Surface Engineering of HEK293 Cell-Derived Extracellular Vesicles for Improved Pharmacokinetic Profile and Targeted Delivery of IL-12 for the Treatment of Hepatocellular Carcinoma

Background: Extracellular vesicles (EVs) are considered a promising drug delivery platform.Naïve EVs face numerous issues that limit their applications, such as fast clearance, hepatic accumulations, and a lack of target-specific tropism.We aimed to explore a series of surface engineering approaches to: 1) reduce the non-specific adhesion of EVs, and 2) improve their enrichment in the target tissue.As a proof-of-concept, we investigated the therapeutic potentials of a multi-modal EVs system carrying a tumor-specific nanobody and the immuno-stimulant interleukin-12 (IL12) using in vivo models of hepatocellular carcinoma.Methods: The major cell adhesion molecule on the HEK293-derived EVs, integrin β1 (ITGB1), was knocked out (KO) by CRISPR/ Cas9-mediated gene editing, followed by deglycosylation to generate ITGB1 -Deg EVs for the subsequent pharmacokinetic and biodistribution analyses.ITGB1 -Deg EVs were further loaded with glypican-3 (GPC3)-specific nanobody (HN3) and mouse singlechain IL12 (mscIL12) to generate ITGB1 -mscIL12 + HN3 + Deg EVs, for evaluation of tumor tropism and therapeutic potential in a mice model of hepatocellular carcinoma.Results: Removal of ITGB1 led to the broad suppression of integrins on the EVs surface, resulting in a decrease in cellular uptake.Deglycosylation of ITGB1 -EVs gave rise to inhibition of the EVs uptake by activated RAW264.7 cells.ITGB1 removal did not significantly alter the pharmacokinetic behaviors of HEK293-EVs, whereas the ITGB1 -Deg EVs exhibited enhanced systemic exposure with reduced hepatic accumulation.Loading of HN3 conferred the ITGB1 -Deg EVs with tumor-specific tropism for both subcutaneous and metastasized tumors in mice.The ITGB1 -mscIL12 + HN3 + Deg EVs activated mouse splenocytes with high potency.Systemic administration of the EVs with the equivalent dose of 1.5µg/kg of exosomal IL12 achieved satisfactory tumor growth inhibition and good tolerability. Conclusion:The combinatorial approach of EVs surface engineering conferred HEK293-EVs with reduced non-specific clearance and enhanced tumor targeting efficacy, which constituted an efficient delivery platform for critical cancer therapeutics like IL12.