Engineering Piezo1‐Mediated Calcium Signalling for Enhanced Extracellular Vesicle Biogenesis and Cargo Loading
ABSTRACT Cell‐derived extracellular vesicles (EVs) are promising nanocarriers for therapeutic delivery platforms owing to their biocompatibility and capacity to protect and efficiently transport bioactive molecules. However, EV‐based therapeutics remain constrained by inefficient cargo loading and low production yields, which limit scalable biomanufacturing. To overcome these limitations, we exploited the use of the mechanosensitive ion channel Piezo1 as a robust regulator of EV biogenesis using HEK293FT cells co‐transfected with Piezo1 and the bioluminescent EV reporter PalmReNL. Activation of Piezo1 with Yoda1 (30 µM) increased PalmReNL‐EV release by 3‐fold, while GsMTx4 inhibited EV release by 80.7%. This effect was unaffected by removal of extracellular Ca 2+ but was suppressed by intracellular Ca 2+ chelation with BAPTA‐AM, indicating a reliance on intracellular Ca 2+ mobilisation. Small EVs (sEVs) from Piezo1‐activated cells were purified by anion exchange chromatography and analysed by proteomics, identifying 48 proteins exclusively in Piezo1‐induced sEVs preparations among 148 total detected, including cytoskeletal and stress‐related factors, while preserving enrichment of extracellular matrix (ECM) structural components prominent in both conditions. Yoda1 treatment increased the release of both large EVs (lEVs) and sEVs, with a particularly pronounced increase in sEV production. As a proof of concept for therapeutic cargo delivery, Yoda1 stimulation increased the incorporation of exogenously expressed interleukin‐10 (IL‐10) into sEVs by up to 4‐fold, and the bioactivity of sEV‐associated IL‐10 was validated using IL‐10‐CyCLoPs reporter cells. However, exposing Piezo1‐overexpressing cells to 30 µM Yoda1 markedly delayed cell adhesion and spreading, indicating that excessive Piezo1 activation may constrain sustained production of therapeutic sEVs. Collectively, these results identify mechanotransduction as a key regulator of sEV biogenesis and underscore the need for precise temporal control, potentially achievable through ultrasound‐based modulation, for the rational engineering of next‐generation sEV therapeutics.