A Myristoylated Cell-Penetrating Peptide Bearing a Transferrin Receptor-Targeting Sequence for Neuro-Targeted siRNA Delivery

[Image: see text] Many neurodegenerative disorders (NDDs) are characterized by aggregation of aberrant proteins and extensive oxidative stress in brain cells. As a treatment option for NDDs, RNA interference (RNAi) is a promising approach to suppress the activation of abnormal genes and negative regulators of antioxidant genes. Efficient neuro-targeted siRNA delivery requires a delicate optimization of nucleic acid carriers, quite distinct from putative pDNA carriers in regard to stable condensation and serum protection of siRNA, blood–brain barrier (BBB) bypass, effective siRNA delivery to brain cells, and functional release of bioactive siRNA at therapeutic levels. Here, we propose that a myristic acid conjugated, cell-penetrating peptide (transportan; TP), equipped with a transferrin receptor-targeting peptide (myr-TP-Tf), will lead to stable encapsulation of siRNA and targeted delivery of siRNA to brain cells overcoming the BBB. Myr-TP-Tf was successfully prepared by solid-phase peptide synthesis with high purity. Myr-TP-Tf–siRNA complexes formulated at a 20:1 (peptide–siRNA) molar ratio provided prolonged siRNA stability against serum and ribonuclease treatment. Fluorescence images clearly indicated that siRNA uptake was successfully achieved by myr-TP-Tf complexes in both a murine brain endothelioma and a human glioma cell line. The luciferase assay and the human placental alkaline phosphatase (hPAP) reporter assay results demonstrated the functional gene silencing effect of myr-TP-Tf–siRNA complexes in a human glioma cell line as well as in primary murine neurons/astrocytes, supportive of successful release of bioactive siRNA into the cytosol. Finally, the transcytosis assay revealed that favorable siRNA transport via receptor-mediated transcytosis was mediated by myr-TP-Tf complexes. In summary, these data suggest that myr-TP-Tf peptides possess promising properties as a vehicle for neuro-targeted siRNA delivery. We will further study this peptide in vitro and in vivo for transport mechanism kinetics and to validate its capability to deliver siRNA to the brain, respectively.