Clostridium neurotoxin fragments as potential targeting moieties for liposomal gene delivery to the CNS

Targeted transfection of the CNS with synthetic, nonviral vectors represents a huge technical challenge. The approach explored here attempts to combine self-assembly ABCD nanoparticles (Kostarelos and Miller, Chem. Soc. Rev. 2005, 34, 970), with the potential of Clostridium neurotoxin fragments to effect receptor-mediated transfection of neuronal cells. Cationic liposome-plasmid DNA complexes were first modified with a PEG stealth layer, before the addition of C-terminal fragments of tetanus toxin (TH(C)), botulinum toxin (BH(C)) or the truncated C-terminal domain of TH(C) as biological "targeting" ligands. First-generation nanoparticles were identified for the transfection of two neuronal cell lines (human SH-5YSY and rat/mouse hybrid N18-RE105); control studies were also performed with HeLa cells. ABCD nanoparticle transfections of the neuronal cell lines were up to 30-fold higher than corresponding control transfections with nanoparticles that lacked the protein ligand. We also demonstrate apparent receptor-mediated uptake by means of competition-binding and real-time confocal experiments. By contrast, nanoparticle transfection of HeLa cells appeared to involve alternative nonspecific enhanced cellular uptake mechanism(s). Receptor-mediated and nonspecific mechanisms appear to be in competition, potentially harming the capacity of receptor-mediated delivery to effect proper targeted delivery of nucleic acids to cells ex vivo and in vivo.