Evaluation of multimeric siRNA conjugates for efficient protamine-based delivery into breast cancer cells

Despite the preferable properties of well-defined cationic peptides for small interfering RNA (siRNA) delivery, their application as siRNA carriers remains limited due to their poor binding affinity with short-chain RNAs. In this study, we investigated the feasibility of a novel strategy for circumventing this limitation, by assessing the utility of multimeric conjugates of siRNA for improving the binding affinity of siRNAs with cationic peptides and the extent of intracellular delivery. Protamine, a natural and arginine-rich peptide, was used to produce stably condensed polyelectrolyte complexes (PECs) with multimeric siRNAs (multi-siRNA) with a size of 120 nm while conventional siRNA/protamine particles are over 500 nm. The formulated multi-siRNA/protamine PECs showed greatly enhanced stability, intracellular uptake, and biocompatibility compared to conventional, monomeric (mono)-siRNA/protamine particles. With the addition of chloroquine, multi-siRNA/protamine PECs successfully inhibited target gene expression in MDA-MB-435 cells, a breast cancer cell line, even in the presence of serum protein. This study demonstrates that multi-siRNA conjugates greatly facilitate the formulation of nano-sized protamine-based carriers and significantly improve intracellular delivery in vitro compared to common siRNAs, and therefore may provide a platform for the design of peptide-based siRNA delivery systems for in vivo applications.