Improvement of antisense oligonucleotides delivery using high hydrostatic pressurized lipoplex
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Improvement of antisense oligonucleotides delivery using high hydrostatic pressurized lipoplex Tsuyoshi Kimura1, Asami Sano1, Kwangwoo Nam1, Kazunari Akiyoshi2, Yoshihiro Sasaki1, Akio Kishida1 1
Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 2-3-10 Kanda-surugadai, Chiyoda-ku Tokyo 101-0062, Japan 2 Department of Polymer Chemistry, Kyoto University, Kyoto daigaku-Katsura, Nishikyo-ku, Kyoto 615-8530 ABSTRACT Cationic liposome (CL) is a promising vector for nucleic acid therapy. In the present study, we investigated the effect of high hydrostatic pressure (HHP) treatment to lipoplex on the lipoplex-based antisense oligodeoxynucleotides (AS-ODNs) delivery in order to improve the transfection efficacy of lipoplex. Cationic liposome consisting of DOTMA and DOPE was used. AS-ODNs were designed to inhibit the expression of firefly luciferase. The complexes of CL and AS-ODN were prepared at various C/A ratios and then pressurized hydrostatically at various atmospheres (~10,000 atm) for 10 min (HHP treatment). After removal of pressure, the pressurized lipoplexes were used. The lipoplex with and without the HHP treatment was transferred into HeLa cells expressing firefly luciferase transiently. The luciferase activity using the HHP-treated lipoplex was decreased compared to that of the non-pressurized lipoplex. Also, for HEK293 cells expressing luciferase stably, the lipoplex with the HHP treatment could effectively suppress the luciferase expression. In order to elucidate relationship between the structure and the transfection efficiency of the HHP-treated lipoplex, the properties of the HHPtreated lipoplex were examined by various physicochemical analyses. The different physicochemical properties between the lipoplexes with and without HHP treatment were showed, suggesting that the nature of lipoplex was changed by the HHP treatment. We believe that this change of lipoplex properties by the HHP treatment affected the efficiency of gene suppression. This HHP treatment for lipoplex appears to be a promising contribution to gene and oligonucleotide delivery. INTRODUCTION As a promising strategy for the treatment of many intractable diseases, gene therapy, which is defined as the genetic modification for therapeutic benefit, has attracted many researchers. For gene therapy, it is required to delivery exogenous gene drugs, such as plasmid DNA and oligonucleotide, into cells healthy. So far, the delivery of plasmid DNA has been attempted using many kinds of gene vectors in order to replace a missing gene. Recently, modulation of gene expression using short nucleic acids, such as antisense oligodeoxynucleotide (AS-ODN) and small interfering RNA (siRNA), is one of attractive methods for gene therapy [1, 2]. Various types of gene, which cause disease, have been target of short nucleic acid based therapy. For this therapy, it is important to deliver nucleic acids into cells effectively and to inhibit expression of target gene selectively [3]. In many cases, cationic liposomes (CLs) are used as
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