The cell type dependent sorting of CD9- and CD81 to extracellular vesicles can be exploited to convey tumor sensitive cargo to target cells

Abstract

Extracellular vesicles (EVs) are lipid membrane-bound particles involved in cell-to-cell communication through a delivery of regulatory molecules essential for physiological processes. Since EVs efficiently vectorize specific cargo molecules, they have been proposed as suitable vehicles for therapeutic agents. Drug loading into EVs can be achieved by active, exogenous strategies or by genetic modifications of vesicle-producing cells. With the aim to produce EVs conveying therapeutic proteins, we genetically engineered and compared HEK293 to tumor cells. Tetraspanin-based RFP fusions were found to be more stable and preferentially sorted into EVs in HEK293. EVs isolated from genetically modified HEK293 cells media were captured by cancer cells, efficiently delivering their cargo. Cathepsin B cleavage site introduced between CD9/CD81 and RFP was recognized by tumor specific proteases allowing the release of the reporter protein. Our results indicate HEK293 cells as a preferential system for the production of EVs and pave the way to the development of nano-platforms for the efficient delivery of therapeutic proteins and prodrugs to tumor cells.

Keywords:

• Extracellular vesicles
• EVs
• cargo sorting
• tetraspanin-based fusion proteins
• cathepsin B-controlled release

Acknowledgments

Part of this work was carried out in the Advanced Light and Electron Microscopy BioImaging Center (ALEMBIC) of San Raffaele Scientific Institute and Vita-Salute University.

Authors’ contributions

SZ, NZ performed the experiments and acquired data; SZ, NZ and RV analyzed and discussed the results; SZ and RV drafted the manuscript; RV supervised and coordinated the project; All authors read and approved the manuscript.

Disclosure statement

The authors report no conflict of interest.

Ethical approval

NA

Additional information

Funding

This work was supported by the Italian Ministry of Health (GR-2011-02351220 to R.V.) and by European Union’s Horizon 2020 research and innovation program (MARVEL project, Grant no. 951768) to RV and NZ.