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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 [email protected]
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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