# Cardenolide‐Engineered Extracellular Vesicles Augment Drug Uptake and Cytotoxicity in Non‐small Cell Lung Cancer Cells

## TL;DR

Researchers developed a targeted drug delivery system using modified extracellular vesicles to deliver chemotherapy to lung cancer cells, improving effectiveness and reducing side effects.

## Contribution

A novel drug delivery system using cardenolide-functionalized extracellular vesicles to target and kill lung cancer cells.

## Key findings

- Cardenolide-modified extracellular vesicles bind to and inhibit Na+/K+-ATPase in A549 lung cancer cells.
- Doxorubicin-loaded, cardenolide-modified vesicles reduced cancer cell viability to 45% after 48 hours.
- Modified vesicles showed enhanced cytotoxicity compared to unmodified vesicles and free drug.

## Abstract

Cancer remains a leading cause of premature mortality worldwide. Targeted drug delivery therapies that selectively attack malignant cells while sparing healthy tissue are essential to minimize side effects and reduce drug dosages. The sodium‐potassium ATPase (Na+/K+‐ATPase), particularly its catalytic α‐subunit, is overexpressed in A549 non‐small cell lung cancer (NSCLC) and has thus emerged as a potential therapeutic target. Cardiac glycosides (CGs), plant‐derived secondary metabolites, specifically bind and inhibit this enzyme providing target engagement. Coupling CGs to a biocompatible carrier provides a promising new approach for a targeted‐orientated drug carrier. Among these nanocarrier systems, cell‐derived extracellular vesicles (EVs) gained attention due to their biocompatibility, tumor‐targeting capability, and ability to encapsulate compounds. Here, we developed a target‐oriented nanocarrier system by linking 3β‐azido‐3‐deoxydigitoxigenin (CA), a semi‐synthetic cardenolide derivative, to the surface of A549 cell‐derived EVs. The EVs were characterized for particle concentration, size and protein markers. Surface modification was achieved via alkyne modification and click chemistry. Successful conjugation was confirmed by inhibition of the Na+/K+‐ATPase activity. Co‐localization of CA‐modified EVs with the Na+/K+‐ATPase was verified by confocal microscopy. Doxorubicin‐loaded, CA‐modified EVs reduced A549 cell viability to 45% after 48 h, demonstrating its potential use as new drug nanocarrier system.

Cardenolide‐functionalized extracellular vesicles enable targeted delivery of the chemotherapeutic doxorubicin to A549 lung cancer cells by binding Na+/K+‐ATPase, which is overexpressed in these cells. Surface‐modified, drug‐loaded EVs exhibit enhanced cytotoxicity compared to unmodified EVs and free drug, suggesting this system may lower the effective dose and thereby reduce dose‐related side effects in cancer treatment.