Engineered Peptide Scrambling for Enhanced Drug Delivery to Resistant Breast Cancer Cells via Small Extracellular Vesicles

Extracellular vesicle (EV)-mediated transfer of biomolecules plays an essential role in intercellular communication and presents promising avenues for targeted drug delivery. Over the past decade, researchers have developed various approaches to modifying EV surfaces for targeting specific cells or tissues, including functionalization with targeting peptides to increase the specificity of drug delivery. Due to technical limitations, methods for characterizing the targeting moieties on the surface of small EVs (sEVs) are considerably restricted. To address these limitations and enhance the throughput capacity of sEV characterization, a dual-reporter platform was utilized to quantitatively assess the binding of tumor homing peptide (THP)-functionalized sEVs to breast cancer cells using bioluminescence assays and fluorescence microscopy. Twenty-four scrambled variants of the uPAR-binding peptide were designed for sEV engineering, and their uptake by MDA-MB-231 cells was evaluated in vitro. Our results revealed that amino acid scrambling generated both enhanced and reduced binding to cancer cells compared to the original peptide sequence. Furthermore, the data demonstrated that mechanical stimulation of EV producer HEK293FT cells enhanced the passive loading of methotrexate (MTX) into sEVs, but not large EVs, by increasing sEV production. By functionalizing MTX-loaded sEVs with a high-binding scrambled peptide, the delivery successfully surpassed the saturated free MTX uptake level in MDA-MB-231 cells, increasing cytotoxicity by 2.1-fold and providing a potent strategy for combating drug-resistant cancers. This study advances synthetic biology approaches to optimize tumor-targeted drug delivery, demonstrating that strategic peptide sequence scrambling can enhance targeting efficiency and drug delivery capabilities.