Elx800

Doxorubicin (Dox) hydrochloride was appropriately diluted by sterile water for injection (SWFI). The 70 μL of Dox HCl (1 mg mL⁻¹) was mixed with 930 μL exosome solution (1 mg mL⁻¹) for 30 min and desalinized with triethylamine for 1 hr at room temperature (RT). The mix solution was then added to dialysis tube prepared by dialysis belt and centrifuge tube and dialyzed against PBS overnight at 4°C to prepare the Exo-Dox complex. The amount of DOX loaded into exosomes was calculated from a standard curve obtained from BioTek ELX800 (Gene Company Limited, USA) by detecting the absorbance value at the wavelength of 490 nm.
In order to measure the in-vitro drug release profile, 3 mL of Exo-Dox solution was transferred into dialysis tubes immersed in PBS at pH 7.4 or pH 4.5. The release of Dox was performed at 37°C and different time intervals. At 1, 2, 4, 8, 12, 24 and 36 h, the concentrations of Dox were determined based on calibration curve by Bioequivalence ELX800 automated enzyme immunoassay analyzer.

Cell viability assays were performed with a Cell Counting Kit-8 (CCK-8, Beyotime, China), which used WST-8, a water-soluble tetrazolium dye, to enhance sensitivity of the WST-8-based assay over conventional 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS)-based assay. Briefly, serial dilutions of C10 in M199 supplemented with 2% FBS, at concentrations of 0.4 mg/L to 25.0 mg/L (in triplicate), were added to EPC cells in 96-well plates. After every 24 h, cell viability assays using CCK-8 were performed as per the manufacturer’s protocols. After 2 h of incubation at 37 °C, colorimetric absorbance from the mitochondrial enzyme substrate reaction was measured at 450 nm using a microplate reader (ELX800, Gene, Hong Kong, China). As DMSO exhibits no cytotoxicity at doses up to 0.1% (v/v) (Liu et al., 2020 (link)), signals were compared to solvent-treated cells (DMSO up to 0.05%, v/v).
Antiviral activity assays were performed as per previous research (Liu et al., 2017 (link)). Briefly, EPC cells seeded on 12-well and 96-well plates were infected with SVCV at 10³×TCID₅₀/mL at 25 °C, followed by the addition of serial dilutions of C10. After 48 h of infection, the antiviral activity of C10 against SVCV replication in EPC cells was tested by quantitative real-time polymerase chain reaction (qRT-PCR) and CCK-8.

Cytotoxicity analyses were performed using Cell Counting Kit-8 (CCK-8) (Beyotime, China) based on a water-soluble tetrazolium dye (WST-8), which is more sensitive than the conventional 3-(4,5-dimethylthiazol-2-yl)-5-(3- carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS)-based assay. Briefly, serial dilutions of TFS at concentrations of 500/2ⁿ μg/L (n=0–6) in triplicate were added to cells cultured on a 96-well plate in M199 supplemented with 5% FBS. At each 24 h period, cell viability following TFS exposure was tested as per the manufacturer’s protocols for CCK-8. Colorimetric absorbance after the mitochondrial enzyme substrate reaction was measured at 450 nm using a microplate reader (ELX800, Gene, Hong Kong, China). As DMSO exhibits no cytotoxicity at doses of up to 0.1% (v/v) (Liu et al., 2020a (link)), TFS-exposed signals were compared to solvent-control signals (DMSO up to 0.001%, v/v).