Nanoparticle size analyzer

BMSCs were purchased from Procell Life Science & Technology Co., Ltd. (CP-M131, Wuhan, China). Cells were cultured in α-MEM complete medium containing 10% fetal bovine serum and 1% penicillin-streptomycin dual antibody. When the density of BMSCs reaches 80-90%, the medium was discarded, and the cells were washed with PBS three times. Then, exosome-free serum medium was added. After cultured for 48 h, the cell supernatant was collected, and transferred to a 50 mL centrifuge tube for gradient centrifugation at 4 °C (500×g, 5 min; 2000×g, 30 min). Subsequently, the supernatant was centrifuged at 120,000×g for 70 min at 4 °C, the supernatant was discarded, and the centrifugation operation was repeated once again. Exosomes were suspended in PBS and frozen at -80 ℃ for later use.
The concentrations of the isolated exosomes were determined using a BCA protein assay kit (Thermo Fisher Scientific, MA, USA) following the manufacturer’s instructions. After that, a NanoSight nanoparticle size analyzer (NS300, Malvern) was used to determine the exosome particle size. Exosome morphology was observed using transmission electron microscopy (JEM-1230, Joel) [19 (link)]. Exosome-specific proteins CD9, CD63 and Alix were detected using western blot with their corresponding antibodies (1:500) [20 (link)].

0.1 mg/mL AC sample suspensions were prepared, and the zeta potential was analyzed using a Malvern Nanoparticle Size Analyzer (ZEN3690, Malvern, UK).

Lip(PFH) was prepared by ultrasonication according to our previously reported study [15 (link)]. Briefly, liposome colloidal suspensions were prepared by dissolving 3.79 mg DSPE-PEG2000 (4.28 mg cholesterol and 24.65 mg lecithin in dichloromethane. The organic solvent was removed through rotary evaporation to form a thin lipids film on the glass vial. Thereafter, the lipid film was hydrated with 1.4 ml pure water by ultrasonication (XO-650D, China) for 10 min in ice bath. After that, 0.6 ml PFH were gradually appended under ultrasonication at 325 w/min for 3 min in ice bath to form 2 ml lip(PFH) (30 v/v % PFH). For in vivo tracking, IR780, a near infrared (NIR) dye, was added in dichloromethane to form lip(PFH + IR780) [15 (link)].
The morphology of lip(PFH) was characterized by transmission electron microscopy (TEM, H-7650, Hitachi, Japan). Particle size distribution of liposomes was detected by Nanoparticle Size Analyzer (ZEN3600, Malvern). B ultrasound was used to further confirm that PFH was encapsulated into liposome.