Genetically Engineered Extracellular Vesicles Harboring Transmembrane Scaffolds Exhibit Differences in Their Size, Expression Levels of Specific Surface Markers and Cell-Uptake
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cells and Reagents
2.2. Vectors and Fusion Genes
2.3. Cell Culture and Transfection
2.4. Isolation of EVs
2.5. Live Cell Microscopy
2.6. On-Chip Capture and Characterization of EVs
2.7. Fluorescein-Labeling of EVs
2.8. Cellular Uptake Assay
2.9. Flow Cytometry Analysis
2.10. Data Analysis and Statistics
3. Results
3.1. Engineering Strategy and Experimental Design
3.2. Transmembrane Scaffolds Incorporate into Biogenic Sites of EVs in Living Human Cells
3.3. Each Transmembrane Scaffold Preferentially Incorporates into Different Subtypes of EVs with Various Efficiency
3.4. Surface Engineering Using Transmembrane Scaffolds Leads to an Increase in EV Size
3.5. CD63 Scaffold Decreases the Expression Levels of CD81 Markers on Modified EVs
3.6. The VSVG Scaffold Increases the Cellular Uptake of EVs
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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CD9+ EVs | CD63+ EVs | CD81+ EVs | |
---|---|---|---|
CD9-GFP-EVs | 47.2% ± 0.8% | 19.7% ± 5.1% | 32.4% ± 4.0% |
CD63-GFP-EVs | 6.7% ± 0.1% | 58.7% ± 1.5% | 7.3% ± 0.5% |
CD81-GFP-EVs | 12.3% ± 6.3% | 4.9% ± 0.6% | 16.6% ± 2.0% |
VSVG-GFP-EVs | 8.3% ± 0.2% | 4.7% ± 0.8% | 8.5% ± 0.1% |
Name of Scaffold | Unmodified EVs (nm, size) | Modified EVs (nm, size) |
---|---|---|
CD9-GFP | 58.40 ± 8.07 | 66.13 ± 16.38 |
CD63-GFP | 62.07 ± 14.20 | |
CD81-GFP | 86.02 ± 34.18 | |
VSVG-GFP | 65.42 ± 15.62 |
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Zhang, J.; Brown, A.; Johnson, B.; Diebold, D.; Asano, K.; Marriott, G.; Lu, B. Genetically Engineered Extracellular Vesicles Harboring Transmembrane Scaffolds Exhibit Differences in Their Size, Expression Levels of Specific Surface Markers and Cell-Uptake. Pharmaceutics 2022, 14, 2564. https://doi.org/10.3390/pharmaceutics14122564
Zhang J, Brown A, Johnson B, Diebold D, Asano K, Marriott G, Lu B. Genetically Engineered Extracellular Vesicles Harboring Transmembrane Scaffolds Exhibit Differences in Their Size, Expression Levels of Specific Surface Markers and Cell-Uptake. Pharmaceutics. 2022; 14(12):2564. https://doi.org/10.3390/pharmaceutics14122564
Chicago/Turabian StyleZhang, Jiayi, Annie Brown, Brendan Johnson, David Diebold, Kyle Asano, Gerard Marriott, and Biao Lu. 2022. "Genetically Engineered Extracellular Vesicles Harboring Transmembrane Scaffolds Exhibit Differences in Their Size, Expression Levels of Specific Surface Markers and Cell-Uptake" Pharmaceutics 14, no. 12: 2564. https://doi.org/10.3390/pharmaceutics14122564