Engineered extracellular vesicles for targeting and treating inflammation

Abstract

Extracellular vesicles (EVs) are nanosized particles released by human and mammalian cells. They are emerging as therapeutic platforms due to their ability to facilitate intercellular communication and their immunomodulatory potential. Their broad clinical translation remains limited by challenges in precise cell targeting, therapeutic efficacy, and scalable manufacturing. This thesis examines various EV engineering strategies designed to enhance targeting and induce immunomodulatory functions, while improving the scalability of isolation. To address these challenges, engineered HEK293F cells were utilized to generate EV-based therapeutics for targeting and treating inflammation. EVs expressing LFA-1 were generated for selective targeting of ICAM-1-positive endothelial cells, providing a strategy for treating endothelial inflammation. Overexpression of CD24 and syndecans was introduced to produce EVs with intrinsic anti-inflammatory properties. These vesicles were further loaded with an anti-inflammatory peptide to potentially augment their therapeutic effects. In addition, a combined workflow employing superabsorbent polymer (SAP) concentration and size exclusion chromatography (SEC) purification was tested to isolate EVs using a non-ultracentrifugation approach. This thesis successfully developed LFA-1-expressing EVs with enhanced targeting capacity to ICAM-1-positive endothelial cells, which enhanced delivery and efficacy of an EV-loaded anti-inflammatory peptide, resulting in synergistic effects (Paper I). Further, expression of either CD24 or syndecans (1, 2, and 4) in EVs induced an anti-inflammatory function, evident both in vitro (Papers II and III) and in vivo (Paper III). Lastly, combining SAP with SEC enabled the isolation and purification of EVs in a possibly scalable manner, retaining their functionality and EV markers (Paper IV). This thesis developed engineered EVs from HEK293F cells to improve targeting, immunomodulation, and scalable production, ultimately generating EV-based drug candidates for treating inflammation. Through surface modifications (LFA-1, CD24, syndecans), therapeutic peptide loading, and a robust SAP-SEC isolation workflow, it supports the development of EV therapeutics with intrinsic anti-inflammatory properties and synergistic efficacy, as well as scalable manufacturing.