Minispin plus

Binding to and internalization of [⁶⁸Ga]Ga-PSMA-11 into LNCaP cells were assessed in vitro as previously described [40 (link)]. Briefly, an aliquot containing 2 × 10⁶ LNCaP cells in RPMI-1640 medium (450 µL) was added to a vial and incubated with [⁶⁸Ga]Ga-PSMA-11 (50 µL/~1 MBq) at 37 °C and under slight agitation for 30 and 60 min (n = 5 for each time interval). Then, the vials were centrifuged (825 g, 5 min) with Minispin^® plus (EppendorfAG—Hamburg, Germany). The cell pellets and supernatants were separated, and their radioactivities were measured in a Wizard²™ 3” 2480 automatic gamma counter (PerkinElmer—Norwalk, CT, USA). Binding to LNCaP cells was calculated as follows (Equation (4)): $\mathrm{Binding}=\frac{\mathrm{cpm} \left(\mathrm{pellet}\right)}{\mathrm{cpm} (\mathrm{pellet}+\mathrm{supernatant})}\times 100\%$
After that, pellets were resuspended in 0.5 mL of acid wash buffer (0.2 M acetic acid in 0.5 M NaCl solution, pH 2.8) and kept at room temperature for 5 min to remove cell-surface-bound [⁶⁸Ga]Ga-PSMA-11. Then, the vials were centrifuged (825 g, 5 min) with Minispin^® plus (EppendorfAG—Hamburg, Germany). The cell pellets and supernatants were separated and their radioactivities were measured in a Wizard²™ 3” 2480 automatic gamma counter (PerkinElmer—Norwalk, CT, USA). Internalization into LNCaP cells was calculated as follows (Equation (5)): $\mathrm{Internalization}=\frac{\mathrm{cpm} \left(\mathrm{pellet}\right)}{\mathrm{cpm} (\mathrm{pellet}+\mathrm{supernatant})}\times 100\%$

Commercial reagents for sEV isolation by PEG precipitation—ExoQuick (System Biosciences, CA) were used according to the manufacturer guidelines. Briefly, 200 µL of MCF7 or MDB-231 sEVs-containing media was combined with 1 mL ExoQuick-TC Solution in sterile Eppendorf tubes, mixed by inverting the tube several times, and incubated at 4 °C for 18 h. The solution was centrifuged at 1,500×g for 30 min in an Eppendorf MiniSpin Plus (Eppendorf, Germany). The supernatant was discarded, and the pellet was suspended in 1 mL PBS and stored at 4 °C.

The CDs were synthesized from citric acid using a solvothermal method. In total, 4.3 g of citric acid (Chip and Dip, Moscow, Russia) and 8.6 g of urea (Vostokreaktiv, Khabarovsk, Russia) were dissolved in 65 mL of formamide (Reakhim, Moscow, Russia) using ultrasonic disperser Elmasonic S40H Elma (Elma Schmidbauer GmbH, Singen, Germany). The clear solution was then transferred to a 90 mL stainless steel polytetrafluoroethylene autoclave (TOPTION Laboratory Store, Xi’an, China) and heated for 12 h at 180 °C. Upon completion of the synthesis, the autoclave was cooled overnight to room temperature. The solution was transferred to a dialysis bag with pores (MWCO) of 12–14 kDa (Spectrum Labs, Moscow, Russia) and placed in a beaker containing deionized water and a magnetic stirrer HS Pro-DT (Stegler, Moscow, Russia) for 24 h. The solution was then filtered using a track membrane with a pore size of 100 nm (JINR, Dubna, Russia). The resulting product was centrifuged using an Eppendorf MiniSpin plus (Eppendorf AG, Hamburg, Germany) at 14,500 rpm for 5 min and decanted over the sediment.