100 kd ultrafiltration centrifuge tube

HEF were cultured in exosome-free RPMI1640 medium. When the cells were at 90% confluence, conditioned medium (CM) was collected and centrifuged at room temperature, 3000 g for 15 min. The collected supernatant was centrifuged at room temperature, 2500 g for 15 min in a 100-kD ultrafiltration centrifuge tube (Millipore, USA). A total of 500 µL of supernatant derived from the above step was centrifuged at 4°C, 17,500 g for 30 min, and then collected and centrifuged for 70 min at 4°C, 110,000 g. Consequently, the supernatant was discarded and phosphate-buffered saline (PBS) was added to resuspend the precipitation and was centrifuged for 70 min at 4°C, 110,000 g again. Finally, 100 μL PBS was added to resuspend the precipitate followed by filtering with 0.22-μm filters (EMD Millipore). The concentration of exosomes was detected using a bicinchoninic acid (BCA) protein concentration determination kit (Solarbio). The samples were dropped onto the copper mesh and negatively stained with 2% phosphotungstic acid for 2 min. After natural air drying for 15 min, the samples were observed under transmission electron microscopy (TEM; Jeol, Japan). Exosome particle size was detected using a dynamic light scattering system (DLS) (Malvin, UK). Exosome-specific proteins were detected by western blot.

125 mg GdW₁₀ were dissolved in 7.5 mL water and stirred to transparency at 1000 rpm. Then, 5 mg DA were added. The mixture was stirred for 1 h at room temperature. The solution was transferred into a 15 mL Teflon-lined autoclave, which was maintained at 160 °C for 16 h. Black solution was obtained after cooling to room temperature. The obtained GdW₁₀@PDA solution was purified by washing 3 times and concentrated using a 50 mL 100 kD ultrafiltration centrifuge tube (Millipore, Billerica, MA, USA) at 4000 rpm for 15 min. The free Na₈H[α-PW₉O₃₄] in the product was separated, and the GdW₁₀@PDA composites in the upper layer were re-suspended in deionized water for further experiments. The characteristic peaks of GdW₁₀@PDA nanocomposites (KBr pellet, cm⁻¹) were 3459, 2373, 1619, 1542, 1486, 1429, 1386, 1354, 1255, 1214, 1132, 948, 896, 804, 644, and 531.