Efficient delivery of small interfering RNA (siRNA) remains a major challenge in gene therapy, particularly due to poor cellular uptake, enzymatic degradation, and endosomal entrapment of the cargo. Cell penetrating peptides (CPPs) offer a promising strategy for intracellular delivery of nucleic acids; however, peptide-based gene delivery carriers possess limited nucleic acid condensation capability, demonstrating reduced transfection efficacies in mammalian cells. This study aimed to enhance siRNA delivery efficacies of CPP-based siRNA therapeutics by combining nucleic acid condensation efficacies of methylated protamine with the cell permeation capabilities of cyclic peptides to improve physiological stability, cellular uptake, and gene silencing efficacies in HER2breast cancer cells. Cyclic CPPs (cCPPs) of different cationic and amphipathic characters (namely, cTAT, cKALA, and cC105Y) were synthesized by solid phase peptide synthesis. Methylated protamine/siRNA complexes of net neutral nanoparticles (NNPs) obtained at nitrogen-to-phosphate ratio of 1 were functionalized with cCPPs at various cCPP/siRNA w/w ratios, and the sizes and net charges of the formulated nanoparticles were analyzed using dynamic light scattering and a zeta potential instrument, respectively. cCPP-functionalized NNPs demonstrated improved nucleic acid condensation efficacy and protection against enzymatic degradation compared with bare NNPs. cCPP-NNPs formulated using various types of peptides overall demonstrated superior siRNA transfection efficacies with ∼60% HER2 gene silencing efficacies in HER2breast cancer cells. cC105Y-NNPs particularly showed improved physiological stability and cellular uptake compared with the other cCPP-functionalized nanoparticles. Endosomal escape remained a limiting factor in the gene expression efficacies of cCPP-NNPs, and incorporation of an endosome-disrupting agent improved HER2 gene knockdown efficacies (∼80%) to levels that were comparable to the Lipofectamine control. Furthermore, the potential of cCPP-NNPs for simultaneous knockdown of HER2 and HER3 was demonstrated to improve the anticancer efficacies of the nanoparticle for HER2breast cancer treatment. SIGNIFICANCE STATEMENT: Cyclic cell penetrating peptide-functionalized nanoparticles with improved physiological stability and optimized nucleic acid condensation and release efficacies formulated by simple deposition of cationic peptides on protamine small interfering RNA complexes demonstrate superior gene knockdown and anticancer efficacies in breast cancer cells. + + +
