SUPPLEMENTARY DATA

ORIGINAL RESEARCH

Abhimanyu Thakur et al

Inhibition of glioma cells’ proliferation by doxorubicin-loaded exosomes via microfluidicsAbhimanyu Thakur1, Rakesh Kumar Sidu1, Heng Zou2, Md Kowsar Alam2, Mengsu Yang2, Youngjin Lee1*

1Department of Neuroscience, City University of Hong Kong, 83 Tat Chee Avenue, Kowloon, Hong Kong SAR

2Department of Biomedical Sciences, City University of Hong Kong, 83 Tat Chee Avenue, Kowloon, Hong Kong SAR

Correspondence:

Youngjin Lee

Department of Neuroscience

1A-205, 2/F, Block 1, To Yuen Building

City University of Hong Kong

Tel +852-3442-4313

Fax +852 3442-0549

Email younglee@cityu.edu.hk

Supplementary Table S1. Efficiency of drug loading into exosomes by various methods.

Method

Source of exosomes

Cargo loaded

Condition

Loading efficiency

Ref.

Incubation

Mouse macrophage cell line (Raw 264.7)

Catalase

Room temperature (RT), 18 hours

4.9 ± 0.5 %

[1]

Incubation with saponin

Raw 264.7

Catalase

RT, 20 min

18.5 ± 1.3 %

[1]

Freeze-thaw cycles

Raw 264.7

Catalase

Rapidly freezed at −80° C, and thawed at RT

14.7 ± 1.1 %

[1]

Sonication

Raw 264.7

Catalase

Sonicated (500 v, 2 kHz, 20% power, 6 cycles by 4 sec pulse/2 sec pause), cooled down on ice for 2 min

26.1 ± 1.2 %

[1]

Extrusion 

Raw 264.7

Catalase

Extruded (×10 times) through Avanti Lipids extruder with 200 nm-pores diameters

22.2 ± 3.1 %

[1]

Incubation 

RAW 264.7

PTX and DOX

37 °C for 1 hour with shaking

1.44± 0.38 %

[2]

Electroporation

RAW 264.7

PTX and DOX

Exosomes were mixed with PTX and added to a chilled 4 mm electroporation cuvette, electroporated using an Eppendorf Eporator (Eppendorf AG, Hamburg, Germany) at 1000 kV for 5 ms, and then incubated at 37 °C for 30 min.

5.3 ± 0.48 %

[2]

Hypotonic dialysis (HP)

MDA

Porphyrin

Performed by transferring EVs and drug into dialysis membranes (cellulose ester, molecular weight cut-off = 100–500 Da, Spectrum Labs) placed in 200 mL of 10 mM phosphate buffer (pH 7.4) and stirred at RT for 4 h.

-

[2]

Sonication

RAW 264.7

PTX and DOX

20% amplitude, 6 cycles of 30 s on/off for three minutes with a two-minute cooling period between each cycle, incubated at 37 °C for 60 min.

28.29 ± 1.38 %

[2]

Incubation

LNCaP and PC-3 PCa cell lines 

PTX

1 h at 22 °C

5.66 ± 10.03 %

[3]

Incubation

MDA-MB231 breast cancer (MDA) cells 

Porphyrin

RT for 10 min

-

[4]

Electroporation

MDA

Porphyrin

200 Ω, 500 μF, 200 mV and pulse time of 20–30 ms 

-

[4]

Extrusion

MDA

Porphyrin

Extrusion at 42 °C using a syringe-based hand-held mini-extruder equipped with a heating block using polycarbonate membranes of 400 nm pore size 

-

[4]

Supp Fig. S1. Design, Simulation, and Fabrication of the Exo-Load device. (A) Representative figure of the design of the Exo-Load device showing inlets (a,b,c) where inlet a is for loading doxorubicin, inlet b is for loading exosomes, and inlet c is for loading saponin. (B) Fluid-flow simulation of two-way inlet microfluidic geometry using COMSOL shows higher fluid velocity in the middle of channel compared to the walls; (C) Soft lithography process used for the fabrication of the Exo-Load device. (D) The Exo-Load device after sealing with microscopic glass slides following plasma treatment; inlets and outlets are secured with needle tips 21 gauge and PET tubing (BD INTRAMEDICTM Polyethylene Tubing, 0.023’’ X 0.038’’). (E) Bright field image of the microchannels.

Supp Table S2. Calculation of doxorubicin loading efficiency in SF7761 GMs- derived exosomes by various methods.

Methods of drug loading into exosomes

Concentration of exosomes

(particles/ml)

Initial conc. of drug taken (µM)

Absorbance of free dox in supernatant

Corresponding conc. of free dox in supernatant

(µM)

Conc. Of Dox loaded in exosome

(µM)

Loading efficiency

(%)

Incubation

4.0 × 106

22

0.310

20.795

1.205

4.82

Incubation with saponin

4.0 × 106

22

0.247

18.853

3.147

12.31

Electroporation

4.0 × 106

22

0.224

17.028

4.972

22.60

Sonication

4.0 × 106

22

0.216

16.439

5.561

25.28

Exo-load device

4.0 × 106

22

0.056

7.056 (outlet-A)

3.966 (Supernatant, outlet-B)

6.644(outlet-C)

18.034

19.7

Supp Table S3. Data used in HPLC for determining the concentration of free doxorubicin in the supernatant which was not loaded into exosomes.

S. No.

Time

Flow

%A

%B

1

0.00

1.0

20.0

80.0

2

12.00

1.0

60.0

40.0

3

17.00

1.0

20.0

80.0

4

20.00

1.0

20.0

80.0

A= 0.1% TFA in Acetonitrile (ACN); B= H2O

Pump mode: Gradient; Pressure: 2588 psi (High limit: 4000.00 psi, Low limit: 0.00 psi)

Flow rate: A=0.35 ml/min, B=0.65 ml/min

Supp Fig. S2. HPLC spectra of standard dilutions of doxorubicin HCl; (A) 15 μg/ml, (C) 25 μg/ml, and (E) 50 μg/ml. (B) Table showing peak area of corresponding standard dilutions of doxorubicin HCl. (D) Standard curve based on the HPLC spectra of standard doxorubicin solutions. (F) HPLC spectra of free doxorubicin present in the supernatant after its loading in SF7761 GMs- derived exosomes by the Exo-Load device. Initial concentration of doxorubicin was 22 μM. The calculated concentration of free doxorubicin in supernatant (through outlet-B) was found to be 3.769 μM (concentration of dox lost through outlet-A = 8.086 μM), and the loading efficiency of doxorubicin in exosomes by the Exo-Load device was estimated as 15.92%.

Supp. Fig. S3. Loading of Paclitaxel in SF7761 GMs-derived exosomes. (A) UV-visible spectra of standard paclitaxel (PTX) dissolved in DMSO (0-50 µM). (B) Calibration curve based on absorbance at five different standard PTX concentrations. (C) Absorbance of free PTX present in the supernatant after its loading into exosomes by the Exo-Load device, with loading efficiency of paclitaxel in SF7761 GMs-derived exosomes as 17.7%.

Supp. Fig. S4. (A-C) Doxorubicin loading in exosomes: Immunofluorescence microscopy for aggregated SF7761 GMs-derived exosomes (red) with doxorubicin (arbitrarily represented with green). Exosomes have been labelled with BODIPY® TR ceramide for membrane staining (Thermo Scientific) as per manufacturer’s protocol. (D) For flow cytometry, SF7761 GMs-derived EXO-DOXs were incubated with latex beads. Shift in fluorescence intensity indicates the entrapment of doxorubicin in SF7761 GMs-derived exosomes.

Supp. Fig. S5. Drug release profile and stability of EXO-DOX. (A, B) Representative release of (A) DOX and (B) PTX from SF7761 GMs-EXO at pH 5 and 7.4. (C-F) Representative size and morphology of SF7761 GMs-derived exosomes (C, D) before and (E, F) after loading with DOX.

Supp. Video S1-S4. A representative video showing the laminar flow and mixing phenomena along the pillars (microchannels) in the Exo-Load device.

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