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Genetically Engineered Extracellular Vesicles Harboring Transmembrane Scaffolds Exhibit Differences in Their Size, Expression Levels of Specific Surface Markers and Cell-Uptake

Background: Human cell-secreted extracellular vesicles (EVs) are versatile nanomaterials suitable for disease-targeted drug delivery and therapy. Native EVs, however, usually do not interact specifically with target cells or harbor therapeutic drugs, which limits their potential for clinical applica...

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Autores principales: Zhang, Jiayi, Brown, Annie, Johnson, Brendan, Diebold, David, Asano, Kyle, Marriott, Gerard, Lu, Biao
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9783873/
https://www.ncbi.nlm.nih.gov/pubmed/36559058
http://dx.doi.org/10.3390/pharmaceutics14122564
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author Zhang, Jiayi
Brown, Annie
Johnson, Brendan
Diebold, David
Asano, Kyle
Marriott, Gerard
Lu, Biao
author_facet Zhang, Jiayi
Brown, Annie
Johnson, Brendan
Diebold, David
Asano, Kyle
Marriott, Gerard
Lu, Biao
author_sort Zhang, Jiayi
collection PubMed
description Background: Human cell-secreted extracellular vesicles (EVs) are versatile nanomaterials suitable for disease-targeted drug delivery and therapy. Native EVs, however, usually do not interact specifically with target cells or harbor therapeutic drugs, which limits their potential for clinical applications. These functions can be introduced to EVs by genetic manipulation of membrane protein scaffolds, although the efficiency of these manipulations and the impacts they have on the properties of EVs are for the most part unknown. In this study, we quantify the effects of genetic manipulations of different membrane scaffolds on the physicochemical properties, molecular profiles, and cell uptake of the EVs. Methods: Using a combination of gene fusion, molecular imaging, and immuno-based on-chip analysis, we examined the effects of various protein scaffolds, including endogenous tetraspanins (CD9, CD63, and CD81) and exogenous vesicular stomatitis virus glycoprotein (VSVG), on the efficiency of integration in EV membranes, the physicochemical properties of EVs, and EV uptake by recipient cells. Results: Fluorescence imaging and live cell monitoring showed each scaffold type was integrated into EVs either in membranes of the endocytic compartment, the plasma membrane, or both. Analysis of vesicle size revealed that the incorporation of each scaffold increased the average diameter of vesicles compared to unmodified EVs. Molecular profiling of surface markers in engineered EVs using on-chip assays showed the CD63-GFP scaffold decreased expression of CD81 on the membrane surface compared to control EVs, whereas its expression was mostly unchanged in EVs bearing CD9-, CD81-, or VSVG-GFP. The results from cell uptake studies demonstrated that VSVG-engineered EVs were taken up by recipient cells to a greater degree than control EVs. Conclusion: We found that the incorporation of different molecular scaffolds in EVs altered their physicochemical properties, surface protein profiles, and cell-uptake functions. Scaffold-induced changes in the physical and functional properties of engineered EVs should therefore be considered in engineering EVs for the targeted delivery and uptake of therapeutics to diseased cells.
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spelling pubmed-97838732022-12-24 Genetically Engineered Extracellular Vesicles Harboring Transmembrane Scaffolds Exhibit Differences in Their Size, Expression Levels of Specific Surface Markers and Cell-Uptake Zhang, Jiayi Brown, Annie Johnson, Brendan Diebold, David Asano, Kyle Marriott, Gerard Lu, Biao Pharmaceutics Article Background: Human cell-secreted extracellular vesicles (EVs) are versatile nanomaterials suitable for disease-targeted drug delivery and therapy. Native EVs, however, usually do not interact specifically with target cells or harbor therapeutic drugs, which limits their potential for clinical applications. These functions can be introduced to EVs by genetic manipulation of membrane protein scaffolds, although the efficiency of these manipulations and the impacts they have on the properties of EVs are for the most part unknown. In this study, we quantify the effects of genetic manipulations of different membrane scaffolds on the physicochemical properties, molecular profiles, and cell uptake of the EVs. Methods: Using a combination of gene fusion, molecular imaging, and immuno-based on-chip analysis, we examined the effects of various protein scaffolds, including endogenous tetraspanins (CD9, CD63, and CD81) and exogenous vesicular stomatitis virus glycoprotein (VSVG), on the efficiency of integration in EV membranes, the physicochemical properties of EVs, and EV uptake by recipient cells. Results: Fluorescence imaging and live cell monitoring showed each scaffold type was integrated into EVs either in membranes of the endocytic compartment, the plasma membrane, or both. Analysis of vesicle size revealed that the incorporation of each scaffold increased the average diameter of vesicles compared to unmodified EVs. Molecular profiling of surface markers in engineered EVs using on-chip assays showed the CD63-GFP scaffold decreased expression of CD81 on the membrane surface compared to control EVs, whereas its expression was mostly unchanged in EVs bearing CD9-, CD81-, or VSVG-GFP. The results from cell uptake studies demonstrated that VSVG-engineered EVs were taken up by recipient cells to a greater degree than control EVs. Conclusion: We found that the incorporation of different molecular scaffolds in EVs altered their physicochemical properties, surface protein profiles, and cell-uptake functions. Scaffold-induced changes in the physical and functional properties of engineered EVs should therefore be considered in engineering EVs for the targeted delivery and uptake of therapeutics to diseased cells. MDPI 2022-11-23 /pmc/articles/PMC9783873/ /pubmed/36559058 http://dx.doi.org/10.3390/pharmaceutics14122564 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Zhang, Jiayi
Brown, Annie
Johnson, Brendan
Diebold, David
Asano, Kyle
Marriott, Gerard
Lu, Biao
Genetically Engineered Extracellular Vesicles Harboring Transmembrane Scaffolds Exhibit Differences in Their Size, Expression Levels of Specific Surface Markers and Cell-Uptake
title Genetically Engineered Extracellular Vesicles Harboring Transmembrane Scaffolds Exhibit Differences in Their Size, Expression Levels of Specific Surface Markers and Cell-Uptake
title_full Genetically Engineered Extracellular Vesicles Harboring Transmembrane Scaffolds Exhibit Differences in Their Size, Expression Levels of Specific Surface Markers and Cell-Uptake
title_fullStr Genetically Engineered Extracellular Vesicles Harboring Transmembrane Scaffolds Exhibit Differences in Their Size, Expression Levels of Specific Surface Markers and Cell-Uptake
title_full_unstemmed Genetically Engineered Extracellular Vesicles Harboring Transmembrane Scaffolds Exhibit Differences in Their Size, Expression Levels of Specific Surface Markers and Cell-Uptake
title_short Genetically Engineered Extracellular Vesicles Harboring Transmembrane Scaffolds Exhibit Differences in Their Size, Expression Levels of Specific Surface Markers and Cell-Uptake
title_sort genetically engineered extracellular vesicles harboring transmembrane scaffolds exhibit differences in their size, expression levels of specific surface markers and cell-uptake
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9783873/
https://www.ncbi.nlm.nih.gov/pubmed/36559058
http://dx.doi.org/10.3390/pharmaceutics14122564
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