A simple method to label vesicles for visualization and in vivo tracking

Background: Labelling of vesicles for their visualization in vitro or in vivo, involves the use of fluorescent dyes. To obtain labelled vesicles free of unincorporated dye, purification steps are necessary. The standard method is density gradient ultracentrifugation which is not only time consuming,...

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Bibliographic Details
Published inJournal of extracellular vesicles Vol. 7; pp. 217 - 218
Main Authors Salas-Huenuleo, Edison, Polakovicova, Iva, Varas-Godoy, Manuel, Lobos-González, Lorena, Bejarano, Julian, Corvalán, Alejandro, Kogan, Marcelo J
Format Journal Article
LanguageEnglish
Published Abingdon John Wiley & Sons, Inc 01.01.2018
Subjects
Cell culture
Dyes
Fluorescence spectroscopy
Fluorescent indicators
Labeling
Light scattering
Liposomes
Methods
Transmission electron microscopy
Ultracentrifugation
Vesicles
Visualization
Zeta potential
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Summary:Background: Labelling of vesicles for their visualization in vitro or in vivo, involves the use of fluorescent dyes. To obtain labelled vesicles free of unincorporated dye, purification steps are necessary. The standard method is density gradient ultracentrifugation which is not only time consuming, but counts with high sample loss and requires expensive equipment. Here, we established a simple and fast method to acquire labelled vesicles for in vivo tracking and visualization. Methods: Extracellular vesicles (EVs) from cell culture supernatant, synthetic exoliposomes (ELIP) and thermosensitive liposomes (TLIP) were obtained and characterized by nanosight, transmission electron microscopy and zeta potential determinations. Subsequently, the nanostructures were incubated with DiR fluorophore. DiR-labelled vesicles were purified by two different methods, using optiprep density gradient ultracentrifugation or commercial exo-spin columns. The eluates obtained from columns and density gradient fractions were characterized by nanosight, dynamic light scattering, zeta potential, protein content, fluorescence spectroscopy and imaging. Obtained yields of labelled vesicles were compared. Next, purified labelled EVs, ELIP and TLIP were administrated via tail vein injection in mice with an equivalent number of particles and visualized at 48 h using In Vivo imaging system. Organs were extracted, visualized and fluorescence intensity was measured. All animal procedures and care were approved by implicated ethic committees. Results: Using exo-spin column, DiR labelled EVs, ELIP and TLIP were obtained. Profound characterization of every step, column and eluate during the process showed that free DiR was not present in labelled samples. Next, we established that the use of column provides reproducible results with low sample loss. The working time is less than 10 min, significantly less than up to 24 h of the density gradient method. Finally, we used these labelled vesicles to determine and compare their biodistribution in organs of mice. Summary/Conclusion: We compared two methods and established the use of exo-spin column as a tool to obtain labelled vesicles in a reproducible, simple and faster manner, with no need of expensive equipment.
ISSN:2001-3078