Versatile tethering system to control cell-specific targeting of bioengineered extracellular vesicles

Researchers at the Université Paris Cité have developed a flexible and powerful system to improve how extracellular vesicles (EVs) deliver therapeutic molecules to specific cells. EVs are tiny particles naturally released by cells that can carry proteins, RNA, and other molecules to communicate with other cells. Scientists are exploring ways to use them as natural delivery vehicles for medicine, but getting EVs to reach only their intended targets has been a major hurdle.

To solve this, the team designed a modular targeting system by genetically engineering EVs to display a special protein on their surface. This protein contains two parts: one allows it to attach to the EV membrane, and the other binds to biotin-tagged molecules such as antibodies or ligands that can recognize specific cell types. When these modified EVs are mixed with target cells, they home in on the intended recipients with much higher precision. The researchers tested the system on several different target cells and found that it boosted EV uptake by as much as 40 times compared to untargeted EVs.

Generation of a versatile tethering system

(A) A versatile tethering system permitting to increase EV docking at acceptor cell surface was designed. EV surface was decorated with an Adapter protein able to capture a biotinylated antibody. The Adapter results from the fusion between a monomeric streptavidin (S), the luminescent enzyme NanoLuciferase (N), the fluorescent protein mCherry (C) and a GPI anchor (G). Two versions of the Adapter were developed: with or without the NanoLuciferase, respectively Streptavidin-NanoLuciferase-mCherry-GPI (SNCG) and Streptavidin-mCherry-GPI (SCG). In this study, the Adapter is coupled with an  -CD8a-Biotin in order to target CD8a-GFP⁺ cells. (B) Confocal images of HeLa transiently expressing the SNCG Adapter labelled with CD8-Biotin and -mouse Alexa 488 (“-mouse 488”) antibodies or the -mouse Alexa 488 only, or HeLa transfected with mCherry labelled with -CD8-Biotin and -mouse Alexa 488 (“-mouse 488”) antibodies. Cell labelling was performed without permeabilization. Merged represents the overlay of the green and red channels. Scale bar, 10 μm. Representative images of two independent experiments. (C) EVs deriving from HeLa transiently expressing the SNCG Adapter or stably expressing the NanoLuciferase-Hsp70 (NLuc-Hsp70) were isolated and submitted to a sucrose gradient (20 to 80%). The gradient was divided into 12 fractions that were analyzed by luminescence. Each dot represents an experimental value. (D) HeLa cells transiently expressing the Streptavidin-NLuc-mCherry-GPI (“SNCG Adapter”) and their derived EVs were characterized by western blot through different positive (CD63, CD9) and negative (Calnexin) EV markers. The expression of the SNCG Adapter was also analyzed using an  -mCherry antibody. Same amounts of proteins were loaded for both “Cells” and “EVs” conditions. (E) EVs emanating from HeLa cells transiently expressing the Adapter (“Adapter⁺ EVs”) or not (“WT EVs”) were isolated and analyzed by Nanoparticle Tracking Analysis (NTA). (F) EVs deriving from HeLa transiently expressing the Streptavidin-NLuc-mCherry-GPI were isolated and incubated with a mouse -CD8-Biotin and -mouse Alexa 488 antibodies for 2 h prior being submitted to size exclusion chromatography. The pick (1) contains complexes of EVs harboring the Adapter and the antibodies, the pick (2) contains the excess of free antibodies. Each dot represents an experimental value.

The platform also includes built-in luminescent and fluorescent markers, allowing scientists to easily track how efficiently EVs reach their targets. Because the system is adaptable, it can be customized for different diseases or delivery needs by simply changing the targeting molecule. This approach could pave the way for safer and more effective EV-based therapies, particularly in cancer, neurology, and gene therapy, where precision delivery is essential.