• Corpus ID: 49389445

The Absence of CpG sequences in Plasmid DNA-Chitosan Polyplexes Increases Transfection Efficiencies and Reduces Inflammatory Responses in Murine Lungs

@inproceedings{Wongrakpanich2014TheAO,
  title={The Absence of CpG sequences in Plasmid DNA-Chitosan Polyplexes Increases Transfection Efficiencies and Reduces Inflammatory Responses in Murine Lungs},
  author={Amaraporn Wongrakpanich and Andrea Adamcakova-Dodd and Vijaya Bharti Joshi and Kranti A Mapuskar and Sean M. Geary and Douglas R. Spitz and Peter S. Thorne and Aliasger K. Salem},
  year={2014}
}

• A. Wongrakpanich, A. Adamcakova-Dodd, +5 authors A. Salem
• Published 2014
• Biology

SUMMARY Chitosan polyplexes containing plasmid DNA (pDNA) have strong potential for pulmonary gene delivery applications. However, before using chitosan/pDNA polyplexes (CSpp) in clinical applications, their potential cytotoxicity needs to be investigated. We formulated CSpp in the size range from 200 to 400 nm with amine to phosphate (N/P) ratios that ranged from 1 100. We compared two types of plasmid DNA within the polyplexes; pDNA that was free of CpG sequences (CpG(-)) and pDNA that contained CpG sequences (CpG(+)). CSpp(CpG(-)) generated higher luciferase expression both in vitro (especially at N/P ratios between 5 – 20) and in vivo, compared with CSpp(CpG(+)). In addition, CSpp(CpG(-)) showed lower inflammatory responses in mice lungs, as determined by proinflammatory cytokine/chemokine levels within the bronchoalveolar lavage (BAL) fluid. Our findings suggest that to achieve optimal gene expression with minimal cytotoxicity, inflammation, and oxidative stress, the N/P ratios and CpG sequences in the pDNA of CSpp need to be considered. These findings will inform the pre-clinical safety assessments of CSpp in pulmonary gene delivery systems. INTRODUCTION Chitosan/pDNA polyplexes (CSpp) are promising as gene delivery formulations for treatment of various pulmonary pathologies. Pure chitosan is non-toxic[1] and CSpp showed no significant impact on cell viability in vitro for various cell types[2, 3]. Aside from chitosan, pDNA itself may affect the cytotoxicity and inflammatory response. Unmethylated CpG sequences in pDNA could induce inflammatory responses in vivo. The goals of this study were to evaluate CSpp cytotoxicity and pulmonary inflammatory response following intranasal delivery to mouse lungs. CpG(-) and CpG(+) pDNA were compared to determine whether unmethylated CpG sequences in pDNA influence transfection efficiencies and inflammatory responses. EXPERIMENTAL METHODS Both types of polyplexes (CSpp(CpG(-) and CSpp(CpG(+)) were formed through ionic interaction between positively charged chitosan and negatively charged pDNA. The polyplexes were formulated at amine to phosphate ratios (N/P) of 1 – 100. CSpp were characterized for size, zeta potential and morphology. The cytotoxicity of CSpp and intracellular superoxide levels in adenocarcinomic human alveolar basal epithelial cells (A549) and human embryonic kidney 293 cells (HEK293) were tested using an MTS assay and dihydroethidium (DHE), respectively. The transfection efficiency was determined by luciferase expression. Nasal instillation was conducted using male C57BL/6 mice. Two doses of 50 μl CSpp (12.5 μg pDNA/dose) were given with an hour interval time. Mice were euthanized 24 hours later. Bronchoalveolar lavage (BAL) fluids were collected. Cells from BAL fluid were stained and counted for different white blood cell types (macrophages, neutrophils, lymphocytes and eosinophils). The BAL supernatants were used for measuring total protein, lactate dehydrogenase (LDH) activity and inflammatory cytokine (TNF-α, IFN-γ, IL-6, IL12, KC and MIP-1α) concentrations. Whole lungs were homogenized, centrifuged and the supernatants were measured for luciferase