery for suppression of postoperative malignant glioma recurrence Jingwei

S22

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.

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

NATURE NANOTECHNOLOGY | www.nature.com/naturenanotechnology 22

SUPPLEMENTARY INFORMATIONDOI: 10.1038/NNANO.2017.54

Page 23: Neutrophil-mediated anticancer drug delivery for …...I o ed. S1 Neutrophil-mediated anticancer drug delivery for suppression of postoperative malignant glioma recurrence Jingwei

S23

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.

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

NATURE NANOTECHNOLOGY | www.nature.com/naturenanotechnology 23

SUPPLEMENTARY INFORMATIONDOI: 10.1038/NNANO.2017.54

Page 24: Neutrophil-mediated anticancer drug delivery for …...I o ed. S1 Neutrophil-mediated anticancer drug delivery for suppression of postoperative malignant glioma recurrence Jingwei

S24

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

NATURE NANOTECHNOLOGY | www.nature.com/naturenanotechnology 24

SUPPLEMENTARY INFORMATIONDOI: 10.1038/NNANO.2017.54

Page 25: Neutrophil-mediated anticancer drug delivery for …...I o ed. S1 Neutrophil-mediated anticancer drug delivery for suppression of postoperative malignant glioma recurrence Jingwei

S25

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.

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

NATURE NANOTECHNOLOGY | www.nature.com/naturenanotechnology 25

SUPPLEMENTARY INFORMATIONDOI: 10.1038/NNANO.2017.54

Page 26: Neutrophil-mediated anticancer drug delivery for …...I o ed. S1 Neutrophil-mediated anticancer drug delivery for suppression of postoperative malignant glioma recurrence Jingwei

S26

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.

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

NATURE NANOTECHNOLOGY | www.nature.com/naturenanotechnology 26

SUPPLEMENTARY INFORMATIONDOI: 10.1038/NNANO.2017.54

Page 27: Neutrophil-mediated anticancer drug delivery for …...I o ed. S1 Neutrophil-mediated anticancer drug delivery for suppression of postoperative malignant glioma recurrence Jingwei

S27

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.

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

NATURE NANOTECHNOLOGY | www.nature.com/naturenanotechnology 27

SUPPLEMENTARY INFORMATIONDOI: 10.1038/NNANO.2017.54

Page 28: Neutrophil-mediated anticancer drug delivery for …...I o ed. S1 Neutrophil-mediated anticancer drug delivery for suppression of postoperative malignant glioma recurrence Jingwei

S28

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.

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

NATURE NANOTECHNOLOGY | www.nature.com/naturenanotechnology 28

SUPPLEMENTARY INFORMATIONDOI: 10.1038/NNANO.2017.54

Page 29: Neutrophil-mediated anticancer drug delivery for …...I o ed. S1 Neutrophil-mediated anticancer drug delivery for suppression of postoperative malignant glioma recurrence Jingwei

S29

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).

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

NATURE NANOTECHNOLOGY | www.nature.com/naturenanotechnology 29

SUPPLEMENTARY INFORMATIONDOI: 10.1038/NNANO.2017.54

Page 30: Neutrophil-mediated anticancer drug delivery for …...I o ed. S1 Neutrophil-mediated anticancer drug delivery for suppression of postoperative malignant glioma recurrence Jingwei

S30

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.

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

NATURE NANOTECHNOLOGY | www.nature.com/naturenanotechnology 30

SUPPLEMENTARY INFORMATIONDOI: 10.1038/NNANO.2017.54

Page 31: Neutrophil-mediated anticancer drug delivery for …...I o ed. S1 Neutrophil-mediated anticancer drug delivery for suppression of postoperative malignant glioma recurrence Jingwei

S31

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.

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

NATURE NANOTECHNOLOGY | www.nature.com/naturenanotechnology 31

SUPPLEMENTARY INFORMATIONDOI: 10.1038/NNANO.2017.54

Page 32: Neutrophil-mediated anticancer drug delivery for …...I o ed. S1 Neutrophil-mediated anticancer drug delivery for suppression of postoperative malignant glioma recurrence Jingwei

S32

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).

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

NATURE NANOTECHNOLOGY | www.nature.com/naturenanotechnology 32

SUPPLEMENTARY INFORMATIONDOI: 10.1038/NNANO.2017.54

Page 33: Neutrophil-mediated anticancer drug delivery for …...I o ed. S1 Neutrophil-mediated anticancer drug delivery for suppression of postoperative malignant glioma recurrence Jingwei

S33

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).

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

NATURE NANOTECHNOLOGY | www.nature.com/naturenanotechnology 33

SUPPLEMENTARY INFORMATIONDOI: 10.1038/NNANO.2017.54

Page 34: Neutrophil-mediated anticancer drug delivery for …...I o ed. S1 Neutrophil-mediated anticancer drug delivery for suppression of postoperative malignant glioma recurrence Jingwei

S34

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).

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

NATURE NANOTECHNOLOGY | www.nature.com/naturenanotechnology 34

SUPPLEMENTARY INFORMATIONDOI: 10.1038/NNANO.2017.54

Page 35: Neutrophil-mediated anticancer drug delivery for …...I o ed. S1 Neutrophil-mediated anticancer drug delivery for suppression of postoperative malignant glioma recurrence Jingwei

S35

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).

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

NATURE NANOTECHNOLOGY | www.nature.com/naturenanotechnology 35

SUPPLEMENTARY INFORMATIONDOI: 10.1038/NNANO.2017.54

Page 36: Neutrophil-mediated anticancer drug delivery for …...I o ed. S1 Neutrophil-mediated anticancer drug delivery for suppression of postoperative malignant glioma recurrence Jingwei

S36

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.

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

NATURE NANOTECHNOLOGY | www.nature.com/naturenanotechnology 36

SUPPLEMENTARY INFORMATIONDOI: 10.1038/NNANO.2017.54

Page 37: Neutrophil-mediated anticancer drug delivery for …...I o ed. S1 Neutrophil-mediated anticancer drug delivery for suppression of postoperative malignant glioma recurrence Jingwei

S37

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).

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

NATURE NANOTECHNOLOGY | www.nature.com/naturenanotechnology 37

SUPPLEMENTARY INFORMATIONDOI: 10.1038/NNANO.2017.54

Page 38: Neutrophil-mediated anticancer drug delivery for …...I o ed. S1 Neutrophil-mediated anticancer drug delivery for suppression of postoperative malignant glioma recurrence Jingwei

S38

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).

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

NATURE NANOTECHNOLOGY | www.nature.com/naturenanotechnology 38

SUPPLEMENTARY INFORMATIONDOI: 10.1038/NNANO.2017.54

Page 39: Neutrophil-mediated anticancer drug delivery for …...I o ed. S1 Neutrophil-mediated anticancer drug delivery for suppression of postoperative malignant glioma recurrence Jingwei

S39

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).

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

NATURE NANOTECHNOLOGY | www.nature.com/naturenanotechnology 39

SUPPLEMENTARY INFORMATIONDOI: 10.1038/NNANO.2017.54

Page 40: Neutrophil-mediated anticancer drug delivery for …...I o ed. S1 Neutrophil-mediated anticancer drug delivery for suppression of postoperative malignant glioma recurrence Jingwei

S40

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).

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

NATURE NANOTECHNOLOGY | www.nature.com/naturenanotechnology 40

SUPPLEMENTARY INFORMATIONDOI: 10.1038/NNANO.2017.54

Page 41: Neutrophil-mediated anticancer drug delivery for …...I o ed. S1 Neutrophil-mediated anticancer drug delivery for suppression of postoperative malignant glioma recurrence Jingwei

S41

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).

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

NATURE NANOTECHNOLOGY | www.nature.com/naturenanotechnology 41

SUPPLEMENTARY INFORMATIONDOI: 10.1038/NNANO.2017.54

Page 42: Neutrophil-mediated anticancer drug delivery for …...I o ed. S1 Neutrophil-mediated anticancer drug delivery for suppression of postoperative malignant glioma recurrence Jingwei

S42

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.

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

NATURE NANOTECHNOLOGY | www.nature.com/naturenanotechnology 42

SUPPLEMENTARY INFORMATIONDOI: 10.1038/NNANO.2017.54

Page 43: Neutrophil-mediated anticancer drug delivery for …...I o ed. S1 Neutrophil-mediated anticancer drug delivery for suppression of postoperative malignant glioma recurrence Jingwei

S43

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.

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

NATURE NANOTECHNOLOGY | www.nature.com/naturenanotechnology 43

SUPPLEMENTARY INFORMATIONDOI: 10.1038/NNANO.2017.54

Page 44: Neutrophil-mediated anticancer drug delivery for …...I o ed. S1 Neutrophil-mediated anticancer drug delivery for suppression of postoperative malignant glioma recurrence Jingwei

S44

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.

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

NATURE NANOTECHNOLOGY | www.nature.com/naturenanotechnology 44

SUPPLEMENTARY INFORMATIONDOI: 10.1038/NNANO.2017.54

Page 45: Neutrophil-mediated anticancer drug delivery for …...I o ed. S1 Neutrophil-mediated anticancer drug delivery for suppression of postoperative malignant glioma recurrence Jingwei

S45

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.

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

NATURE NANOTECHNOLOGY | www.nature.com/naturenanotechnology 45

SUPPLEMENTARY INFORMATIONDOI: 10.1038/NNANO.2017.54

Page 46: Neutrophil-mediated anticancer drug delivery for …...I o ed. S1 Neutrophil-mediated anticancer drug delivery for suppression of postoperative malignant glioma recurrence Jingwei

S46

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.

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

NATURE NANOTECHNOLOGY | www.nature.com/naturenanotechnology 46

SUPPLEMENTARY INFORMATIONDOI: 10.1038/NNANO.2017.54

Page 47: Neutrophil-mediated anticancer drug delivery for …...I o ed. S1 Neutrophil-mediated anticancer drug delivery for suppression of postoperative malignant glioma recurrence Jingwei

S47

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

© 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

NATURE NANOTECHNOLOGY | www.nature.com/naturenanotechnology 47

SUPPLEMENTARY INFORMATIONDOI: 10.1038/NNANO.2017.54