The Potential of Chitosan Nanoparticles as a Delivery System for the Expression of Dengue Virus' rE Gene with pEGFP-N1 Vector in HeLa Cells
Keywords:
chitosan, gene delivery, Dengue virus, rE gene, HeLa cells
Abstract
Dengue virus (DENV) infection remains a significant global health concern, necessitating the development of effective preventative strategies. DNA vaccines hold great potential for addressing viral infections, but they often face challenges in gene delivery. Chitosan nanoparticles have emerged as a biocompatible, non-toxic carrier for enhancing DNA delivery. This study evaluates chitosan nanoparticles as a delivery system for the recombinant envelope (rE) gene from the four DENV serotypes. The pEGFP-N1-rE plasmid (N1-rE) was transformed into Escherichia coli DH5α, with colony PCR, restriction analysis, and sequencing confirming accurate gene insertion. N1-rE was complexed with chitosan nanoparticles at various DNA mass ratios (wt./wt.), with optimal complex formation at a 1:0.5 ratio as confirmed by gel retardation assays. The complex exhibited stability against DNase I and fetal bovine serum and non-toxicity to HeLa cells, with 91.24% cell viability. The chitosan-DNA complex had an average size of 217.4 nm and a zeta potential of -21.9 mV. Confocal microscopy, RT-PCR, and qPCR analyses confirmed successful transfection of the N1-rE/CH complex into HeLa cells, with fluorescence detection, a 134 bp amplicon, and a 10.63-fold increase in gene expression, respectively. These results highlight the potential of chitosan nanoparticles as an effective delivery platform for targeted gene delivery, paving the way for novel therapeutic approaches against DENV infection.
References
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Cao, Y., Tan, Y. F., Wong, Y. S., Liew, M. W. J., & Venkatraman, S. (2019). Recent Advances in Chitosan-Based Carriers for Gene Delivery. Marine Drugs, 17(6), 381. https://doi.org/10.3390/md17060381
Castells-Sala, C., Martorell, J., & Balcells, M. (2017). A human plasma derived supplement preserves function of human vascular cells in absence of fetal bovine serum. Cell & Bioscience, 7(1), 41. https://doi.org/10.1186/s13578-017-0164-4
Ekowati, N., Mumpuni, A., & Muljowati, J. S. (2017). Effectiveness of Pleurotus ostreatus Extract Through Cytotoxic Test and Apoptosis Mechanism of Cervical Cancer Cells. Biosaintifika: Journal of Biology & Biology Education, 9(1), 148. https://doi.org/10.15294/biosaintifika.v9i1.7546
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Faraji, H., Ramezani, M., Sadeghnia, H. R., Abnous, K., Soltani, F., & Mashkani, B. (2017). High-level expression of a biologically active staphylokinase in Pichia pastoris. Preparative Biochemistry & Biotechnology, 47(4), 379–387. https://doi.org/10.1080/10826068.2016.1252924
Gomes-Cornélio, A. L., Rodrigues, E. M., Salles, L. P., Mestieri, L. B., Faria, G., Guerreiro-Tanomaru, J. M., & Tanomaru-Filho, M. (2017). Bioactivity of MTA Plus, Biodentine and an experimental calcium silicate-based cement on human osteoblast-like cells. International Endodontic Journal, 50(1), 39–47. https://doi.org/10.1111/iej.12589
Halstead, S. B. (2024). Three Dengue Vaccines — What Now? New England Journal of Medicine, 390(5), 464–465. https://doi.org/10.1056/NEJMe2314240
Huang, M., Fong, C.-W., Khor, E., & Lim, L.-Y. (2005). Transfection efficiency of chitosan vectors: Effect of polymer molecular weight and degree of deacetylation. Journal of Controlled Release, 106(3), 391–406. https://doi.org/10.1016/j.jconrel.2005.05.004
Irawati, A. D., & Salim, H. M. (2019). Dengue Vaccine Development at the Dengue virus serotypes. International Islamic Medical Journal, 1(1), 9–15. https://doi.org/10.33086/iimj.v1i1.1360
Januar, I., Unsunnidhal, L., Martien, R., & Kusumawati, A. (2019). In vitro evaluation of chitosan-DNA plasmid complex encoding Jembrana disease virus Env-TM protein as a vaccine candidate. Gadjah Mada University.
Kaur, M., Sharma, A., Puri, V., Aggarwal, G., Maman, P., Huanbutta, K., Nagpal, M., & Sangnim, T. (2023). Chitosan-Based Polymer Blends for Drug Delivery Systems. Polymers, 15(9), 2028. https://doi.org/10.3390/polym15092028
Kean, T., & Thanou, M. (2010). Biodegradation, biodistribution and toxicity of chitosan. Advanced Drug Delivery Reviews, 62(1), 3–11. https://doi.org/10.1016/j.addr.2009.09.004
Kementrian Kesehatan RI. (2024, June 24). Waspada DBD di Musim Kemarau. Kementrian Kesehatan RI. https://kemkes.go.id/id/waspada-dbd-di-musim-kemarau.
Kozak, M. (1989). The scanning model for translation: an update. The Journal of Cell Biology, 108(2), 229–241. https://doi.org/10.1083/jcb.108.2.229
Lai, S.-C., Huang, Y.-Y., Shu, P.-Y., Chang, S.-F., Hsieh, P.-S., Wey, J.-J., Tsai, M.-H., Ben, R.-J., Hsu, Y.-M., Fang, Y.-C., Hsiao, M.-L., & Lin, C.-C. (2019). Development of an Enzyme-Linked Immunosorbent Assay for Rapid Detection of Dengue Virus (DENV) NS1 and Differentiation of DENV Serotypes during Early Infection. Journal of Clinical Microbiology, 57(7). https://doi.org/10.1128/JCM.00221-19
Lauková, L., Konečná, B., Janovičová, Ľ., Vlková, B., & Celec, P. (2020). Deoxyribonucleases and Their Applications in Biomedicine. Biomolecules, 10(7), 1036. https://doi.org/10.3390/biom10071036
MacLaughlin, F. C., Mumper, R. J., Wang, J., Tagliaferri, J. M., Gill, I., Hinchcliffe, M., & Rolland, A. P. (1998). Chitosan and depolymerized chitosan oligomers as condensing carriers for in vivo plasmid delivery. Journal of Controlled Release, 56(1–3), 259–272. https://doi.org/10.1016/S0168-3659(98)00097-2
Mao, S., Sun, W., & Kissel, T. (2010). Chitosan-based formulations for delivery of DNA and siRNA. Advanced Drug Delivery Reviews, 62(1), 12–27. https://doi.org/10.1016/j.addr.2009.08.004
Maruti, A. A., Amalia, F., & Meiyanto, E. (2017). Synergistic Effect of Arecoline in Combination with Doxorubicin on HeLa Cervical Cancer Cells. Indonesian Journal of Cancer Chemoprevention, 6(2), 64. https://doi.org/10.14499/indonesianjcanchemoprev6iss2pp64-70
Matica, A., Menghiu, G., & Ostafe, V. (2017). Biodegradability of Chitosan Based Products. New Frontiers in Chemistry, 26(1), 75–86.
Maulina, N. T. A., Kusumawati, A., & Umniyati, S. R. (2023). Ekspresi Gen rE Virus Dengue pada Sel HeLa menggunakan Vektor pEGFP-N1. Universitas Gadjah Mada.
Németh, Z., Csóka, I., Semnani Jazani, R., Sipos, B., Haspel, H., Kozma, G., Kónya, Z., & Dobó, D. G. (2022). Quality by Design-Driven Zeta Potential Optimisation Study of Liposomes with Charge Imparting Membrane Additives. Pharmaceutics, 14(9), 1798. https://doi.org/10.3390/pharmaceutics14091798
Qiu, S., Wang, Y., Cao, B., Guo, Z., Chen, Y., & Yang, G. (2015). The suppression and promotion of DNA charge inversion by mixing counterions. Soft Matter, 11(20), 4099–4105. https://doi.org/10.1039/C5SM00326A
Rahayu, A., Saraswati, U., Supriyati, E., Kumalawati, D. A., Hermantara, R., Rovik, A., Daniwijaya, E. W., Fitriana, I., Setyawan, S., Ahmad, R. A., Wardana, D. S., Indriani, C., Utarini, A., Tantowijoyo, W., & Arguni, E. (2019). Prevalence and Distribution of Dengue Virus in Aedes aegypti in Yogyakarta City before Deployment of Wolbachia Infected Aedes aegypti. International Journal of Environmental Research and Public Health, 16(10), 1742. https://doi.org/10.3390/ijerph16101742
Rahayu, I. L., Kusumawati, A., & Martien, R. (2019). Optimasi Formula Kandidat Vaksin DNA (env-Tm) Penyakit Jembrana Berbasis Kitosan dan Poly(D-L- Lactic Co-Glicolide Acid) . Universitas Gadjah Mada.
Rennick, J. J., Johnston, A. P. R., & Parton, R. G. (2021). Key principles and methods for studying the endocytosis of biological and nanoparticle therapeutics. Nature Nanotechnology, 16(3), 266–276. https://doi.org/10.1038/s41565-021-00858-8
Santos-Carballal, B., Fernández Fernández, E., & Goycoolea, F. (2018). Chitosan in Non-Viral Gene Delivery: Role of Structure, Characterization Methods, and Insights in Cancer and Rare Diseases Therapies. Polymers, 10(4), 444. https://doi.org/10.3390/polym10040444
Seleci, M., Ag Seleci, D., Joncyzk, R., Stahl, F., Blume, C., & Scheper, T. (2016). Smart multifunctional nanoparticles in nanomedicine. BioNanoMaterials, 17(1–2), 33–41. https://doi.org/10.1515/bnm-2015-0030
Srinivas, K. P. (2021). Recent developments in vaccines strategies against human viral pathogens. In Recent Developments in Applied Microbiology and Biochemistry (pp. 3–12). Elsevier. https://doi.org/10.1016/B978-0-12-821406-0.00001-1
Suryani, S., Chaerunisaa, A., Joni, I. M., Ruslin, R., Aspadiah, V., Anton, A., Sartinah, A., & Ramadhan, L. O. A. (2024). The Chemical Modification to Improve Solubility of Chitosan and Its Derivatives Application, Preparation Method, Toxicity as a Nanoparticles. Nanotechnology, Science and Applications, Volume 17, 41–57. https://doi.org/10.2147/NSA.S450026
Unsunnidhal, L., Ishak, J., & Kusumawati, A. (2019). Expression of gag-CA Gene of Jembrana Disease Virus with Cationic Liposomal and Chitosan Nanoparticle Delivery Systems as DNA Vaccine Candidates. Tropical Life Sciences Research, 30(3), 15–36. https://doi.org/10.21315/tlsr2019.30.3.2
Vancevska, A., & Nikolic, A. (2013). Assessment of Deoxyribonuclease Activity in Biological Samples by a Fluorescence Detection-Based Method. Laboratory Medicine, 44(2), 125–128. https://doi.org/10.1309/LMD9INNMFDO5XGIW
World Health Organization. (2024, April 23). Dengue and severe dengue. https://www.who.int/news-room/fact-sheets/detail/dengue-and-severe-dengue.
Yang, C., He, B., Dai, W., Zhang, H., Zheng, Y., Wang, X., & Zhang, Q. (2021). The role of caveolin-1 in the biofate and efficacy of anti-tumor drugs and their nano-drug delivery systems. Acta Pharmaceutica Sinica B, 11(4), 961–977. https://doi.org/10.1016/j.apsb.2020.11.020
Published
2026-03-02
How to Cite
Muzakki, M. R., Sekar Cita, V., Fahlevi, N. A., Untari, T., & Kusumawati, A. (2026). The Potential of Chitosan Nanoparticles as a Delivery System for the Expression of Dengue Virus’ rE Gene with pEGFP-N1 Vector in HeLa Cells. Indonesian Journal of Pharmacy, 37(1), 83-95. https://doi.org/10.22146/ijp.16822
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Research Article
