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 β 1 (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 1.5μg/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.
Keywords: exosome, glycosylation, glycan, drug delivery