Enhancement of Liposomal Plasmid DNA and siRNA Delivery by Itraconazole through Intracellular Cholesterol Accumulation
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RESEARCH PAPER
Enhancement of Liposomal Plasmid DNA and siRNA Delivery by Itraconazole through Intracellular Cholesterol Accumulation Ira Shrestha 1 & Jong-Soo Choi 1 & Yun-Ui Bae 1 & Kyung-Oh Doh 1
Received: 28 January 2020 / Accepted: 26 May 2020 # Springer Science+Business Media, LLC, part of Springer Nature 2020
ABSTRACT Purpose Efficient and safe vehicle that can enhance gene transfer is still needed. Since intracellular cholesterol is known to have an important role in gene delivery and itraconazole alters intracellular cholesterol trafficking, we investigated the effect of itraconazole on pDNA and siRNA delivery. Methods The pDNA and Bcl2 siRNA transfection efficiency was measured by luciferase assay and cytotoxicity. Cellular cholesterol was observed using filipin staining, and intracellular uptake was analyzed by flow cytometry. Lipoplex localization was observed by fluorescent labeling of DNA and lysosome after treatment of itraconazole or co-treatment of itraconazole and bafilomycin A1. Results Itraconazole enhanced the transfection efficiency of pDNA and siRNA compared to that of control through the accumulation of cholesterol. Bafilomycin A1 diminished the effect of itraconazole on gene delivery and the increment of cholesterol. Itraconazole did not increase the cellular uptake of lipoplex, but increased free pDNA during the endosomelysosome pathway was observed during the endosomelysosome pathway. Treating cells with both imipramine and itraconazole caused an additive effect in pDNA and siRNA delivery. Conclusions Itraconazole enhanced gene delivery of pDNA and siRNA, and it can be used to potentiate nucleic acid therapeutics.
Ira Shrestha and Jong-Soo Choi contributed equally to this work. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s11095-020-02846-4) contains supplementary material, which is available to authorized users. * Kyung-Oh Doh [email protected] 1
Department of Physiology, College of Medicine, Yeungnam University, Daegu 42415, Republic of Korea
KEY WORDS Bcl2 siRNA . endosomal escape . gene delivery . itraconazole
ABBREVIATIONS Baf Imi ITZ SC siRNA
Bafilomycine A1 Imipramine Itraconazole Scrambled siRNA
INTRODUCTION Gene therapy is the administration of genetic materials (DNAs or RNAs) to cure a disease or at least alleviate its symptoms [1]. The simplest way to deliver a gene into a host cell is to inject a naked therapeutic DNA into the target cell. However, naked genes are degraded during circulation as well as by intracellular nucleases. Besides, their uptake is very poor because of their large size and negative charge, thereby leading to low transfection efficiency. Delivery vehicles are therefore required for their transportation [2]. Although viral vectors provide very high transfection efficiency, the residual viral element can generate an immune response in patients [1, 3]. Furthermore, the cytotoxic possibility, immune response, nontarget effects of viral vectors and even lethal complications are major concerns of their cli
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