Microfluidic fabrication of cell-derived nanovesicles as endogenous RNA carriers

Abstract

Exosomes/microvesicles are known to shuttle biological signals between cells, possibly by transferring biological signal components such as encapsulated RNAs and proteins, plasma membrane proteins, or both. Therefore exosomes are being considered for use as RNA and protein delivery vehicles for various therapeutic applications. However, living cells in nature secrete only a small number of exosomes, and procedures to collect them are complex; these complications impede their use in mass delivery of components to targeted cells. We propose a novel and efficient method that forces cells through hydrophilic microchannels to generate artificial nanovesicles. These mimetic nanovesicles contain mRNAs, intracellular proteins and plasma membrane proteins, and are shaped like cell-secreted exosomes. When recipient cells are exposed to nanovesicles from embryonic stem cells, mRNAs of Oct 3/4 and Nanog are transferred from embryonic stem cells to the target cells. This result suggests that mimetic nanovesicles can be used as vehicles to deliver RNA. This nanovesicle formation method is expected to be used in exosome research and to have applications in drug and RNA-delivery systems.

Microfluidics‐based on‐a‐chip systems for isolating and analysing extracellular vesicles

“…One potential avenue of exploration is the microfluidic extrusion method. In 2014, Jo et al developed a microfluidic instrument to mechanically break down cells into EV-mimetics (EVMs), which have similar properties to naturally secreted EVs [124]. Then, EVMs were used to load siRNA via random packaging and re-assembly after the breakdown [125].…”

Therapeutic biomaterials based on extracellular vesicles: classification of bio‐engineering and mimetic preparation routes

“…Those strategies rely on the principle of self-assembly of lipids and lipid membranes into spherical structures and the encapsulation of surrounding material into the aqueous cavity of generated nanovesicles. Current methods include extrusion over membrane filters [16,17,87], hydrophilic microchannels [88] or cell slicing by Si x N y blades [18] (see Table 4). …”

“…Current methods include extrusion over membrane filters [16,17,87], hydrophilic microchannels [88] or cell slicing by Si x N y blades [18] (see Table 4). …”

“…The analysis of these NVs [88] revealed that the formation of exosome-like NVs through hydrophilic channels produced a delivery system of endogenous material with identical results to those with exosomes.…”

Therapeutic and diagnostic applications of extracellular vesicles

“…Additionally, the idea of creating EV mimicking vesicles, e.g. by means of sequential extrusion of cells through micro-and nanoporous filters [185,186] or by mixing synthetic components attempting to reproduce the most important EV characteristics [79,187], is gaining interest. However, the latter approach is difficult to implement as long as the knowledge on which components are essential for EV functionality is lacking or incomplete.…”