Simple and Efficient Approach for siRNA Encapsulation into PCLs by Freeze-thawing Method

[Introduction] Small interfering RNA (siRNA) has been widely studied for the treatment of intractable diseases, such as cancer. Since naked siRNA is unstable in plasma and has low transfection efficiency, the siRNA delivery... [ view full abstract ]

[Introduction] Small interfering RNA (siRNA) has been widely studied for the treatment of intractable diseases, such as cancer. Since naked siRNA is unstable in plasma and has low transfection efficiency, the siRNA delivery system is indispensable for the establishment of systemic siRNA therapy. In the present study, we report an effective and easy-to-use approach for siRNA encapsulation into liposomes by freeze-thawing. We found that freeze-thawing of single-layer polycation liposomes (PCLs)/siRNA complex forms multi-layer structures, suggesting siRNA was effectively packaged between the lipid layers of multi-layer PCLs.

[Method] Dicetyl phosphate-diethylenetriamine conjugate-based PCLs were incubated with siRNA to form PCLs/siRNA complexes (conventional lipoplex). The lipoplex was frozen in liquid nitrogen and thawed in a water bath with vortexing to prepare freeze-thawed lipoplex. Structure of the freeze-thawed lipoplex was observed by transmission electron microscopy. To confirm stability of the siRNA, conventional or freeze-thawed lipoplexes were incubated with fetal bovine serum for 72 h, and then undegraded siRNA was extracted and detected by electrophoretic assay. Gene silencing effect of the freeze-thawing lipoplex was evaluated by reporter gene assay using B16F10 murine melanoma cells transduced with firefly luciferase gene. In order to determine the biodistribution of siRNA in the freeze-thawed lipoplexes, alexa750-modified siRNA (alexa-siRNA) formulated in PEGylated freeze-thawed lipoplex was administrated intravenously to tumor-bearing mice. Then, accumulation of the alexa-siRNA in each organ and tumor was detected by in vivo imaging system.

[Result and Discussion] The transmission electron microscopic observation showed that the freeze-thawed lipoplex formed a multi-layer structure after the freeze-thawing of single-layered PCLs/siRNA complex. The siRNA formulated in freeze-thawed lipoplexes was not degraded even after incubation with 90% fetal bovine serum for 72 h while siRNA formulated in the conventional lipoplexes was degraded. These results indicate that siRNA was packed between the lipid layers and prevented from exposure to nucleases. The freeze-thawed lipoplex showed significantly higher gene-silencing efficacy compared with the conventional ones. Additionally, more amount of alexa-siRNA formulated in PEGylated freeze-thawed lipoplexes showed long-term blood circulation and accumulated in tumor compared with the PEGylated conventional lipoplexes. Our results provide an effective strategy for systemic siRNA delivery with a quite simple procedure.