Jamal (2020) Design of experiment (DoE)-driven invitro and invivo uptake studies of exosomes for pancreatic cancer delivery enabled by copper-free click chemistry-based labelling,

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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.

Stem cell‐derived exosomes are emerging as novel and clinically relevant cell‐free therapeutics for regenerative therapy. This work focused on investigating the stimulation of fibroblasts by exosomes derived from umbilical cord‐derived mesenchymal stem cells (ucMSC) in a defined serum‐free three‐dimensional (3D) culture. 3D culture of ucMSC was carried out in medium supplemented with KnockOut serum replacement (KO‐medium) using the Aggrewell system. ucMSC in KO‐medium formed spheroids with maintained size and integrity throughout culture. This enabled the isolation of vesicles from ucMSC spheroids in KO‐medium with sizes that fall within the exosomal size range and were positive for the expression of canonical exosomal markers CD63, CD9, CD81, Alix, and TSG101. The ucMSC‐derived exosomes (ExoucMSC) were shown to significantly increase the migration and proliferation of murine fibroblasts in vitro. To conclude, 3D culture of ucMSC in defined serum‐free KO‐medium formed viable spheroids which enabled the isolation of ExoucMSC with the potential of accelerating wound healing.

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