PET Imaging of Small Extracellular Vesicles via [⁸⁹Zr]Zr(oxinate)₄ Direct Radiolabeling

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 wholebody 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 [ 89 Zr]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 89 Zrlabeled 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 89 Zr-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.

Radiovesicolomics-new approach in medical imaging

“…NOTA) can be conjugated with the amine group of the membrane proteins on EVs ( Royo et al, 2019 ; Shi et al, 2019 ; Jung et al, 2020 ; Lázaro-Ibáñez et al, 2021 ). In the intraluminal radiolabeling strategy, a lipophilic radiotracer can easily penetrate to the EV lumen or ionophores allow radionuclides to be transported across the lipid membrane where they can be trapped as their loose lipophilicity ( Son et al, 2020 ; Khan et al, 2022 ).…”

“…The in vivo PET imaging of EVs is achievable by the gallium 68 Ga ( t 1/2 = 68 min), cooper 64 Cu ( t 1/2 = 12.7 h), zirconium 89 Zr ( t 1/2 = 78.4 h) and iodine 124 I ( t 1/2 = 100 h) isotopes, with the increasing half-life ( Royo et al, 2019 ; Jung et al, 2020 ; Khan et al, 2022 ). The advent of high sensitivity total-body PET scanners opens possibility for an efficient in vivo tracking of EVs in the whole human body simultaneously ( Stępień et al, 2021b ; Vandenberghe et al, 2020 ) ( Figure 2C ).…”

“…Another advantage of SPECT or PET imaging modalities is their very high sensitivity reaching a factor 10 −6 with compare to MRI. This allows application of the radionuclide in a dose from very small dose 0.2–1 MBq per mouse for whole-body imaging with the use of EVs in a concentration 10 10 of particles/Gram body weight (p/g) ( Lázaro-Ibáñez et al, 2021 ; Khan et al, 2022 ). Depending on imaging radionuclides and the detection system, the applied dose can vary from 37 kBq ( 125 I-biotin) ( Matsumoto et al, 2017 ) to 5–10 MBq ([ 111 In]DTPA) ( Lázaro-Ibáñez et al, 2021 ), 7 MBq ( 64 Cu-NOTA) ( Banerjee et al, 2019 ), 3.7 MBq ( 99m Tc) ( Son et al, 2020 ), 2 MBq ( 64 Cu-NOTA) ( Shi et al, 2019 ), and 0.2–1 89 Zr-PANC1 ( Khan et al, 2022 ) to track/image EVs using either SPECT or PET in a mouse.…”

“…This allows application of the radionuclide in a dose from very small dose 0.2–1 MBq per mouse for whole-body imaging with the use of EVs in a concentration 10 10 of particles/Gram body weight (p/g) ( Lázaro-Ibáñez et al, 2021 ; Khan et al, 2022 ). Depending on imaging radionuclides and the detection system, the applied dose can vary from 37 kBq ( 125 I-biotin) ( Matsumoto et al, 2017 ) to 5–10 MBq ([ 111 In]DTPA) ( Lázaro-Ibáñez et al, 2021 ), 7 MBq ( 64 Cu-NOTA) ( Banerjee et al, 2019 ), 3.7 MBq ( 99m Tc) ( Son et al, 2020 ), 2 MBq ( 64 Cu-NOTA) ( Shi et al, 2019 ), and 0.2–1 89 Zr-PANC1 ( Khan et al, 2022 ) to track/image EVs using either SPECT or PET in a mouse. Radiovesicolomics may benefit also from the new multi-photon PET scanners ( Moskal et al, 2020 ; Moskal et al, 2021a ; Moskal et al, 2021b ; Moskal et al, 2021c ) which enable simultaneous multi-tracer imaging ( Moskal and Stępień, 2022 ) and hence studying simultaneously the kinetics of two different types of EVs by marking them with different isotopes.…”

Dual-labeled nanoparticles based on small extracellular vesicles for tumor detection

“…Different approaches have been proposed for the development of novel EV-based probes. Among these approaches, those based on radioactive isotope labeling, such as positron emission tomography (PET) [20,21] or single photon emission computed tomography (SPECT), stand out [22][23][24][25]. In addition, other strategies have been developed to label EVs with non-ionizing sources, such as dyes and fluorophores, including lipophilic [26], luminal [27], and engineered optical reporters [28], or by covalent bond [29].…”