Sulfonated Amphiphilic Poly(α)glutamate Amine—A Potential siRNA Nanocarrier for the Treatment of Both Chemo-Sensitive and Chemo-Resistant Glioblastoma Tumors
Abstract
:1. Introduction
2. Materials and Methods
2.1. Materials
2.2. Cell Culture
2.2.1. Establishment of mCherry and iRFP Stably-Expressing U251 Cells
2.2.2. Establishment of mCherry U251 Chemo-Resistant Cell Line (U251 TMZ-R)
2.3. IC50 Determination Assay
2.4. RNA Isolation and Quantitative Real-Time RT-PCR (qRT-PCR)
2.5. Animal Models
Animals Ethics Statement
2.6. Frozen OCT Tissue Fixation
2.7. Immunostaining
2.8. Survival Analysis Based on TCGA Data
2.9. Synthesis of APA-Sulfonate (APAS)
2.10. Multi-Angle Static Light Scattering (MALS)
2.11. Scanning Electron Microscope (SEM)
2.12. Dynamic Light Scattering (DLS) and Phase Analysis Light Scattering (PALS)
2.13. Elemental Analysis (EDS)
2.14. Proliferation Assay
2.15. Multicellular Tumor Spheroids (MCTS)
2.16. SELP Expression in 3D vs. 2D Cell Culture
2.17. Infra-Red Spectroscopy
2.18. Electrophoretic Shift Assay (EMSA)
2.19. Western Blot
2.20. H-Nuclear Magnetic Resonance (NMR)
2.21. Internalization of APA/APAS:Cy5-siRNA Polyplexes into U251 Cells (2D)
2.22. Statistical Analysis
3. Results and Discussion
3.1. TMZ-Induced Resistance in U251 GB Cells Enhances MGMT mRNA Levels but Does Not Alter Plk1 mRNA Levels
3.2. APA:siPlk1 Complexes Efficiently Silence Plk1 Expression in Both U251 and U251 TMZ-R Cells
3.3. Treatment with APA:siPlk1 Polyplexes Reduces the Viability of Both U251 Cells and U251 TMZ-R Clone
3.4. SELP Is Expressed on the Membranes of GB Cells and Represents a Suitable Candidate for Active Targeting for the Delivery of siRNA Polyplexes
3.5. Sulfonation of Amphiphilic Poly(α)glutamate Amine (APA)
3.6. APAS forms Active Complexes with siRNA and Enables Silencing of GB-Relevant Genes
3.7. Sulfonate Modification Facilitated Internalization of Cy5-siRNA into U251 and U251 TMZ-R Spheroids
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Krivitsky, A.; Pozzi, S.; Yeini, E.; Israeli Dangoor, S.; Zur, T.; Golan, S.; Krivitsky, V.; Albeck, N.; Pisarevsky, E.; Ofek, P.; et al. Sulfonated Amphiphilic Poly(α)glutamate Amine—A Potential siRNA Nanocarrier for the Treatment of Both Chemo-Sensitive and Chemo-Resistant Glioblastoma Tumors. Pharmaceutics 2021, 13, 2199. https://doi.org/10.3390/pharmaceutics13122199
Krivitsky A, Pozzi S, Yeini E, Israeli Dangoor S, Zur T, Golan S, Krivitsky V, Albeck N, Pisarevsky E, Ofek P, et al. Sulfonated Amphiphilic Poly(α)glutamate Amine—A Potential siRNA Nanocarrier for the Treatment of Both Chemo-Sensitive and Chemo-Resistant Glioblastoma Tumors. Pharmaceutics. 2021; 13(12):2199. https://doi.org/10.3390/pharmaceutics13122199
Chicago/Turabian StyleKrivitsky, Adva, Sabina Pozzi, Eilam Yeini, Sahar Israeli Dangoor, Tal Zur, Sapir Golan, Vadim Krivitsky, Nitzan Albeck, Evgeny Pisarevsky, Paula Ofek, and et al. 2021. "Sulfonated Amphiphilic Poly(α)glutamate Amine—A Potential siRNA Nanocarrier for the Treatment of Both Chemo-Sensitive and Chemo-Resistant Glioblastoma Tumors" Pharmaceutics 13, no. 12: 2199. https://doi.org/10.3390/pharmaceutics13122199
APA StyleKrivitsky, A., Pozzi, S., Yeini, E., Israeli Dangoor, S., Zur, T., Golan, S., Krivitsky, V., Albeck, N., Pisarevsky, E., Ofek, P., Madi, A., & Satchi-Fainaro, R. (2021). Sulfonated Amphiphilic Poly(α)glutamate Amine—A Potential siRNA Nanocarrier for the Treatment of Both Chemo-Sensitive and Chemo-Resistant Glioblastoma Tumors. Pharmaceutics, 13(12), 2199. https://doi.org/10.3390/pharmaceutics13122199