Smart Polyion Complex Micelles for Targeted Intracellular Delivery of PEGylated Antisense Oligonucleotides Containing Acid‐Labile Linkages

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Abstract
A novel pH‐sensitive and targetable antisense ODN delivery system based on multimolecular assembly into polyion complex (PIC) micelles of poly(L‐lysine) (PLL) and a lactosylated poly(ethylene glycol)–antisense ODN conjugate (Lac‐PEG–ODN) containing an acid‐labile linkage (β‐propionate) between the PEG and ODN segments has been developed. The PIC micelles thus prepared had clustered lactose moieties on their peripheries and achieved a significant antisense effect against luciferase gene expression in HuH‐7 cells (hepatoma cells), far more efficiently than that produced by the nonmicelle systems (ODN and Lac‐PEG–ODN) alone, as well as by the lactose‐free PIC micelle. In line with this pronounced antisense effect, the lactosylated PIC micelles showed better uptake than the lactose‐free PIC micelles into HuH‐7 cells; this suggested the involvement of an asialoglycoprotein (ASGP) receptor‐mediated endocytosis process. Furthermore, a significant decrease in the antisense effect (27 % inhibition) was observed for a lactosylated PIC micelle without an acid‐labile linkage (thiomaleimide linkage); this suggested the release of the active (free) antisense ODN molecules into the cellular interior in response to the pH decrease in the endosomal compartment is a key process in the antisense effect. Use of branched poly(ethylenimine) (B‐PEI) instead of the PLL for PIC micellization led to a substantial decrease in the antisense effect, probably due to the buffer effect of the B‐PEI in the endosome compartment, preventing the cleavage of the acid‐labile linkage in the conjugate. The approach reported here is expected to be useful for the construction of smart intracellular delivery systems for antisense ODNs with therapeutic value.