General Approach to Engineering Extracellular Vesicles for Biomedical Analysis

Abstract

Extracellular vesicles (EVs) are cell-derived nanoscale vesicles that play critical roles in numerous pathophysiological processes. Enrichment and detection of EVs are technically challenging due to the lack of appropriate modification strategies. Herein, we propose a general, facile, and robust approach to engineering EVs by installation of maleimide (Mal) moieties onto EV surfaces based on a hydrophobic insertion strategy. Mal serves as a high-efficiency clickable handle for functionalizing EVs without influencing their structural integrity and biological activity. The Mal-installed EVs were applied into three biomedical applications: (i) labeling with a fluorescent dye for monitoring the EV-mediated cellular communication, (ii) rapid enrichment by magnetic particles (MPs) for high-efficiency EVs isolation, and (iii) conjugation with gold nanoparticles (AuNPs) for Raman detection of the surface components of EVs in situ. This technique would greatly facilitate the applications of EVs in both basic studies and clinical uses.

Nanozyme-assisted sensitive profiling of exosomal proteins for rapid cancer diagnosis

“…To immobilize the 2 nm AuNP nanozymes on the surface of the Exos, thiol-terminated 1,2-distearoyl-sn-glycero-3-phosphethanolamine-poly(ethylene glycol) (DSPE-PEG-SH) was inserted into the exosomal membrane via hydrophobic interactions. The DSPE-PEG-SH acted as a handle, as the exposed SH moieties could capture AuNPs tightly via Au-S bonds 37 . After incubating the DSPE-PEG-SH-treated Exos with HAuCl 4 and NaBH 4 , 2 nm AuNPs were formed and uniformly deposited over the vesicle surfaces without altering the Exo morphology (Figure 1 C), as confirmed by the elemental mapping results (Figure 1 D) .…”

Inorganic Nanoparticle-Loaded Exosomes for Biomedical Applications

“…Direct surface conjugation was mainly achieved by attaching chemical linkers to exosome surfaces via covalent binding to surface proteins [25] or hydrophobic tail insertion of molecules with click chemistry to lipid bilayer of exosomes [26]. These functional linkers subsequently reacted with the functional surfaces of nanoparticles, leading to a "raspberry" nanoconstruct where the exosomes were decorated with inorganic nanoparticles.…”

“…In addition to linker conjugation, linkers can be displayed on exosome surfaces through linkers with a hydrophobic tail, which can be directly inserted into the membrane of exosomes. For example, a hydrophobic insertion strategy was applied to insert maleimide-terminated 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine (DSPE)-PEG linker with a hydrophobic chain (DSPE), a hydrophilic linker (PEG), and a Mal reactive end (Figure 2b) [26]. DSPE is able to insert into membrane structures spontaneously through hydrophobic interactions, while PEG possesses good biocompatibility and hydrophilicity for improved solubility.…”

Extracellular Vesicles-based Pre-Targeting Strategy Enables Multi-Modal Imaging of Orthotopic Colon Cancer and Image-Guided Surgery

“…The modi cation of extracellular vesicles was adapted from a literature reported method [31]. Brie y, we introduced 1mg DSPE-PEG per 1mg extracellular vesicles.…”

“…Using hydrophobic insertion approach [32], DSPE-PEG 2000 -Cy7 (Ruixi, Xi'an, China) and DSPE-PEG 2000 -N 3 (Ruixi, Xi'an, China) were incubated with ADSCs-EV for 30 min at 37°C to form N 3 (Cy7)-PEG 2000 -DSPE-ADSCs-EV (Cy7-EV-N 3 ). The samples were passed through centrifugal lter devices (100 kDa molecular weight, Amicon®Ultra-15) before further use.…”