Effects of PEG anchors in PEGylated siRNA lipoplexes on in vitro gene­silencing effects and siRNA biodistribution in mice.

Polyethylene glycol (PEG)­modifications (PEGylations) of cationic liposome/small interfering RNA complexes (siRNA lipoplexes) can enhance their systemic stability. The present study determined the effects of PEG anchors in PEGylated siRNA lipoplexes on in vitro gene­silencing effects and siRNA biodistribution after intravenous injection. Three types of dialkyl or trialkyl cationic lipids were used in the current study for the preparation of cationic liposomes. Additionally, various PEGylated siRNA lipoplexes that contained PEG­1,2­distearoyl­sn­-glycero­-3­phosphoethanolamine (DSPE), PEG­1,2­distearoyl­rac­glycero­3­-methylpolyoxyethylene (DSG), PEG­cholesterol (PEG­Chol) and PEG­chondroitin sulfate conjugate (PEG­CS) were prepared. The results revealed that PEGylation of siRNA lipoplexes with PEG­DSPE strongly decreased gene­silencing effects in cells. In contrast, those with PEG­DSG, PEG­Chol and PEG­CS did not largely decrease gene-silencing effects. However, regardless of the PEG­derivative type, PEGylation of siRNA lipoplexes decreased their agglutination with erythrocytes. Furthermore, intravenous injection of PEGylated siRNA lipoplexes markedly decreased the accumulation of siRNA in the lungs, regardless of the type of PEG­derivative. However, non­PEGylated siRNA lipoplexes accumulated mainly in the lungs regardless of the siRNA lipoplex cationic lipid type. The results indicated that PEGylation of siRNA lipoplexes with PEG­DSG, PEG­Chol and PEG­CS may improve systemic stability without losing transfection activity by PEGylation.

Effect of cationic lipid in cationic liposomes on siRNA delivery into the lung by intravenous injection of cationic lipoplex.

Cationic liposomes composed of dialkyl cationic lipid such as 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) can efficiently deliver siRNA to the lungs following the intravenous injection of cationic liposome/siRNA complexes (lipoplexes). In this study, we examined the effect of cationic lipid of cationic liposomes on siRNA delivery to the lungs after intravenous injection. We used six kinds of cationic cholesterol derivatives and 11 kinds of dialkyl or trialkyl cationic lipids as cationic lipids, and prepared 17 kinds of cationic liposomes composed of a cationic lipid and 1,2-dioleoyl-L-α-glycero-3-phosphatidylethanolamine (DOPE) for evaluation of siRNA biodistribution and in vivo gene silencing effects. Among cationic liposomes, those composed of N-hexadecyl-N,N-dimethylhexadecan-1-aminium bromide (DC-1-16), N,N-dimethyl-N-octadecyloctadecan-1-aminium bromide (DC-1-18), 2-((1,5-bis(octadecyloxy)-1,5-dioxopentan-2-yl)amino)-N,N,N-trimethyl-2-oxoethan-1-aminium chloride (DC-3-18D), 11-((1,3-bis(dodecanoyloxy)-2-((dodecanoyloxy)methyl)propan-2-yl)amino)-N,N,N-trimethyl-11-oxoundecan-1-aminium bromide (TC-1-12), or cholesteryl (3-((2-hydroxyethyl)amino)propyl)carbamate hydroiodide (HAPC-Chol) with DOPE exhibited high accumulation of siRNA in the lung and significant suppression of Tie2 mRNA expression after the intravenous injection of cationic lipoplexes with Tie2 siRNA. Furthermore, DC-1-16/DOPE and DC-1-18/DOPE lipoplexes with protein kinase N3 (PKN3) siRNA could suppress the tumour growth when intravenously injected into mice with lung LLC metastasis. These findings indicate that the siRNA biodistribution and in vivo knockdown efficiency after the intravenous injection of cationic lipoplexes were strongly affected by the type of cationic lipid of cationic liposomes.