Heterogeneous Subcellular Origin of Exosome-Mimetic Nanovesicles Engineered from Cells

Lee, Hyunjin; Kang, Hyejin; Kang, Minsu; Han, Chul–Hi; Yi, Johan; Kwon, Yongmin

doi:10.1021/acsbiomaterials.0c01157

Cited by 11 publications

(8 citation statements)

References 39 publications

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“…Finally, we characterized unique surface protein markers of CDVs in comparison with EVs. As previously reported, CDVs formed by the extrusion of cells are expected to be generated from more diverse lipid sources such as plasma and subcellular organelle membranes and therefore exhibit slightly different marker patterns from EVs 21,37 . To obtain more precise knowledge of CDV membrane protein markers, we combined the results of three distinct analytical methods: proteome analysis, western blotting, and nFCM.…”

Section: Discussionmentioning

confidence: 74%

Unraveling the surface marker signature of cell-derived vesicles via proteome analysis and nanoparticle flow cytometry

Lau,

Passalacqua,

Jung

et al. 2024

Sci Rep

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The cell-derived vesicles (CDVs) obtained using a proprietary extrusion process are the foundation of BioDrone platform technology. With superior productivity and versatility, this technology has garnered increasing attention in broad applications, particularly as a drug delivery vehicle. Previously, we showed that CDVs exhibited varying levels of expression for tetraspanin and organelle membrane markers while revealing no discernible differences in physical characteristics compared to naturally produced extracellular vesicles (EVs). To further understand and utilize the therapeutic potentials of CDVs, a more comprehensive study of membrane protein profiles is necessary. In addition, it is crucial to validate that the CDVs produced from extrusion are indeed intact lipid vesicles rather than other impurities. Here, we produced multiple batches of CDVs and EVs from HEK293 cells. CDVs and EVs were subjected to the same purification processes for subsequent proteome and particle analyses. The proteome analyses revealed unique proteome signatures between CDVs, EVs, and parental cells. Extensive proteome analyses identified the nine most prominent membrane markers that are abundant in CDVs compared to cells and EVs. Subsequent western blotting and nanoparticle flow cytometry analyses confirmed that CD63, lysosome-associated membrane glycoprotein 1 (LAMP1), and nicastrin (NCSTN) are highly enriched in CDVs, whereas CD81, CD9, and prostaglandin F2 receptor negative regulator (PTGFRN) are more abundant in EVs. This highlights the unique membrane composition and marker signature of CDVs that are distinct from EVs. Lastly, we demonstrated that more than 90% of the CDVs are genuine lipid vesicles by combining two different classes of vesicle labeling dyes and detergents to disrupt lipid membranes. This indicates that our proprietary extrusion technology is highly compatible with other well-characterized EV production methods. The robust CDV markers identified in this study will also facilitate the engineering of CDVs to achieve enhanced therapeutic effects or tissue-selective cargo delivery.

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Section: Discussionmentioning

confidence: 74%

Unraveling the surface marker signature of cell-derived vesicles via proteome analysis and nanoparticle flow cytometry

Lau,

Passalacqua,

Jung

et al. 2024

Sci Rep

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“…CDVs are EV‐mimetic nano‐vesicles produced by extruding source cells through small pores. Since they inherit different sets of membrane proteins and lipids from parental cells (Lee et al., 2020 ), a group of CDVs were prepared to test the general applicability of our method. For proof‐of‐principle experiments, we measured their interactions with ICAM‐1, a major cell adhesion molecule that controls adhesion between leukocytes and endothelial cells.…”

Section: Resultsmentioning

confidence: 99%

Quantitative imaging of vesicle–protein interactions reveals close cooperation among proteins

Cha

Jeong

Jung

et al. 2023

J of Extracellular Vesicle

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Membrane-bound vesicles such as extracellular vesicles (EVs) can function as biochemical effectors on target cells. Docking of the vesicles onto recipient plasma membranes depends on their interaction with cell-surface proteins, but a generalizable technique that can quantitatively observe these vesicle-protein interactions (VPIs) is lacking. Here, we describe a fluorescence microscopy that measures VPIs between single vesicles and cell-surface proteins, either in a surface-tethered or in a membrane-embedded state. By employing cell-derived vesicles (CDVs) and intercellular adhesion molecule-1 (ICAM-1) as a model system, we found that integrin-driven VPIs exhibit distinct modes of affinity depending on vesicle origin. Controlling the surface density of proteins also revealed a strong support from a tetraspanin protein CD9, with a critical dependence on molecular proximity. An adsorption model accounting for multiple protein molecules was developed and captured the features of density-dependent cooperativity. We expect that VPI imaging will be a useful tool to dissect the molecular mechanisms of vesicle adhesion and uptake, and to guide the development of therapeutic vesicles. K E Y WO R D SCD9, cell-derived vesicles, ICAM-1, integrin, total internal reflection fluorescence microscopy, vesicleprotein interactions  INTRODUCTIONMembrane-bound vesicles can effectively elicit biochemical changes in cells. For example, extracellular vesicles (EVs), which are naturally secreted by virtually all types of cells, can adhere to the plasma membranes of target cells and trigger intracellular signalling, often accompanied by cargo transfer upon internalization. This process is therefore believed to enable effective Minkwon Cha and Sang Hyeok Jeong contributed equally to this work.

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“…However, this difference significantly improved the characteristics of the resulting nanovesicles, specifically the homogeneity in protein expression and size. Cell‐extruded nanovesicles inevitably contain components not only from plasma membranes but also membranous subcellular organelles, leading to highly heterogeneous vesicle populations that express different protein markers (Lee et al., 2020 ). Furthermore, due to their rigid cytoskeleton and dense genomic material, cells can only be serially extruded through 1 µm‐pore filters, resulting in nanovesicles with highly heterogeneous sizes.…”

Section: Discussionmentioning

confidence: 99%

Cell‐engineered virus‐mimetic nanovesicles for vaccination against enveloped viruses

Han,

Kim,

Seo

et al. 2024

J of Extracellular Vesicle

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Enveloped viruses pose a significant threat to human health, as evidenced by the recent COVID‐19 pandemic. Although current vaccine strategies have proven effective in preventing viral infections, the development of innovative vaccine technologies is crucial to fortify our defences against future pandemics. In this study, we introduce a novel platform called cell‐engineered virus‐mimetic nanovesicles (VNVs) and demonstrate their potential as a vaccine for targeting enveloped viruses. VNVs are generated by extruding plasma membrane‐derived blebs through nanoscale membrane filters. These VNVs closely resemble enveloped viruses and extracellular vesicles (EVs) in size and morphology, being densely packed with plasma membrane contents and devoid of materials from other membranous organelles. Due to these properties, VNVs express viral membrane antigens more extensively and homogeneously than EVs expressing the same antigen. In this study, we produced severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) VNVs expressing the SARS‐CoV‐2 Spike glycoprotein (S) on their surfaces and assessed their preclinical efficacy as a COVID‐19 vaccine in experimental animals. The administration of VNVs successfully stimulated the production of S‐specific antibodies both systemically and locally, and immune cells isolated from vaccinated mice displayed cytokine responses to S stimulation.

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Heterogeneous Subcellular Origin of Exosome-Mimetic Nanovesicles Engineered from Cells

Cited by 11 publications

References 39 publications

Unraveling the surface marker signature of cell-derived vesicles via proteome analysis and nanoparticle flow cytometry

Unraveling the surface marker signature of cell-derived vesicles via proteome analysis and nanoparticle flow cytometry

Quantitative imaging of vesicle–protein interactions reveals close cooperation among proteins

Cell‐engineered virus‐mimetic nanovesicles for vaccination against enveloped viruses

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