Chitosan Oleate Coated PLGA Nanoparticles as siRNA Drug Delivery System
Abstract
:1. Introduction
2. Materials and Methods
2.1. Materials
2.2. Methods
2.2.1. Preparation of Chitosan Oleate PLGA Nanoparticles (CS-NPs)
2.2.2. Anti–Tat/Rev siRNAs
2.2.3. siRNAs/CS-NPs Functionalization
2.2.4. Characterization of CS-NPs and SiRNAs/CS-NPs
2.2.5. Qualitative Determination of siRNA-NPs Interaction
Gel Electrophoresis
Fluorescence Titration Assay
Quantification of Primary Amine Groups
2.2.6. In Vitro Evaluation
- HEPG2 cell line
- PBMCs cell line
Internalization and Flow Cytometric Analyses on HepG2 and PBMCs
- HepG2 cells were washed twice with DPBS 1X, then trypsinized with 0.5 mL/well of Trypsin EDTA 1 × solution in HBSS (Irvine Scientific, Santa Ana, CA, USA), collected in tubes and centrifuged at 160× g for 5 min. Pellets were washed in DPBS 1 X for two times. Finally, cells were fixed with 0.4 mL/tube with IC fixation buffer (Invitrogen, Carlsbad CA, CA, USA) diluted 1:1 in cold DPBS 1× (4 °C). Samples were stored in the fridge, protected from light until FACS (fluorescence activated cell sorting) analysis.
- PBMCs were mechanically detached from the bottom of the well, collected in tubes, centrifuged at 300× g for 5 min, the supernatant discarded, and cells were washed once in cold DPBS 1× (4 °C), followed by another centrifuge step. Then, PBMCs were fixed using the same procedure described above.
Cytotoxicity Test on Human CD14+ Monocytes from Peripheral Blood (hMoCD14+-PB) and Morphological Evaluation
Characterization of Immune Responses of Human PBMCs
3. Results and Discussion
3.1. Characterization of CS-NPs and siRNA/CS-NPs
3.2. Qualitative Determination of siRNA-NPs Interaction
3.2.1. Gel Electrophoresis
3.2.2. Fluorescence Titration Assay
3.2.3. Quantification of Primary Amine Groups
3.3. Internalization and Flow Cytometric Analyses on HepG2 and PBMCs
3.4. Cytotoxicity Test on Human CD14+ Monocytes from Peripheral Blood
3.5. Characterization of Inflammatory Response of Human PBMCs on CS-NPs
4. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Before Dialysis (pH 4.5) | After Dialysis (pH 7.4) | |
---|---|---|
PS (nm) | 236.7 ± 4.0 | 333.6 ± 11.5 |
PI | 0.18 ± 0.03 | 0.14 ± 0.04 |
Zeta potential, ζ (mV) | 36.49 ± 8.55 | 15.95 ± 3.40 |
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Miele, D.; Xia, X.; Catenacci, L.; Sorrenti, M.; Rossi, S.; Sandri, G.; Ferrari, F.; Rossi, J.J.; Bonferoni, M.C. Chitosan Oleate Coated PLGA Nanoparticles as siRNA Drug Delivery System. Pharmaceutics 2021, 13, 1716. https://doi.org/10.3390/pharmaceutics13101716
Miele D, Xia X, Catenacci L, Sorrenti M, Rossi S, Sandri G, Ferrari F, Rossi JJ, Bonferoni MC. Chitosan Oleate Coated PLGA Nanoparticles as siRNA Drug Delivery System. Pharmaceutics. 2021; 13(10):1716. https://doi.org/10.3390/pharmaceutics13101716
Chicago/Turabian StyleMiele, Dalila, Xin Xia, Laura Catenacci, Milena Sorrenti, Silvia Rossi, Giuseppina Sandri, Franca Ferrari, John J. Rossi, and Maria Cristina Bonferoni. 2021. "Chitosan Oleate Coated PLGA Nanoparticles as siRNA Drug Delivery System" Pharmaceutics 13, no. 10: 1716. https://doi.org/10.3390/pharmaceutics13101716
APA StyleMiele, D., Xia, X., Catenacci, L., Sorrenti, M., Rossi, S., Sandri, G., Ferrari, F., Rossi, J. J., & Bonferoni, M. C. (2021). Chitosan Oleate Coated PLGA Nanoparticles as siRNA Drug Delivery System. Pharmaceutics, 13(10), 1716. https://doi.org/10.3390/pharmaceutics13101716