Alkyne-Functionalized Cationic Polysiloxane Polymers Conjugated with Targeting Molecules by Click Reactions for DNA Delivery

Cell-specific gene delivery through conjugating targeting molecules to gene carriers is a promising strategy for gene therapy. Click chemistry is a convenient tool for such conjugations. We have developed siloxane-based amphiphilic polymers with alkyne-functionalized and quaternized imidazolium salts (PIm) for forming nanoemulsions capable of conjugating azide-functionalized targeting molecules by click chemistry. Being positively charged, these polymers were expected to be applicable to targeted gene delivery. In this study, PIm was conjugated with lactose, which is recognized by asialoglycoprotein receptors (ASGP-Rs) on hepatocytes, using click chemistry and was examined for DNA binding, cytotoxicity, and in vitro transfection in HepG2 and NIH3T3 cells. The agarose gel retardation assay using a 5.3-kbp plasmid DNA (pDNA) confirmed complex formation between the lactose-conjugated polymers and DNA at a nitrogen/phosphorous (N/P) ratio of 8. The polymers exhibited no significant hemolytic activity up to 50 mu g/mL. The polymer-pDNA complexes have low cytotoxicity, which maintained a cell survival rate greater than 70% at N/P ratios of up to 12 under transfection conditions. The luciferase assay revealed that PIm with a low level of lactose conjugation showed a 29-fold higher transfection efficiency than unmodified PIm at an N/P ratio of 12 in HepG2 cells, but not in NIH3T3 cells that do not express ASGP-Rs. These results demonstrate that the lactose conjugation confers significantly enhanced gene transfer capability for HepG2 cells to PIm most likely due to binding of the polymer to ASGP-Rs on HepG2 cells. Therefore, PIm could be a promising gene delivery vehicle clickable for conjugating cell-specific targeting molecules.