Agitation during lipoplex formation improves the gene knockdown effect of siRNA
| Autor: | Tomohiro Asai, Hiroshi Kiwada, Tatsuaki Tagami, Jose Mario Barichello, Naoto Oku, Hiroshi Kikuchi, Tatsuhiro Ishida, Shinji Kizuki |
|---|---|
| Rok vydání: | 2011 |
| Předmět: |
Gene knockdown
Liposome Cell Pharmaceutical Science Biology Endocytosis Lipids Molecular biology Clathrin Cell biology medicine.anatomical_structure Cell culture Cations Cell Line Tumor Gene Knockdown Techniques Liposomes medicine Humans Gene silencing lipids (amino acids peptides and proteins) Cationic liposome Gene Silencing RNA Small Interfering Intracellular |
| Zdroj: | International Journal of Pharmaceutics. 410:153-160 |
| ISSN: | 0378-5173 |
| DOI: | 10.1016/j.ijpharm.2011.03.001 |
| Popis: | The successful delivery of therapeutic siRNA to the designated target cells and their availability at the intracellular site of action are crucial requirements for successful RNAi therapy. In the present study, we focused on the siRNA-lipoplex preparation procedure and its effect on the gene-knockdown efficiency of siRNA in vitro. Agitation (vortex-mixing) during siRNA-lipoplex (vor-LTsiR) preparation and its effect on the gene-knockdown efficiency of stably expressed cell GFP was investigated, and their efficiency was compared with that of spontaneously formed lipoplex (spo-LTsiR). A dramatic difference in size between lipoplexes was observed at the N/P ratio of 7.62 (siRNA dose of 30 nM), even though both lipoplexes were positively charged. With the siRNA dose of 30 nM, vor-LTsiR accomplished a 50% gene-knockdown, while spo-LTsiR managed a similar knockdown effect at the 120 nM level, suggesting that the preparation procedure remarkably affects the gene-knockdown efficacy of siRNA. The uptake of vor-LTsiR was mainly via clathrin-mediated endocytosis, whereas that of spo-LTsiR was via membrane fusion. In addition, by inhibiting clathrin-mediated endocytosis, the gene-knockdown efficiency was significantly lowered. The size of the lipoplex, promoted by the preparation procedure, is likely to define the entry pathway, resulting in an increased amount of siRNA internalized in cells and an enhanced gene-knockdown efficacy. The results of the present study definitively show that a proper siRNA-lipoplex preparation procedure makes a significant contribution to the efficiency of cellular uptake, and thereby, to the gene-knockdown efficiency of siRNA. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|
Full Text from ScienceDirect