Hyper-Branched Cationic Cyclodextrin Polymers for Improving Plasmid Transfection in 2D and 3D Spheroid Cells
Abstract
:1. Introduction
- ➢
- To study the ability of positively charged NPs to retard the pDNA-eGFP at proper N/P ratios;
- ➢
- To achieve enhanced transfection efficiency and cellular uptake of the pDNA-eGFP in complex with positively charged PPoly and to compare it with lipofectamine in 2D and 3D cell cultures;
- ➢
- To understand the safety of NPs in both 2D and 3D cell cultures;
- ➢
- To understand the differences among 2D and 3D spheroid cells in terms of the conditions for gene delivery.
2. Material and Methods
2.1. Material and Reagents
2.2. Polymer Synthesis
2.2.1. Elemental Analysis Characterization
2.2.2. Size Exclusion Chromatography
2.3. Plasmid Encoding eGFP Preparation
2.4. Polymer–DNA Complex Formation and Gel Retardation Assay
2.5. Complex Characterization
2.5.1. Complex Size and Zeta Potential Measurement
2.5.2. Scanning Electron Microscopy (SEM) Studies
2.5.3. Fourier Transformed Infrared Study
2.5.4. Determination of pDNA Encapsulation Efficiency
2.6. Cell Culture Protocol for 2D Cell Culture
Cell Culture Protocol for 3D Spheroid
2.7. Cells Cytotoxicity Assay
2.7.1. MTT (3-(4, 5-Dimethylthiazol-2-yl) 2, 5-diphenyl tetrazolium bromide) Assay
2.7.2. Lactate Dehydrogenase (LDH) Assay in 2D and 3D Cell Cultures
2.8. Transfection Assessment in 2D and 3D Cell Cultures
2.8.1. Measuring the DNA-Polymer Transfection Using Fluorescence Microscopy in 2D and 3D Cell Cultures
2.8.2. Measuring the DNA-Polymer Uptake Using Flow Cytometry Analysis
2.9. Histopathology Method for Evaluation DNA-Polymer Penetration in 3D Spheroid Cells
2.10. Hemocompability
2.11. Statistical Analysis
3. Results and Discussion
3.1. Synthesis and Characterization of Soluble Cyclodextrin-Based Polymer
3.2. Preparation and Characterization of PPoly-DNA Complexes
3.3. Encapsulation Efficacy and Hemocompatibility
3.4. Cytotoxicity Assay
3.4.1. MTT Assay in 2D Cell Culture
3.4.2. LDH Assay in 2D and 3D Cell Cultures
3.5. Cellular Uptake and Transfection Analysis in 2D Culture
3.6. Cellular Uptake and Transfection Analysis in 3D Spheroid Culture
3.7. Evaluation the Penetration Capacity of PPoly–pDNA Complexes inside the 3D Spheroid Cells on Paraffin-Embedded Cell Block
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
References
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Element | wt% |
---|---|
N | 3.28 ± 0.32 |
C | 39.26 ± 0.55 |
H | 6.56 ± 0.16 |
O | 50.9 ± 0.12 |
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Monfared, Y.K.; Mahmoudian, M.; Cecone, C.; Caldera, F.; Haiaty, S.; Heidari, H.R.; Rahbarghazi, R.; Matencio, A.; Zakeri-Milani, P.; Trotta, F. Hyper-Branched Cationic Cyclodextrin Polymers for Improving Plasmid Transfection in 2D and 3D Spheroid Cells. Pharmaceutics 2022, 14, 2690. https://doi.org/10.3390/pharmaceutics14122690
Monfared YK, Mahmoudian M, Cecone C, Caldera F, Haiaty S, Heidari HR, Rahbarghazi R, Matencio A, Zakeri-Milani P, Trotta F. Hyper-Branched Cationic Cyclodextrin Polymers for Improving Plasmid Transfection in 2D and 3D Spheroid Cells. Pharmaceutics. 2022; 14(12):2690. https://doi.org/10.3390/pharmaceutics14122690
Chicago/Turabian StyleMonfared, Yousef Khazaei, Mohammad Mahmoudian, Claudio Cecone, Fabrizio Caldera, Sanya Haiaty, Hamid Reza Heidari, Reza Rahbarghazi, Adrián Matencio, Parvin Zakeri-Milani, and Francesco Trotta. 2022. "Hyper-Branched Cationic Cyclodextrin Polymers for Improving Plasmid Transfection in 2D and 3D Spheroid Cells" Pharmaceutics 14, no. 12: 2690. https://doi.org/10.3390/pharmaceutics14122690