Reprogramming Extracellular Vesicles for Protein Therapeutics Delivery
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cell Lines
2.2. Plasmids
2.3. Protein Expression in E. coli and Purification
2.4. Dynamic Light Scattering
2.5. Isolation and Purification of EVs
2.6. Western Blot Analysis
2.7. Transmission Electron Microscopy (TEM)
2.8. Protein Delivery of EV Cargoes to Target Cells
2.9. LC-MS/MS and Bioinformatics
2.10. Molecular Modeling
2.11. Estimation of Protein Numbers in EVs
2.12. Quantification and Statistical Analysis
3. Results
3.1. Improving Loading Capacity of Protein Nanocages Preserves Their Capacity to Self-Assemble
3.2. Production of Engineered EVs in Mammalian Cells
3.3. EV Production by Lentiviral Construction
3.4. Isolation and Characterization of Genetically Encoded EVs
3.5. Delivery of Protein Cargo to Target Cells
3.6. Proteome Profiling of Engineered EVs
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Sample | EV_C | EV_M | EV_p6 | EV_EPN(-) | EV_C(lenti) |
---|---|---|---|---|---|
Size determined by DLS, nm | 174 ± 45 | 176 ± 59 | 171 ± 57 | 150 ± 38 | 168 ± 45 |
EV Samples | EV_C | EV_M | EV_p6 | EV_(EPN-) | EV_C(lenti) |
---|---|---|---|---|---|
VSV-G, molecules | 450 ± 50 | 700 ± 80 | 1000 ± 90 | 850 ± 80 | 450 ± 40 |
NanoLuc, molecules | 430 ± 50 | 520 ± 50 | 110 ± 30 | 70 ± 30 | 1000 ± 80 |
EPN, molecules | 1700 ± 600 | 1500 ± 500 | 4300 ± 1000 | - | 1500 ± 400 |
Assembled nanocages | 28 ± 10 | 25 ± 8 | 71 ± 16 | - | 25 ± 7 |
NanoLuc, molecules in a single nanocage | 15 ± 6 | 21 ± 7 | 1.5 ± 0.5 | - | 40 ± 12 |
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Ovchinnikova, L.A.; Terekhov, S.S.; Ziganshin, R.H.; Bagrov, D.V.; Filimonova, I.N.; Zalevsky, A.O.; Lomakin, Y.A. Reprogramming Extracellular Vesicles for Protein Therapeutics Delivery. Pharmaceutics 2021, 13, 768. https://doi.org/10.3390/pharmaceutics13060768
Ovchinnikova LA, Terekhov SS, Ziganshin RH, Bagrov DV, Filimonova IN, Zalevsky AO, Lomakin YA. Reprogramming Extracellular Vesicles for Protein Therapeutics Delivery. Pharmaceutics. 2021; 13(6):768. https://doi.org/10.3390/pharmaceutics13060768
Chicago/Turabian StyleOvchinnikova, Leyla A., Stanislav S. Terekhov, Rustam H. Ziganshin, Dmitriy V. Bagrov, Ioanna N. Filimonova, Arthur O. Zalevsky, and Yakov A. Lomakin. 2021. "Reprogramming Extracellular Vesicles for Protein Therapeutics Delivery" Pharmaceutics 13, no. 6: 768. https://doi.org/10.3390/pharmaceutics13060768
APA StyleOvchinnikova, L. A., Terekhov, S. S., Ziganshin, R. H., Bagrov, D. V., Filimonova, I. N., Zalevsky, A. O., & Lomakin, Y. A. (2021). Reprogramming Extracellular Vesicles for Protein Therapeutics Delivery. Pharmaceutics, 13(6), 768. https://doi.org/10.3390/pharmaceutics13060768