Effect of Fluorination Modification on Transfection Efficiency of Non-Viral Gene Carrier Systems Based on Chitosan

Abstract

The aim of this study was to examine the transfection efficiency of the fluorination modification without the addition of any cationic charge on the chitosan (Chi) molecule. The fluorination reaction on Chi (ChiF) was carried out with using 1H,1H,2H,2H-Perfluorooctyltriethoxysilane (SiF). The characterization of ChiF was realized by Fourier transform infrared (FTIR) analysis and its molecular weight (Mw) and polydispersity index (PDI_Mw) were determined using GPC-SEC system. The physical properties of nanoparticles (nChiF) obtained by ionic gelation method were determined. The gel electrophoresis analysis was applied to the nanoparticles for determine the gene complexing capacity. The cytotoxicity of ChiF onto Human Embryonic Kidney (HEK293) cells was determined via MTT colorimetric assay. The cell confluency (after transfection) and transfection efficiency of nChiF on HEK293 cells were evaluated. The results showed that the nChiF2:pEGFN1 complex (ratio of 35:1) with a particle size of 98.1±2.2 nm and zeta potential of 34.7 ± 6.5 mV, is more superior agent for transfection efficiency in HEK293 cells due to its high transfection effect and higher cell confluency. As a result, it has been showed that the fluorination reaction onto Chi without any cationic charge modification enhance the transfection efficiency for HEK293 cell lines.

Keywords

• Chitosan,Fluorination,Transfection efficiency,Gene therapy

Kitosan Esaslı Viral Olmayan Gen Taşıyıcı Sistemlerin Transfeksiyon Verimliliği Üzerine Florlama Modifikasyonunun Etkisi

Abstract

Bu çalışmanın amacı, kitosan (Chi) molekülü üzerine herhangi bir katyonik yük eklenmeden florinasyon modifikasyonunun transfeksiyon etkinliğini incelemektir. Chi üzerindeki florlama reaksiyonu (ChiF), 1H, 1H, 2H, 2H-Perflorooktiltrietoksisilan (SiF) kullanılarak gerçekleştirildi. ChiF'in karakterizasyonu, Fourier Transform Infrared Spektroskopisi (FTIR) analizi ile gerçekleştirilmiş ve molekül ağırlığı (Mw) ve polidispersite indeksi (PDI_Mw), GPC-SEC sistemi kullanılarak belirlenmiştir. İyonik jelleşme yöntemiyle elde edilen nanopartiküllerin (nChiF) fiziksel özellikleri belirlenmiştir. Gen kompleksleme kapasitesini belirlemek için nanoparçacıklara jel elektroforez analizi uygulandı. ChiF'nin İnsan Embriyonik Böbrek (HEK293) hücreleri üzerindeki sitotoksisitesi, MTT kolorimetrik deneyi ile belirlendi. HEK293 hücreleri üzerine nChiF'nin transfeksiyon etkinliği ve hücre yayılımı (transfeksiyon sonrası) sonuçları değerlendirildi. Sonuçlar, 98.1 ± 2.2 nm partikül büyüklüğüne ve 34.7 ± 6.5 mV zeta potansiyeline sahip nChiF2: pEGFN1 kompleksinin (35: 1 oranı), yüksek transfeksiyon etkisi ve daha yüksek hücre yayılımı gösterdiğinden dolayı HEK293 hücrelerinde transfeksiyon etkinliği için daha üstün bir ajan olduğunu göstermiştir. Sonuç olarak, Chi üzerine herhangi bir katyonik yük modifikasyonu olmadan florinasyon reaksiyonunun, HEK293 hücre hatları için transfeksiyon verimliliğini arttırdığı görülmektedir.

Keywords

• Kitosan,Florlama,Transfeksiyon etkinliği,Gen terapi

References

1. [1] Luo, T.Y., Zhang, H.J., Chen, P., Liu, Y.H., Wang H.J., Yu, X.Q. 2018. Photoluminescent F-doped carbon dots prepared by ring-opening reaction for gene delivery and cell imaging. RSC Advances, 8, 6053-6062.
2. [2] Cai, X., Jin, R., Wang, J., Yue, D., Jiang, Q., Wu, Y., Gu, Z. 2016. Bioreducible fluorinated peptide dendrimers capable of circumventing various physiological barriers for highly efficient and safe gene delivery. Applied Materials & Interfaces, 8, 5821-5832.
3. [3] Özgümüş, S., Gök, M.K., Pabuccuoğlu, S. 2015. Chitosan: Gene Delivery. ss 1735-1749. Mishra, M., ed. 2015. Encyclopedia of Biomedical Polymers and Polymeric Biomaterials, CRCPress, Taylor & Francis, USA, 10444 s.
4. [4] Mintzer, M.A., Simanek, E.E. 2008. Nonviral vectors for gene delivery. Chemical Reviews, 109(2), 259-302.
5. [5] Thomas, C.E., Ehrhardt, A., Kay, M.A. 2003. Progress and problems with the use of viral vectors for gene therapy. Nature Reviews Genetics, 4(5), 346-358.
6. [6] Saranya, N., Moorthi, A., Saravanan, S., Devi, M.P., Selvamurugan, N. 2011. Chitosan and its derivatives for gene delivery. International Journal of Biological Macromolecules, 48(2), 234-238.
7. [7] Thomas, M., Klibanov, A. 2003. Non-viral gene therapy: polycation-mediated DNA delivery. Applied Microbiology and Biotechnology, 62(1), 27-34.
8. [8] Opanasopit, P., Sajomsang, W., Ruktanonchai, U., Mayen, V., Rojanarata, T., Ngawhirunpat, T. 2008. Methylated N-(4-pyridinylmethyl) chitosan as a novel effective safe gene carrier. International Journal of Pharmaceutics, 364(1), 127-134.

9. [9] Gong, J.H., Wang, Y., Xing, L., Cui P.F., Qiao, J.B., He, Y.J., Jiang, H.L. 2018. Biocampatible fluorinated poly(β-amino ester)s for safe and efficient gene tehrapy. International Journal of Pharamceutics, 535, 180-193.
10. [10] Chen G., Wang, K., Hu, Q., Ding, L., Yu, F., Zhou, Z., Zhou, Y., Li, J., Sun, M., Oupicky, D. 2017. Combining fluorination and bioreducibility for ımproved sirna polyplex delivery. Applied Materials & Interfaces, 9, 4457-4466.
11. [11] Zuo, G., Xie, A., Pan, X., Su, T., Li, J., Dong, W. 2018. Fluorine-Doped cationic carbon dots for efficient gene delivery. Applied Nano Materials, 1, 2376-2385.
12. [12] Wu, T., Wang, L., Ding, S., You, Y. 2017. Fluorinated PEG-polypeptide polyplex micelles have good serum-resistance and low cytotoxicity for gene delivery. Macromolecular Bioscience, 17, 1-8.
13. [13] Belabassi, Y., Moreau, J., Gheran, V., Henoumont, C., Robert A., Callewaert, M., Rigaux, G., Cadiou, C., Elst, L.V., Laurent, S., Muller, R.N., Dinischiotu, A., Voicu, S.N., Chuburu, F. 2017. Synthesis and characterization of PEGylated and fluorinated chitosans: Application to the synthesis of targeted naoparticles for drug delivery. Biomacromolecules, 18, 2756-2766.
14. [14] ASTM, ASTM F2602-08, Standard Test Method for Determining the Molar Mass of Chitosan and Chitosan Salts by Size Exclusion Chromatography with Multi angle Light Scattering Detection (SEC-MALS), ASTM International, West Conshohocken, PA, 2008.
15. [15] Duran, H., Alkan, F.Ü., Ulkay, M.B., Karakuş, S., Aktaş, A., Şişmanoğlu, T. 2019. Investigation of the in vitro cytotoxic effects and wound healing activity of ternary composite substance (hollow silica sphere/gum arabic/methylene blue). International Journal of Biological Macromolecules, 121, 1194-1202.
16. [16] Sharma, D., Singh, J. 2017. Synthesis and characterization of fatty acid grafted chitosan polymer and their nanomicelles for nonviral gene delivery applications. Bioconjugate Chemistry, 28(11), 2772-2783.
17. [17] Shahabadi, S.M.S., Rabiee, H., Seyedi, S.M., Mokhtare, A., Brant, J.A. 2017. Superhydrophobic dual layer functionalized titanium dioxide/polyvinylidene fluoride-co-hexafluoropropylene (TiO2/PH) nanofibrous membrane for high flux membrane distillation. Journal of Membrane Science, 537, 140-150.
18. [18] Zhang, K., Wu, J., Chu, P., Ge, Y., Zhao, R., Li, X. 2015. A novel CVD method for rapid fabrication of superhydrophobic surface on aluminium alloy coated nanostructured cerium-oxide and its corrosion resistance. International Journal of Electrochemical Science, 10, 6257-6272.
19. [19] Sabnis, S., Block, L.H. 1997. Improved infrared spectroscopic method for the analysis of degree of N-deacetylation of chitosan. Polymer Bulletin, 39(1), 67-71.
20. [20] Gök, M.K. 2019. In vitro evaluation of synergistic effect of primary and tertiary amino groups in chitosan used as non-viral gene carrier system. European Polymer Journal, 115, 375-383.
21. [21] Cho, K., Wang, X., Nie, S., Chen, Z., Shin, D.M. 2008. Therapeutic nanoparticles for drug delivery in cancer. Clinical Cancer Research, 14(5), 1310-1316.
22. [22] Lavertu, M., Methot, S., Tran-Khanh, N., Buschmann, M.D. 2006. High efficiency gene transfer using chitosan/DNA nanoparticles with specific combinations of molecular weight and degree of deacetylation. Biomaterials, 27(27), 4815-4824.
23. [23] Li, Z.T., Guo, J., Zhang, J.S., Zhao, Y.P., Lv, L., Ding, C., Zhang, X.Z. 2010. Chitosan-graft-polyethylenimine with improved properties as a potential gene vector. Carbohydrate Polymers, 80(1), 254-259.
24. [24] Wong, S.Y., Pelet, J.M., Putnam, D. 2007. Polymer systems for gene delivery-past, present, and future. Progress in Polymer Science, 32(8-9), 799-837.
25. [25] Felgner, P.L., Gadek, T.R., Holm, M., Roman, R., Chan, H.W., Wenz, M., Northrop, J.P., Ringold, G.M., Danielsen, M. 1987. Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure. Proceedings of the National Academy of Sciences, 84(21), 7413-7417.
26. [26] Wang, M., Liu H., Li, L., Cheng, Y. 2014. A fluorinated dendrimer achieves excellent gene transfection efficacy at extremely low nitrogen to phosphorus ratios. Nature Communications, 5(3053), 1-8.
27. [27] Kim, T.H., Jiang, H.L., Jere, D., Park, I.K., Cho, M.H., Nah, J.W., Choi, Y.J., Akaike, T., Cho, C.S. 2007. Chemical modification of chitosan as a gene carrier in vitro and in vivo. Progress in Polymer Science, 32(7), 726-753.