Comparison of cationic and amphipathic cell penetrating peptides for siRNA delivery and efficacy.

Cell penetrating peptides (CPPs) are short strands of arginine- and/or lysine-rich peptides (<30 amino acids) that use their cationic nature for efficient intracellular accumulation. CPPs have been used for small interfering RNA (siRNA) delivery by direct complexation with the siRNA anionic phosphate backbone. During this process, however, part of the CPP cationic charges are neutralized, and the resultant loss of free positive charges may substantially compromise CPP's internalization capabilities and eventually reduce siRNA delivery efficiency. The purpose of this study was to design a novel type of polyplex for siRNA delivery to overcome the CPP neutralization issue. This novel polyplex consists of three components: siRNA, 21mer oligolysine (K21) chemically modified to incorporate CPP conjugation sites (K21-PDP), and CPP delivery moiety. The siRNA was first neutralized by cationic charges of K21-PDP to form a polyplex. Then a cationic (hexaarginine, R6) or an amphipathic (model amphipathic peptide, MAP) CPP was conjugated to the polyplex. Agarose gel shift assays indicated that the siRNA could be released from the polyplex after K21-PDP degradation or polyplex dilution. Furthermore, the total intracellular internalization of these two CPP-polyplexes was studied. Compared with R6-polyplex, MAP-polyplex exhibited 170- and 600-fold greater uptake of fluorescently labeled siRNA at 1 and 6 h post-transfection, respectively. MAP-polyplex also exhibited comparable GFP silencing effects as Lipofectamine 2000 complex in Huh7.5 cells stably transfected to express GFP-light chain 3 protein, whereas R6-polyplex did not demonstrate significant silencing activity. Further studies indicated that the K21-PDP-siRNA polyplex formation and conjugation of MAP to the polyplex were essential for siRNA polyplex uptake and gene silencing. MAP-polyplex was also shown to be unaffected by the presence of 10% FBS during transfection. In addition, MAP-polyplex uptake was dependent on vesicle formation and fusion due to 70 and 54% loss of uptake at 4 and 16 °C, respectively, compared to incubation at 37 °C. Therefore, the amphipathic CPP is a more suitable carrier moiety for delivery of siRNA polyplex.