Sorting Extrusion-Prepared Coreless Cell Membrane Vesicles of Mixed Orientations by Functional DNA Probes.

Cell membrane vesicles (CMVs) have been extensively used as delivery vehicles for a variety of cargos, which are generally prepared via membrane extrusion. Extruded CMVs are not necessarily to have the outer membrane facing outward due to the randomness of membrane wrapping. Nanoparticles have been used to serve as cores to direct the membrane orientation of CMVs; nevertheless, there is a lack of methods to efficiently sort coreless CMVs of desired orientations. Herein, we utilized a group of functional DNA probes to reveal the random distribution of membrane orientations of coreless CMVs after extrusion, producing either right-side-out vesicles (RSVs) or inside-out vesicles (ISVs). More importantly, DNA probes that protrude out from the outer membrane can serve as handles for efficiently sorting out RSVs from ISVs to produce vesicles with a dominant right-side-out orientation. We investigated three methods to enrich RSVs, including strand displacement reaction, photo cleavage (PC), and enzymatic cleavage. Among them, PC exhibits the highest enrichment efficiency (∼93%) and RSVs purity (∼85.4%), which therefore is recommended for future applications. This work revealed the mixed orientations of coreless CMVs and provided a technical platform to efficiently enrich CMVs of wanted membrane orientations that shall be useful toward a vast array of biomedical applications.