Abstract

Exosomes or small extracellular vesicles (sEVs) are increasingly gaining attention for their potential as drug delivery systems and biomarkers of disease. Therefore, it is important to understand their in vivo biodistribution using imaging techniques that allow tracking over time and at the whole-body level. Positron emission tomography (PET) allows short- and long-term whole-body tracking of radiolabeled compounds in both animals and humans and with excellent quantification properties compared to other nuclear imaging techniques. In this report, we explored the use of [⁸⁹Zr]Zr(oxinate)₄ (a cell and liposome radiotracer) for direct and intraluminal radiolabeling of several types of sEVs, achieving high radiolabeling yields. The radiosynthesis and radiolabeling protocols were optimized for sEV labeling, avoiding sEV damage, as demonstrated using several characterizations (cryoEM, nanoparticle tracking analysis, dot blot, and flow cytometry) and in vitro techniques. Using pancreatic cancer sEVs (PANC1) in a healthy mouse model, we showed that it is possible to track ⁸⁹Zr-labeled sEVs in vivo using PET imaging for at least up to 24 h. We also report differential biodistribution of intact sEVs compared to intentionally heat-damaged sEVs, with significantly reduced spleen uptake for the latter. Therefore, we conclude that ⁸⁹Zr-labeled sEVs using this method can reliably be used for in vivo PET tracking and thus allow efficient exploration of their potential as drug delivery systems.

Results from the nanoScan^® PET/CT

• In vivo PET-CT imaging in healthy mice showed that 89Zr-labeled sEVs are stable for 24 h and thus can reliably be tracked within this timeframe
• The differential spleen:bone uptake ratio for intact versus heat-damaged 89Zr-PANC1 sEVs led to the proposition of using this parameter as an imaging biomarker for sEV stability when using this radiolabeling method

Figure 5. PET imaging and ex vivo biodistribution of 89Zr-PANC1 sEVs. (A) Maximum intensity projection PET-CT images of (i) intact 89ZrPANC1 sEVs, (ii) heat-damaged 89Zr-PANC1 sEVs, and (iii) neutralized 89Zr4+ biodistribution in a C57BL/6j mouse at 1 h and 24 h postintravenous injection; white arrowheads = representative LNs (see Figure S6) and B = bladder; the PET imaging scale for the 89Zr-control was adjusted for image clarity. (B) PET-CT images (axial, sagittal, and coronal slices) of a mouse injected with intact 89Zr-PANC1 sEVs showing uptake within the brain; the image scale is the same as in (A).

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