Nonviral cationic polymers like chitosan can be combined with DNA to protect it from degradation. The chitosan is a biocompatible, biodegradable, nontoxic, and cheap polycationic polymer with low immunogenicity. The objective of this study was to synthesize and then assess different chitosan-DNA nanoparticles and to select the best ones for selective in vitro transfection in human epidermoid carcinoma (KB) cell lines. It revealed that different combinations of molecular weight, the presence or absence of folic acid ligand, and different plasmid DNA sizes can lead to nanoparticles with various diameters and diverse transfection efficiencies. The intracellular trafficking, nuclear uptake, and localization are also studied by confocal microscopy, which confirmed that DNA was delivered to cell nuclei to be expressed. 1. Introduction Gene therapy is being applied to various health problems, such as cancer, acquired immunodeficiency syndrome, and cardiovascular diseases. The main challenge is to develop a method that delivers the transgene to selected cells, where a proper gene expression can be achieved. Several trials have aimed at introducing genes straight into human cells, focusing on diseases caused by single-gene defects, such as cystic fibrosis [1], hemophilia [2], adenosine deaminase deficiency [3], muscular dystrophy [4], and sickle cell anemia [5]. Ideally, gene therapy must protect DNA against degradation by nucleases in intercellular matrices so that the disposition of macromolecules is not affected. Transgenes should be brought across the plasma membrane and into the nucleus of targeted cells but should have no detrimental effects. Hence, interaction with blood components, vascular endothelial cells, and uptake by the reticuloendothelial system must be avoided [6]. For gene therapy to succeed, small-sized systems must internalize into cells and pass to the nucleus. Also, flexible tropisms allow applicability to a range of disease targets. Last but not least, such systems should be able to escape endosome-lysosome processing for endocytosis [7]. Viral gene therapy consists of using viral vectors which, given their structure and mechanisms of action, are good candidates or models to carry therapeutic genes efficiently, leading to long-term expression [8, 9]. They have the natural ability to enter cells and express their own proteins. Nowadays, most viral vectors used are retroviruses, herpes virus, adenoviruses, and lentiviruses [10]. However, viral vectors can cause several problems to patients, namely, toxicity, oncogenic effects, and immune and
References
[1]
D. J. Porteous, J. R. Dorin, G. McLachlan et al., “Evidence for safety and efficacy of DOTAP cationic liposome mediated CFTR gene transfer to the nasal epithelium of patients with cystic fibrosis,” Gene Therapy, vol. 4, no. 3, pp. 210–218, 1997.
[2]
S. Youjin and Y. Jun, “The treatment of hemophilia A: from protein replacement to AAV-mediated gene therapy,” Biotechnology Letters, vol. 31, no. 3, pp. 321–328, 2009.
[3]
P. Liu, I. Santisteban, L. M. Burroughs et al., “Immunologic reconstitution during PEG-ADA therapy in an unusual mosaic ADA deficient patient,” Clinical Immunology, vol. 130, no. 2, pp. 162–174, 2009.
[4]
S. Braun, “Muscular gene transfer using nonviral vectors,” Current Gene Therapy, vol. 8, no. 5, pp. 391–405, 2008.
[5]
P. Ulug, N. Vasavda, R. Kumar et al., “Hydroxyurea therapy lowers circulating DNA levels in sickle cell anemia,” American Journal of Hematology, vol. 83, no. 9, pp. 714–716, 2008.
[6]
X. Gao, K. S. Kim, and D. Liu, “Nonviral gene delivery: what we know and what is next,” The American Association of Pharmaceutical Scientists Journal, vol. 9, no. 1, pp. E92–E104, 2007.
[7]
S. Mansouri, P. Lavigne, K. Corsi, M. Benderdour, E. Beaumont, and J. C. Fernandes, “Chitosan-DNA nanoparticles as non-viral vectors in gene therapy: strategies to improve transfection efficacy,” European Journal of Pharmaceutics and Biopharmaceutics, vol. 57, no. 1, pp. 1–8, 2004.
[8]
M. Marsh and A. Helenius, “Virus entry: open sesame,” Cell, vol. 124, no. 4, pp. 729–740, 2006.
[9]
E. M. Campbell and T. J. Hope, “Gene therapy progress and prospects: viral trafficking during infection,” Gene Therapy, vol. 12, no. 18, pp. 1353–1359, 2005.
[10]
T. J. Oligino, Q. Yao, S. C. Ghivizzani, and P. Robbins, “Vector systems for gene transfer to joints,” Clinical Orthopaedics and Related Research, supplement 379, pp. S17–S30, 2000.
[11]
T. Niidome and L. Huang, “Gene therapy progress and prospects: nonviral vectors,” Gene Therapy, vol. 9, no. 24, pp. 1647–1652, 2002.
[12]
M. Nishikawa and L. Huang, “Nonviral vectors in the new millennium: delivery barriers in gene transfer,” Human Gene Therapy, vol. 12, no. 8, pp. 861–870, 2001.
[13]
X. Zhang, C. Yu, XuShi, C. Zhang, T. Tang, and K. Dai, “Direct chitosan-mediated gene delivery to the rabbit knee joints in vitro and in vivo,” Biochemical and Biophysical Research Communications, vol. 341, no. 1, pp. 202–208, 2006.
[14]
G. Borchard, “Chitosans for gene delivery,” Advanced Drug Delivery Reviews, vol. 52, no. 2, pp. 145–150, 2001.
[15]
H. Q. Mao, K. Roy, V. L. Troung-Le et al., “Chitosan-DNA nanoparticles as gene carriers: synthesis, characterization and transfection efficiency,” Journal of Controlled Release, vol. 70, no. 3, pp. 399–421, 2001.
[16]
J. C. Fernandes, M. J. Tiera, and F. M. Winnik, “DNA-chitosan nanoparticles for gene therapy: current knowledge and future trends,” in Biological and Pharmaceutical Nanomaterial, C. S. S. R. Kumar, Ed., pp. 68–99, Wiley-VCH, Weinheim, Germany, 2006.
[17]
M. J. Turk, G. J. Breur, W. R. Widmer et al., “Folate-targeted imaging of activated macrophages in rats with adjuvant-induced arthritis,” Arthritis and Rheumatism, vol. 46, no. 7, pp. 1947–1955, 2002.
[18]
S. Sabharanjak and S. Mayor, “Folate receptor endocytosis and trafficking,” Advanced Drug Delivery Reviews, vol. 56, no. 8, pp. 1099–1109, 2004.
[19]
J. Sudimack and R. J. Lee, “Targeted drug delivery via the folate receptor,” Advanced Drug Delivery Reviews, vol. 41, no. 2, pp. 147–162, 2000.
[20]
S. Mima, M. Miya, R. Iwamoto, and S. Yoshikawa, “Highly deacetylated chitosan and its properties,” Journal of Applied Polymer Science, vol. 28, no. 6, pp. 1909–1917, 1983.
[21]
K. C. Cho, S. H. Kim, J. H. Jeong, and T. G. Park, “Folate receptor-mediated gene delivery using folate-poly(ethylene glycol)-poly(L-lysine) conjugate,” Macromolecular Bioscience, vol. 5, no. 6, pp. 512–519, 2005.
[22]
T. Kiang, J. Wen, H. W. Lim, and K. W. Leong, “The effect of the degree of chitosan deacetylation on the efficiency of gene transfection,” Biomaterials, vol. 25, no. 22, pp. 5293–5301, 2004.
K. Corsi, F. Chellat, L. Yahia, and J. C. Fernandes, “Mesenchymal stem cells, MG63 and HEK293 transfection using chitosan-DNA nanoparticles,” Biomaterials, vol. 24, no. 7, pp. 1255–1264, 2003.
[25]
J. C. Fernandes, H. Wang, C. Jreyssaty et al., “Bone-protective effects of nonviral gene therapy with folate-chitosan DNA nanoparticle containing interleukin-1 receptor antagonist gene in rats with adjuvant-induced arthritis,” Molecular Therapy, vol. 16, no. 7, pp. 1243–1251, 2008.
[26]
A. Bozkir and O. M. Saka, “Chitosan-DNA nanoparticles: effect on DNA integrity, bacterial transformation and transfection efficiency,” Journal of Drug Targeting, vol. 12, no. 5, pp. 281–288, 2004.
[27]
H. G. Jo, K. H. Min, T. H. Nam, S. J. Na, J. H. Park, and S. Y. Jeong, “Prolonged antidiabetic effect of zinc-crystallized insulin loaded glycol chitosan nanoparticles in type 1 diabetic rats,” Archives of Pharmacal Research, vol. 31, no. 7, pp. 918–923, 2008.
[28]
M. Lavertu, S. Méthot, N. Tran-Khanh, and M. D. Buschmann, “High efficiency gene transfer using chitosan/DNA nanoparticles with specific combinations of molecular weight and degree of deacetylation,” Biomaterials, vol. 27, no. 27, pp. 4815–4824, 2006.
[29]
K. Turan and K. Nagata, “Chitosan-DNA nanoparticles: the effect of cell type and hydrolysis of chitosan on in vitro DNA transfection,” Pharmaceutical Development and Technology, vol. 11, no. 4, pp. 503–512, 2006.
[30]
J. A. Reddy, L. S. Haneline, E. F. Srour, A. C. Antony, D. W. Clapp, and P. S. Low, “Expression and functional characterization of the β-isoform of the folate receptor on CD34+ cells,” Blood, vol. 93, no. 11, pp. 3940–3948, 1999.
[31]
D. Lee, R. Lockey, and S. Mohapatra, “Folate receptor-mediated cancer cell specific gene delivery using folic acid-conjugated oligochitosans,” Journal of Nanoscience and Nanotechnology, vol. 6, no. 9-10, pp. 2860–2866, 2006.
[32]
N. Nakashima-Matsushita, T. Homma, S. Yu et al., “Selective expression of folate receptor β and its possible role in methotrexate transport in synovial macrophages from patients with rheumatoid arthritis,” Arthritis and Rheumatism, vol. 42, no. 8, pp. 1609–1616, 1999.
