Radioiodine labeling and in vivo trafficking of extracellular vesicles

Abstract

Biodistribution and role of extracellular vesicles (EVs) are still largely unknown. Reliable tracking methods for EVs are needed. In this study, nuclear imaging using radioiodine were developed and applied for tracking EVs derived from cell lines. EVs were obtained from supernatant of thyroid cancer cell (Cal62) and natural killer cells (NK92-MI) using sequential ultracentrifuges. Sulfosuccinimidyl-3-(4-hydroxypheynyl) propionate were labeled to membrane of Cal62 and NK92-MI cell derived EVs, then the EVs were labeled with radioiodine (I-131 and I-125) using pre-coated iodination tubes (RI-EVs). In vivo gamma camera images were obtained after intravenous injection of the RI-EVs, and ex vivo biodistribution study was also performed. EVs were labeled with radioiodine and radiochemical purity of the RI-EV was more than 98%. Results of nanoparticle tracking analysis and electron microscopy showed that there was no significant difference in EVs before and after the radioiodine labeling. After intravenous injection of RI-EVs to mice, gamma camera imaging well visualized the real-time biodistribution of the RI-EVs. RI-EVs were mainly visualized at liver, spleen, and lung. Nuclear imaging system of EVs derived from thyroid cancer and NK cells using radioiodine labeling of the EVs was established. Thus, this system might be helpful for in vivo tracking of EVs.

Figure 1

Schematic diagram of radioiodine labeling of extracellular vesicles. Created with BioRender.com.

Figure 2

Radiochemical purity and stability of radioiodine labeled EVs. (A) Instant thin layer chromatography (ITLC) showed good radiochemical purity after radioiodine labeling. Radiochemical purity of I-131-Cal62-EVs and I-131-NK-EVs after purification were 98.9 ± 1.8% (n = 3) and 98.6 ± 0.9% (n = 3). (B) Stability of I-131-Cal62-EVs was examined in 20% fetal bovine serum (n = 3). The level of stability was analyzed by ITLC. Data are presented as mean ± standard deviation.

Figure 3

Characterization of I-131-Cal62-EVs. (A) Size distribution of naïve-Cal62-EVs and I-131-Cal62-EVs were examined by nanoparticle tracking analysis. (B) There was no significant difference of the average sizes between naïve-Cal62-EVs and I-131-Cal62-EVs. (C) Scanning electron microscopy images revealed that there were no significant differences of morphology and size between naïve-Cal62-EVs and I-131-Cal62-EVs. Data are presented as mean ± standard deviation.

Figure 4

Characterization of I-131-NK-EVs. (A) Size distributions of naïve-NK-EVs and I-131-NK-EVs were examined by nanoparticle tracking analysis. (B) There was no significant difference of average sizes between naïve-NK-EVs and I-131-NK-EVs. (C) There were no significant differences of morphology and size between naïve-NK-EVs and I-131-NK-EVs at transmission electron microscopy images. Data are presented as mean ± standard deviation.

Figure 5

In vivo imaging of I-131-Cal62-EVs. After intravenous injection of I-131-Cal-62-EVs (3.7 GBq), gamma camera images were acquired at 1 h, 3 h, 5 h and 24 h in BALB/c nude mice. The gamma camera images showed intense uptake in liver and spleen area. And there shows intense trace accumulation at bladder.

Figure 6

Ex vivo biodistribution of I-125-Cal62-EVs. I-125-Cal62-EVs (111 kBq) were intravenously injected to mice, and mice were sacrificed at 1 h, 3 h, 5 h (n = 3 for each group) and 24 h (n = 4) after injection. (A) %ID/organ represents counts of radioactivity normalized by injected dose per organ. (B) %ID/g represents counts of radioactivity normalized by injected dose per gram of organ. Data are presented as mean ± standard deviation.