Abstract
Extracellular vesicle (EV)-mediated transfer of biomolecules plays an essential role in intercellular communication and may improve targeted drug delivery. In the past decade, various approaches to EV surface modification for targeting specific cells or tissues have been proposed, including genetic engineering of parental cells or postproduction EV engineering. However, due to technical limitations, targeting moieties of engineered EVs have not been thoroughly characterized. Here, we report the bioluminescence resonance energy transfer (BRET) EV reporter, PalmReNL-based dual-reporter platform for characterizing the cellular uptake of tumor-homing peptide (THP)-engineered EVs, targeting PDL1, uPAR, or EGFR proteins expressed in MDA-MB-231 breast cancer cells, simultaneously by bioluminescence measurement and fluorescence microscopy. Bioluminescence analysis of cellular EV uptake revealed the highest binding efficiency of uPAR-targeted EVs, whereas PDL1-targeted EVs showed slower cellular uptake. EVs engineered with two known EGFR-binding peptides via lipid nanoprobes did not increase cellular uptake, indicating that designs of EGFR-binding peptide conjugation to the EV surface are critical for functional EV engineering. Fluorescence analysis of cellular EV uptake allowed us to track individual PalmReNL-EVs bearing THPs in recipient cells. These results demonstrate that the PalmReNL-based EV assay platform can be a foundation for high-throughput screening of tumor-targeted EVs.
Highlights
Extracellular vesicles (EVs) provide a natural delivery system that transfers various pieces of cellular cargo to both adjacent and distant cells in the body [1]
We previously revealed that bioluminescence signals in PalmReNL-EVs rapidly decreased after cellular uptake due to their sensitivity to acidic cellular compartments [30]
We developed a novel approach for screening tumor-homing peptide (THP)-engineered EVs using PalmReNL
Summary
Extracellular vesicles (EVs) provide a natural delivery system that transfers various pieces of cellular cargo (protein, mRNA, non-coding RNA, and DNA) to both adjacent and distant cells in the body [1]. EVs are heterogeneous populations of nanoscale membrane-enclosed vesicles released virtually from all cell types. EVs offer advantages for surface engineering while possessing inherent immune evasion and tissue-penetrating characteristics [3,6,7]. Several approaches have achieved targeted delivery of engineered EVs using various targeting moieties, including antibodies, nanobodies, and peptides [8]. EVs have been modified with tissue-targeting peptides by genetically engineering As shown in pioneering studies, EVs have been modified with tissue-targeting peptides by genetically engineering
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