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. Na?ve 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 single-chain 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 1.5?g/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.

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