Extracellular vesicles (EVs) are small vesicles secreted by almost all cells with a size range of 30-150 nm, containing abundant bioactive substances. Depending on the cell of origin, EVs show high heterogeneity, possessing multiple components specific to parent cells¹. EVs are released into body fluids and transported to distant sites where they are taken up by target cells for action², which can be utilized to deliver a wide range of bioactive molecules and drugs for tissue repairing, tumor diagnosis and treatment, and immune modulation³^,⁴. However, other biological nanoparticles (e.g., lipoproteins) and nanovesicles (e.g., EVs derived from non-endosomal pathways) with similar biophysical properties in body fluids inevitably affect EV isolation and purification. To date, ultracentrifugation remains the gold standard for EV isolation, and other isolation methods, including sucrose density gradient centrifugation, ultrafiltration, polyethylene glycol precipitation, chromatography, and immunomagnetic bead isolation, have been developed⁵. The current bottleneck limiting clinical translation and commercialization of EV therapeutics is the severe lack of isolation techniques that allow for highly scalable and reproducible isolation of EVs⁶^,⁷^,⁸. Traditional EV isolation techniques (e.g., ultracentrifugation and size exclusion chromatography) suffer from low yield (1 x 10⁷-1 x 10⁸/1 x 10⁶ cells), long production cycle (24-48 h), poor reproducibility of product quality, and require expensive and energy-intensive production equipment that cannot meet the current clinical demand for EVs⁶.

Exosome mimics (EMs), synthetic surrogates of native EVs, have attracted important attention due to their highly similar structure, function, and scalability in production. The main source of EMs is from the direct extrusion of whole parental cells with continuous sectioning⁹^,¹⁰, demonstrating potent biological functions as native EVs¹¹^,¹². For instance, EMs derived from human umbilical cord mesenchymal stem cells (hUCMSCs) exert similar wound-healing effects as native EVs and are richer in protein composition¹³. Though EMs derived from whole cells have the biological complexity of EVs, their main drawback is the heterogeneity of products because they are inevitably contaminated by various cellular organelles and cell debris. Protein localization analysis further revealed that EMs derived from whole-cell extrusion contain many non-EVs-specific proteins from mitochondria and the endoplasmic reticulum¹³. Moreover, most methods for manufacturing EMs still require ultracentrifugation, a highly time and energy-consuming process¹⁴. Considering the fact that exosomes are exclusively derived from cellular endosomes, we hypothesized that bioengineered endosome-derived nanovesicles may better recapitulate the biological homology between exosomes and EMs in comparison with the well-established cell membrane-derived EMs produced by whole cell extrusion method¹⁴. Nevertheless, the manufacture of endosome-derived nanovesicles is difficult due to the lack of viable approaches.

Clinical studies have been carried out by utilizing EVs as a surrogate of cell-free therapy and a nanoscale drug delivery system for the treatment of various diseases. For instance, EVs derived from bone marrow mesenchymal stem cells have been used to treat severe pneumonia caused by COVID-19 and have achieved promising results. Recently, genetically engineered EVs carrying CD24 proteins have also demonstrated potent therapeutic benefits for treating COVID-19 patients¹⁵^,¹⁶. However, the clinical requirement of EV therapy still cannot be met with traditional isolation methods because of the low yield and cost. This study reports the large-scale production of endosome-derived nanovesicles via a magnetic separation-assisted high-speed homogenization approach. It takes advantage of the endocytosis pathway of MNPs to isolate MNP-loaded endosomes via magnetic separation, followed by high-speed homogenization to formulate endosomes into monodisperse nanovesicles. Since the types of endosomes collected by this protocol are diverse, further in-depth research is still required to establish good manufacturing practices (GMP) in the industry. This novel EM preparation approach is more time efficient (5 min of high-speed homogenization) to obtain nanovesicles homologous to native EVs. It produces exponentially more vesicles from the same amounts of cells than ultracentrifugation, which can be generally applied to various cell types.

Isolation and Preparation of Endosomes Containing Magnetic Nanoparticles from Cultured Cells