title: Effect of lyophilization and freeze-thawing on the stability of siRNA-liposome complexes.
abstract: The purpose of this research was to describe the application of lyophilization in the delivery of siRNA using cationic lipids by addressing the long-term formulation/stability issues associated with cationic lipids and to understand the mechanism of lyoprotection. siRNA liposomes complexes were formed in different potential cyro/lyoprotectants and subjected to either lyophilization or freeze thaw cycles. siRNA, liposomes and/or lipoplexes were tested for activity, SYBR Green I binding, cellular uptake and particle size. The lipoplexes when lyophilized in the presence of sugars as lyoprotectants could be lyophilized and reconstituted without loss of transfection efficacy but in ionic solutions they lost 65-75% of their functionality. The mechanism of this loss of activity was further investigated. The lyophilization process did not alter siRNA's intrinsic biological activity as was evident by the ability of lyophilized siRNA to retain functionality and SYBR green I binding ability. While the lipoplex size dramatically increased ( approximately 50-70 times) after lyophilization in the absence of non-ionic lyoprotectants. This increase in size correlated to the decrease in cellular accumulation of siRNA and a decrease in activity. In conclusion, siRNAs can be applied in cationic lipid lyophilized formulations and these complexes represent a potential method of increasing the stability of pre-formed complex.
authors.affiliation: Department of Pharmaceutics, University of Florida, 1600 SW Archer Road, Box 100494, Gainesville, FL 32610, USA.
authors.forename: Preeti
authors.initials: P
authors.lastname: Yadava
authors.forename: Melissa
authors.initials: M
authors.lastname: Gibbs
authors.forename: Carlos
authors.initials: C
authors.lastname: Castro
authors.forename: Jeffrey A
authors.initials: JA
authors.lastname: Hughes
mesh_terms: D000818:Animals; D052160:Benzothiazoles; D002241:Carbohydrates / Q000737:chemistry*; D045744:Cell Line, Tumor; D003451:Cryoprotective Agents / Q000737:chemistry*; D003959:Diamines; D005229:Fatty Acids, Monounsaturated / Q000737:chemistry; D005456:Fluorescent Dyes / Q000378:metabolism; D005612:Freeze Drying; D005615:Freezing; D005947:Glucose / Q000737:chemistry; D007785:Lactose / Q000737:chemistry; D008055:Lipids / Q000737:chemistry*; D008081:Liposomes; D051379:Mice; D009930:Organic Chemicals / Q000378:metabolism; D010316:Particle Size; D010714:Phosphatidylethanolamines / Q000737:chemistry; D000644:Quaternary Ammonium Compounds / Q000737:chemistry; D011804:Quinolines; D020871:RNA Stability; D034741:RNA, Small Interfering / Q000737:chemistry* / Q000378:metabolism; D013395:Sucrose / Q000737:chemistry; D014162:Transfection; D014199:Trehalose / Q000737:chemistry
chemical_list: C410262:1,2-dioleoyl-glycero-3-phosphatidyl ethanolamine; D052160:Benzothiazoles; D002241:Carbohydrates; D003451:Cryoprotective Agents; D003959:Diamines; D005229:Fatty Acids, Monounsaturated; D005456:Fluorescent Dyes; D008055:Lipids; D008081:Liposomes; D009930:Organic Chemicals; D010714:Phosphatidylethanolamines; D000644:Quaternary Ammonium Compounds; D011804:Quinolines; D034741:RNA, Small Interfering; C098022:SYBR Green I; D013395:Sucrose; D014199:Trehalose; D005947:Glucose; D007785:Lactose; C070046:1,2-dioleoyloxy-3-(trimethylammonium)propane