EGFR-targeted extracellular vesicles loaded with doxorubicin for enhanced chemotherapy in non-small cell lung cancer via bioorthogonal click chemistry.

Abstract

In the field of non-small cell lung cancer (NSCLC) treatment, targeted drug delivery to epidermal growth factor receptor (EGFR)-overexpressing tumors remains a significant challenge. This study aims to tackle this issue by engineering extracellular vesicles (EVs) derived from 293 T cells. Through azide-alkyne cycloaddition, EVs were functionalized with GE11 peptides (GE11-EVs), and loaded with doxorubicin to form GE11&DOX@EVs. We conduted Ac4ManNAz metabolic labeling to facilitate the site-specific conjugation of GE11 peptides and fluorescent probes onto the membranes of EVs. Then we comprehensively assessed the targeting efficiency, drug-loading capacity, and therapeutic efficacy of GE11-EVs in A549 NSCLC cells and xenograft models. In vitro experiments demonstrated significantly enhanced uptake of GE11-EVs by EGFR-positive A549 cells compared to unmodified EVs, along with markedly improved cytotoxicity of GE11&DOX@EVs against tumor cells (IC₅₀: 1.6 μg/mL vs. 3.96 μg/mL for free doxorubicin, P < 0.01) and induction of mitochondrial membrane depolarization. Mechanistically, GE11&DOX@EVs upregulated pro-apoptotic proteins (caspase-3/8/9 and Bax) and downregulated anti-apoptotic BCL-2, significantly enhancing apoptosis compared to free DOX. In vivo imaging revealed a two-fold greater tumor accumulation of GE11-EVs than unmodified EVs at 24 h post-injection. GE11&DOX@EVs treatment suppressed tumor growth by 73.7 % in A549 xenografts, significantly outperforming both free doxorubicin (24.9 %) and DOX@EVs (58.6 %) groups (P < 0.05), with minimal systemic toxicity. Future research could further explore the clinical translation potential of this bioorthogonal platform for precision engineering of EVs in NSCLC therapy.