Development of a Novel Method for Formulating Stable siRNA-Loaded Lipid Particles for In vivo Use
A simple yet novel method was developed to prepare stable PEGylated siRNA-loaded lipid particles which are suitable for in vivo use.
PEGylated siRNA-loaded lipid particles were formulated by hydration of a freeze-dried matrix. The effect of various formulation parameters on the size and homogeneity of resulting particles was studied. Particles prepared using this method were compared to those prepared using an established post-insertion procedure for the entrapment efficiency, stability, in vitro biological activity as well as in vivo biodistribution.
Using this hydration method, a particle size of less than 200 nm can be obtained with high siRNA entrapment efficiency (>90%) and high gene-silencing efficiency. Following intravenous administration into mice, these particles achieved a similar degree of accumulation in subcutaneous tumours but displayed less liver uptake compared to the post-insertion formulations. Importantly, in contrast to post-insertion preparations, particles made by hydration method retained 100% of their gene-silencing efficiency after storage at room temperature for 1 month.
This paper describes a simple method of formulating PEGylated siRNA-loaded lipid particles. Given the ease of preparation, long term stability and favourable characteristics for in vivo delivery, our work represents an advance in lipid formulation of siRNA for in vivo use.
KEY WORDScancer liposomes PEGylation siRNA systemic gene delivery
Dulbecco’s modified Eagle medium
Dioleoyl trimethylammonium propane
Fluorescence activated cell sorting
Fetal bovine serum
Green fluorescence protein
Hydration of freeze-dried matrix
Phosphate buffered saline
Small interfering RNA
This work was funded by National Health and Medical Research Council (NHMRC) and we thank Australian Institute for Bioengineering & Nanotechnology for providing Malvern Nano Zetasizer for this study. The authors also gratefully acknowledge Dr Montree Jaturanpinyo for technical assistance and Dr Wenyi Gu for providing cell lines.
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