Breaking the Barrier of Polynucleotide Size, Type, and Topology in Smad2 Antisense Therapy Using a Cationic Cholesterol Dimer with Flexible Spacer.

A liposomal formulation comprising a dicationic cholesterol based lipid, Chol + -(CH 2 ) 5 -Chol + , and a helper zwitterionic lipid, DOPE (1:4), was prepared to deliver polynucleotides of different topologies, molecular weights, and backbones. This formulation was used to transfect HeLa cells with circular and linearized plasmid pEGFP-C3. The transfection efficiency of the dicationic cholesterol based coliposomal formulation Chol + -(CH 2 ) 5 -Chol + /DOPE (1:4) was observed to be better when compared against different commercial delivery agents, Lipofectamine2000, Effectene, and a known oligonucleotide delivery agent, Oligofectamine. The efficacy was also compared with the respective monocationic cholesterol based liposomal formulations. Western blot analysis for Smad2 protein detection showed almost 100% downregulation of the Smad2 protein by polynucleotides delivered by Chol + -(CH 2 ) 5 -Chol + /DOPE (1:4), which was better than that with Oligofectamine and Effectene. Similarly, semiquantitative RT-PCR showed the downregulation of Smad2 RNA along with that of a downstream target of Smad2, Id2. The higher efficiency of different types of nucleic acid delivery was also evident with Chol + -(CH 2 ) 5 -Chol + /DOPE (1:4) in A549 cells. As an added benefit, the formulation Chol + -(CH 2 ) 5 -Chol + /DOPE (1:4) was found to be highly biocompatible at all the compositions investigated herein.