Introduction: Acceleration and improvement of penetration across cell-membrane interfaces of active targeted nanotherapeutics into tumor cells would improve tumor-therapy efficacy by overcoming the issue of poor drug penetration. Cell-penetrating peptides, especially synthetic polyarginine, have shown promise in facilitating cargo delivery. However, it is unknown whether polyarginine can work to overcome the membrane interface in an inserted pattern for cyclic peptide ligand-mediated active targeting drug delivery. Here, we conducted a study to test the hypothesis that tandem-insert nona-arginine (tiR9) can act as an accelerating component for intracellular internalization, enhance cellular penetration, and promote antitumor efficacy of active targeted cyclic asparagine-glycine-arginine (cNGR)-decorated nanoliposomes.

Methods: Polyarginine was coupled with the polyethylene glycol (PEG) chain and the cNGR moiety, yielding a cNGR-tiR9-PEG2,000-distearoylphosphatidylethanolamine conjugate.

Results: The accelerating active targeted liposome (Lip) nanocarrier (cNGR-tiR9-Lip-doxorubicin [Dox]) constructed in this study held suitable physiochemical features, such as appropriate particle size of ~150 nm and sustained-release profiles. Subsequently, tiR9 was shown to enhance cellular drug delivery of Dox-loaded active targeted systems (cNGR-Lip-Dox) significantly. Layer-by-layer confocal microscopy indicated that the tandem-insert polyarginine accelerated active targeted system entry into deeper intracellular regions based on observations at marginal and center locations. tiR9 enhanced the penetration depth of cNGR-Lip-coumarin 6 through subcellular membrane barriers and caused its specific accumulation in mitochondria, endoplasmic reticulum, and Golgi apparatus. It was also obvious that cNGR-tiR9-Lip-Dox induced enhanced apoptosis and activated caspase 3/7. Moreover, compared with cNGR-Lip-Dox, cNGR-tiR9-Lip-Dox induced a significantly higher antiproliferative effect and markedly suppressed tumor growth in HT1080-bearing nude mice.

Conclusion: This active tumor-targeting nanocarrier incorporating a tandem-insert polyarginine (tiR9) as an accelerating motif shows promise as an effective drug-delivery system to accelerate translocation of drugs across tumor-cell/subcellular membrane barriers to achieve improved specific tumor therapy.