Stability of Erythrocyte-Derived Nanovesicles Assessed by Light Scattering and Electron Microscopy
Extracellular vesicles (EVs) are gaining increasing amounts of attention due to their potential use in diagnostics and therapy, but the poor reproducibility of the studies that have been conducted on these structures hinders their breakthrough into routine practice. We believe that a better understa...
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MDPI AG
2021-11-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/22/23/12772 |
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| author | Darja Božič Matej Hočevar Matic Kisovec Manca Pajnič Ljubiša Pađen Marko Jeran Apolonija Bedina Zavec Marjetka Podobnik Ksenija Kogej Aleš Iglič Veronika Kralj-Iglič |
| author_facet | Darja Božič Matej Hočevar Matic Kisovec Manca Pajnič Ljubiša Pađen Marko Jeran Apolonija Bedina Zavec Marjetka Podobnik Ksenija Kogej Aleš Iglič Veronika Kralj-Iglič |
| author_sort | Darja Božič |
| collection | DOAJ |
| description | Extracellular vesicles (EVs) are gaining increasing amounts of attention due to their potential use in diagnostics and therapy, but the poor reproducibility of the studies that have been conducted on these structures hinders their breakthrough into routine practice. We believe that a better understanding of EVs stability and methods to control their integrity are the key to resolving this issue. In this work, erythrocyte EVs (hbEVs) were isolated by centrifugation from suspensions of human erythrocytes that had been aged in vitro. The isolate was characterised by scanning (SEM) and cryo-transmission electron microscopy (cryo-TEM), flow cytometry (FCM), dynamic/static light scattering (LS), protein electrophoresis, and UV-V spectrometry. The hbEVs were exposed to various conditions (pH (4–10), osmolarity (50–1000 mOsm/L), temperature (15–60 °C), and surfactant Triton X-100 (10–500 μM)). Their stability was evaluated by LS by considering the hydrodynamic radius (<i>R</i><sub>h</sub>), intensity of scattered light (<i>I</i>), and the shape parameter (<i>ρ</i>). The morphology of the hbEVs that had been stored in phosphate-buffered saline with citrate (PBS–citrate) at 4 °C remained consistent for more than 6 months. A change in the media properties (50–1000 mOsm/L, pH 4–10) had no significant effect on the <i>R</i><sub>h</sub> (=100–130 nm). At pH values below 6 and above 8, at temperatures above 45 °C, and in the presence of Triton X-100, hbEVs degradation was indicated by a decrease in <i>I</i> of more than 20%. Due to the simple preparation, homogeneous morphology, and stability of hbEVs under a wide range of conditions, they are considered to be a suitable option for EV reference material. |
| first_indexed | 2024-03-10T04:53:22Z |
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| id | doaj.art-7e3a77d7e5d44a098451c03bcfd8bd4f |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-10T04:53:22Z |
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| series | International Journal of Molecular Sciences |
| spelling | doaj.art-7e3a77d7e5d44a098451c03bcfd8bd4f2023-11-23T02:27:47ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672021-11-0122231277210.3390/ijms222312772Stability of Erythrocyte-Derived Nanovesicles Assessed by Light Scattering and Electron MicroscopyDarja Božič0Matej Hočevar1Matic Kisovec2Manca Pajnič3Ljubiša Pađen4Marko Jeran5Apolonija Bedina Zavec6Marjetka Podobnik7Ksenija Kogej8Aleš Iglič9Veronika Kralj-Iglič10Laboratory of Clinical Biophysics, Faculty of Health Sciences, University of Ljubljana, SI-1000 Ljubljana, SloveniaDepartment of Physics and Chemistry of Materials, Institute of Metals and Technology, SI-1000 Ljubljana, SloveniaDepartment of Molecular Biology and Nanobiotechnology, National Institute of Chemistry, SI-1000 Ljubljana, SloveniaLaboratory of Clinical Biophysics, Faculty of Health Sciences, University of Ljubljana, SI-1000 Ljubljana, SloveniaLaboratory of Clinical Biophysics, Faculty of Health Sciences, University of Ljubljana, SI-1000 Ljubljana, SloveniaLaboratory of Clinical Biophysics, Faculty of Health Sciences, University of Ljubljana, SI-1000 Ljubljana, SloveniaDepartment of Molecular Biology and Nanobiotechnology, National Institute of Chemistry, SI-1000 Ljubljana, SloveniaDepartment of Molecular Biology and Nanobiotechnology, National Institute of Chemistry, SI-1000 Ljubljana, SloveniaFaculty of Chemistry and Chemical Technology, University of Ljubljana, SI-1000 Ljubljana, SloveniaLaboratory of Physics, Faculty of Electrical Engineering, University of Ljubljana, SI-1000 Ljubljana, SloveniaLaboratory of Clinical Biophysics, Faculty of Health Sciences, University of Ljubljana, SI-1000 Ljubljana, SloveniaExtracellular vesicles (EVs) are gaining increasing amounts of attention due to their potential use in diagnostics and therapy, but the poor reproducibility of the studies that have been conducted on these structures hinders their breakthrough into routine practice. We believe that a better understanding of EVs stability and methods to control their integrity are the key to resolving this issue. In this work, erythrocyte EVs (hbEVs) were isolated by centrifugation from suspensions of human erythrocytes that had been aged in vitro. The isolate was characterised by scanning (SEM) and cryo-transmission electron microscopy (cryo-TEM), flow cytometry (FCM), dynamic/static light scattering (LS), protein electrophoresis, and UV-V spectrometry. The hbEVs were exposed to various conditions (pH (4–10), osmolarity (50–1000 mOsm/L), temperature (15–60 °C), and surfactant Triton X-100 (10–500 μM)). Their stability was evaluated by LS by considering the hydrodynamic radius (<i>R</i><sub>h</sub>), intensity of scattered light (<i>I</i>), and the shape parameter (<i>ρ</i>). The morphology of the hbEVs that had been stored in phosphate-buffered saline with citrate (PBS–citrate) at 4 °C remained consistent for more than 6 months. A change in the media properties (50–1000 mOsm/L, pH 4–10) had no significant effect on the <i>R</i><sub>h</sub> (=100–130 nm). At pH values below 6 and above 8, at temperatures above 45 °C, and in the presence of Triton X-100, hbEVs degradation was indicated by a decrease in <i>I</i> of more than 20%. Due to the simple preparation, homogeneous morphology, and stability of hbEVs under a wide range of conditions, they are considered to be a suitable option for EV reference material.https://www.mdpi.com/1422-0067/22/23/12772light scatteringvesicle characterizationvesicle stabilityextracellular vesicle reference materialnanovesiclescellular vesicles |
| spellingShingle | Darja Božič Matej Hočevar Matic Kisovec Manca Pajnič Ljubiša Pađen Marko Jeran Apolonija Bedina Zavec Marjetka Podobnik Ksenija Kogej Aleš Iglič Veronika Kralj-Iglič Stability of Erythrocyte-Derived Nanovesicles Assessed by Light Scattering and Electron Microscopy International Journal of Molecular Sciences light scattering vesicle characterization vesicle stability extracellular vesicle reference material nanovesicles cellular vesicles |
| title | Stability of Erythrocyte-Derived Nanovesicles Assessed by Light Scattering and Electron Microscopy |
| title_full | Stability of Erythrocyte-Derived Nanovesicles Assessed by Light Scattering and Electron Microscopy |
| title_fullStr | Stability of Erythrocyte-Derived Nanovesicles Assessed by Light Scattering and Electron Microscopy |
| title_full_unstemmed | Stability of Erythrocyte-Derived Nanovesicles Assessed by Light Scattering and Electron Microscopy |
| title_short | Stability of Erythrocyte-Derived Nanovesicles Assessed by Light Scattering and Electron Microscopy |
| title_sort | stability of erythrocyte derived nanovesicles assessed by light scattering and electron microscopy |
| topic | light scattering vesicle characterization vesicle stability extracellular vesicle reference material nanovesicles cellular vesicles |
| url | https://www.mdpi.com/1422-0067/22/23/12772 |
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