Efficient Labeling of Vesicles with Lipophilic Fluorescent Dyes via the Salt-Change Method

Cha, Minkwon; Jeong, Sang Hyeok; Bae, Seoyoon; Park, Jun Hyuk; Baeg, Yoonjin; Han, Dong Woo; Kim, Sang Soo; Shin, Jaehyeon; Park, Jeong Eun; Oh, Seung Wook; Gho, Yong Song; Shon, Min Ju

doi:10.1021/acs.analchem.2c05166

Cited by 15 publications

(5 citation statements)

References 23 publications

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“…The three CDVs (NK‐, U937‐, and UCMSC‐CDVs) were next labelled with DiI (DiIC 18 (3)), a lipophilic fluorescent dye that stains lipid membranes ( Video S1 ) (Cha et al, 2023 ). Finally, the labelled CDVs were introduced to the surface preparations of ICAM‐1.…”

Section: Resultsmentioning

confidence: 99%

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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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Section: Resultsmentioning

confidence: 99%

“…How to cite this article: Cha, M., Jeong, S. H., Jung, J., Baeg, Y., Park, S. -S., Bae, S., Lim, C. S., Park, J. H., Lee, J. -O., Gho, Y. S., Oh, S. W., & Shon, M. J. (2023).…”

Section: S U P P O R T I N G I N F O R M At I O Nmentioning

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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“…Various lipophilic tracer dyes, such as PKH67 and DiR/DiL/DiD, cover a wide range of emission wavelengths for better in vivo penetration through tissues [ 392 , 393 , 394 ]. Although these dyes are capable of rapid and efficient labeling of EVs without altering the EV-producing cells, they tend to aggregate into micelles similar in size to EVs and also potentially label non-EV particles [ 395 ]. In addition, the non-covalent bond promotes a high risk of transferring the EV-bound dye to the plasma membrane, as well as altering the properties of EVs, thereby affecting the biodistribution of EVs in vivo [ 394 , 396 ].…”

Section: Pharmacology Of Extracellular Vesiclesmentioning

confidence: 99%

Unraveling the Multifaceted Roles of Extracellular Vesicles: Insights into Biology, Pharmacology, and Pharmaceutical Applications for Drug Delivery

Al-Jipouri,

Eritja,

Bozic

2023

IJMS

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Extracellular vesicles (EVs) are nanoparticles released from various cell types that have emerged as powerful new therapeutic option for a variety of diseases. EVs are involved in the transmission of biological signals between cells and in the regulation of a variety of biological processes, highlighting them as potential novel targets/platforms for therapeutics intervention and/or delivery. Therefore, it is necessary to investigate new aspects of EVs’ biogenesis, biodistribution, metabolism, and excretion as well as safety/compatibility of both unmodified and engineered EVs upon administration in different pharmaceutical dosage forms and delivery systems. In this review, we summarize the current knowledge of essential physiological and pathological roles of EVs in different organs and organ systems. We provide an overview regarding application of EVs as therapeutic targets, therapeutics, and drug delivery platforms. We also explore various approaches implemented over the years to improve the dosage of specific EV products for different administration routes.

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“…Meanwhile, the uneven staining of lipids under nonoptimal and inconsistent conditions lead to inaccurate assessments . Additional factors contributing to false-positive analysis of exosome tracking include the polarity of the solvent used for dye dissolution and the dye aggregation in salt-containing solutions that are commonly used to suspend exosomes . Moreover, the binding or incorporation of lipophilic dyes potentially alters the membrane properties, which, in turn, triggers signal transduction within cells .…”

Section: Introductionmentioning

confidence: 99%

Fluorescent Chiral Quantum Dots to Unveil Origin-Dependent Exosome Uptake and Cargo Release

Kim,

Zhu,

Zhang

et al. 2024

ACS Appl. Bio Mater.

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Exosomes are promising nanocarriers for drug delivery. Yet, it is challenging to apply exosomes in clinical use due to the limited understanding of their physiological functions. While cellular uptake of exosomes is generally known through endocytosis and/or membrane fusion, the mechanisms of origin-dependent cellular uptake and subsequent cargo release of exosomes into recipient cells are still unclear. Herein, we investigated the intricate mechanisms of exosome entry into recipient cells and intracellular cargo release. In this study, we utilized chiral graphene quantum dots (GQDs) as representatives of exosomal cargo, taking advantage of the superior permeability of chiral GQDs into lipid membranes as well as their excellent optical properties for tracking analysis. We observed that the preferential cellular uptake of exosomes derived from the same cell-of-origin (intraspecies exosomes) is higher than that of exosomes derived from different cell-oforigin (cross-species exosomes). This uptake enhancement was attributed to receptor−ligand interaction-mediated endocytosis, as we identified the expression of specific ligands on exosomes that favorably interact with their parental cells and confirmed the higher lysosomal entrapment of intraspecies exosomes (intraspecies endocytic uptake). On the other hand, we found that the uptake of cross-species exosomes primarily occurred through membrane fusion, followed by direct cargo release into the cytosol (cross-species direct fusion uptake). We revealed the underlying mechanisms involved in the cellular uptake and subsequent cargo release of exosomes depending on their cell-of-origin and recipient cell types. Overall, this study envisions valuable insights into further advancements in effective drug delivery using exosomes, as well as a comprehensive understanding of cellular communication, including disease pathogenesis.

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Efficient Labeling of Vesicles with Lipophilic Fluorescent Dyes via the Salt-Change Method

Cited by 15 publications

References 23 publications

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

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

Unraveling the Multifaceted Roles of Extracellular Vesicles: Insights into Biology, Pharmacology, and Pharmaceutical Applications for Drug Delivery

Fluorescent Chiral Quantum Dots to Unveil Origin-Dependent Exosome Uptake and Cargo Release

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