Complex Size and Surface Charge Determine Nucleic Acid Transfer by Fusogenic Liposomes
Abstract
:1. Introduction
2. Results
2.1. Neutralization of Nucleic Acids Is Essential for Their Efficient Transfer by Fusogenic Liposomes
2.2. Effect of the Liposomal Formulation on Nucleic Acid Transfer Efficiency
2.3. Quantification of mRNA Transfer on Single Cell Level
2.4. mRNA Delivery Into Primary Cortical Neurons and Freshly Isolated Neural Tissue
3. Discussion
4. Materials and Methods
4.1. Cell Culture
4.2. Preparation of Liposomes
4.3. Preparation of Neutralization Reagents (NR)
4.4. Preparation of Nucleic Acid Containing Fusogenic Liposomes
4.5. Quantification of Knock Down Efficiency of Transiently Expressed eGFP
4.6. mRNA Transfer Into Isolated Cortical Brain Tissue
4.7. Zeta Potential and Size Distribution Measurements
4.8. Light Microscopy
4.9. Flow Cytometry
4.10. Quantitative Analysis of Membrane Fusion Events
4.11. RNA Isolation and cDNA Synthesis
4.12. RT-PCR Assays
4.13. Statistical Analysis
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
References
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NA | NA Length (Nucleotides) | Molar Ratio of NR/NA | Nucleotides/NR |
---|---|---|---|
siRNA | 20–30 | 5/1 | 4–6/1 |
mRNA | 1000 | 50/1 | 20/1 |
pDNA | 4700 | 450/1 | 16/1 |
NR | Size [kDa] | NR/NA [mol/mol] |
---|---|---|
PEI branched | 25 | 0.4/1 |
chitosan | 100 | 0.8/1 |
PEI linear | 10 | 10/1 |
protamine | 3–6 | 50/1 |
pH of NR Buffer | Zeta Potential ± s.d. (mV) | Size ± s.d. (nm) | eGFP Transfer Efficiency ± s.d. (%) | eGFP Fluorescence Intensity ± s.d. (counts) |
---|---|---|---|---|
11 | 60.5 ± 0.3 | 141.5 ± 36 | 54 ± 7 | 475 ± 117 |
8 | 54.5 ± 3 | 344.8± 20 | 46 ± 3 | 230 ± 88 |
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Hoffmann, M.; Hersch, N.; Gerlach, S.; Dreissen, G.; Springer, R.; Merkel, R.; Csiszár, A.; Hoffmann, B. Complex Size and Surface Charge Determine Nucleic Acid Transfer by Fusogenic Liposomes. Int. J. Mol. Sci. 2020, 21, 2244. https://doi.org/10.3390/ijms21062244
Hoffmann M, Hersch N, Gerlach S, Dreissen G, Springer R, Merkel R, Csiszár A, Hoffmann B. Complex Size and Surface Charge Determine Nucleic Acid Transfer by Fusogenic Liposomes. International Journal of Molecular Sciences. 2020; 21(6):2244. https://doi.org/10.3390/ijms21062244
Chicago/Turabian StyleHoffmann, Marco, Nils Hersch, Sven Gerlach, Georg Dreissen, Ronald Springer, Rudolf Merkel, Agnes Csiszár, and Bernd Hoffmann. 2020. "Complex Size and Surface Charge Determine Nucleic Acid Transfer by Fusogenic Liposomes" International Journal of Molecular Sciences 21, no. 6: 2244. https://doi.org/10.3390/ijms21062244
APA StyleHoffmann, M., Hersch, N., Gerlach, S., Dreissen, G., Springer, R., Merkel, R., Csiszár, A., & Hoffmann, B. (2020). Complex Size and Surface Charge Determine Nucleic Acid Transfer by Fusogenic Liposomes. International Journal of Molecular Sciences, 21(6), 2244. https://doi.org/10.3390/ijms21062244