Efficient Delivery of SARS-CoV-2 Plasmid DNA in HEK-293T Cells Using Chitosan Nanoparticles
Abstract
:1. Introduction
2. Results
2.1. Construction of DNA Plasmid pIRES2-eGFP-Spike
2.2. Retardation and Release Assay of CNPs-pDNA Polyplexes
2.3. DNase I Protection Assay of CNPs-pDNA Polyplexes
2.4. Cell Viability Assay by MTT
2.5. Characterization of CNPs-pDNA Polyplexes
2.6. Transfection of HEK-293T Cells
2.7. Spike mRNA Expression in HEK-293T Cells by RT-qPCR
2.8. Verification of Spike Protein Expression in HEK-293T Cells by Western Blot
3. Discussion
4. Materials and Methods
4.1. Construction of DNA Plasmid pIRES2-eGFP-Spike
4.1.1. Synthesis of Spike cDNA by RT-PCR from Positive Samples
4.1.2. Amplification of the Spike Sequence
4.1.3. Cloning of the Spike Sequence into pIRES2-eGFP
4.2. Synthesis of Chitosan Nanoparticles and Preparation of pDNA Polyplexes
4.3. Retardation and Release Assay of pDNA Polyplexes
4.4. DNase I Protection Assay of CNPs-pDNA Polyplexes
4.5. Cell Viability Assay by MTT
4.6. Characterization of CNPs-pDNA Polyplexes
4.6.1. Spectroscopic Measurement by UV-Vis
4.6.2. Chemical Composition by Fourier-Transform Infrared Spectroscopy (FT-IR)
4.6.3. Size and Morphology by Transmission Electron Microscopy (TEM)
4.6.4. Size, PDI, and Zeta Potential by Dynamic Light Scattering (DLS)
4.7. Transfection of HEK-293T Cells
4.7.1. Spike mRNA Expression in HEK-293T Cells by RT-qPCR
4.7.2. Spike mRNA Expression in HEK-293T Cells by Western Blot
4.8. Statistical Analysis
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Characterization | CNPs | CNPs-pDNA |
---|---|---|
TEM diameter (nm) | 11.1 ± 5.2 | 159.0 ± 33.1 |
Hydrodynamic diameter (nm) | 397.6 ± 51.6 | 422.1 ± 12.4 |
PDI | 0.6 ± 0.1 | 0.6 ± 0.1 |
Zeta potential (mV) | 20.4 ± 0.6 | 19.7 ± 0.3 |
Target Sequence (pb) | Primer | Sequence 5′-3′ | Cycling Conditions (40 Cycles) |
---|---|---|---|
Spike F1 (1974 bp) | Forward F1 | AGGGGTACTGCTGTTATGTC | 98 °C—15 s 56 °C—15 s 72 °C—120 s |
Reverse F1 | ACACCCTGATAAAGAACAGC | ||
Spike F2 (2208 bp) | Forward F2 | TCCAACAATTTGGCAGAGAC | 98 °C—15 s 57 °C—15 s 72 °C—120 s |
Reverse F2 | TTGATTTCACCTTGCTTCAAAGTTAC | ||
Spike F1 with NheI site (1850 bp) (a) | Forward S-RE | TGTTCTTGCTAGCAACTAAACGAAC | 98 °C—15 s 58 °C—20 s 72 °C—120 s |
Reverse F1 | ACACCCTGATAAAGAACAGC | ||
Spike F2 with SacI site (2153 bp) (b) | Forward F2 | TCCAACAATTTGGCAGAGAC | 98 °C—15 s 58 °C—20 s 72 °C—120 s |
Reverse S-ER | ATCCATGAGCTCGTTTATGTGTAATG | ||
Sequencing (397 bp) | Forward CMV | GCCCAGTACATGACCTTATGGG | 98 °C—15 s 60 °C—15 s 72 °C—20 s |
Reverse CMV | GCCCAGTACATGACCTTATGGG | ||
qPCR Spike (160 bp) | Forward qPCR-Spike | TAGGGCGTGATCTCCCTCAG | 98 °C—15 s 63 °C—17 s 72 °C—15 s |
Reverse qPCR-Spike | TAAGCTGCAGCACCAGCTGT | ||
qPCR GAPDH (146 bp) | Forward qPCR-GAPDH | AGGTCGGAGTCAACGGATTT | 98 °C—15 s 63 °C—17 s 72 °C—15 s |
Reverse qPCR-GAPDH | ATGGGTGGAATCATATTGGAAC |
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Mendoza-Guevara, C.C.; Martinez-Escobar, A.; Ramos-Godínez, M.d.P.; Muñoz-Medina, J.E.; Ramon-Gallegos, E. Efficient Delivery of SARS-CoV-2 Plasmid DNA in HEK-293T Cells Using Chitosan Nanoparticles. Pharmaceuticals 2025, 18, 683. https://doi.org/10.3390/ph18050683
Mendoza-Guevara CC, Martinez-Escobar A, Ramos-Godínez MdP, Muñoz-Medina JE, Ramon-Gallegos E. Efficient Delivery of SARS-CoV-2 Plasmid DNA in HEK-293T Cells Using Chitosan Nanoparticles. Pharmaceuticals. 2025; 18(5):683. https://doi.org/10.3390/ph18050683
Chicago/Turabian StyleMendoza-Guevara, Citlali Cecilia, Alejandro Martinez-Escobar, María del Pilar Ramos-Godínez, José Esteban Muñoz-Medina, and Eva Ramon-Gallegos. 2025. "Efficient Delivery of SARS-CoV-2 Plasmid DNA in HEK-293T Cells Using Chitosan Nanoparticles" Pharmaceuticals 18, no. 5: 683. https://doi.org/10.3390/ph18050683
APA StyleMendoza-Guevara, C. C., Martinez-Escobar, A., Ramos-Godínez, M. d. P., Muñoz-Medina, J. E., & Ramon-Gallegos, E. (2025). Efficient Delivery of SARS-CoV-2 Plasmid DNA in HEK-293T Cells Using Chitosan Nanoparticles. Pharmaceuticals, 18(5), 683. https://doi.org/10.3390/ph18050683