Intelligent “ PeptideGathering PeptideGathering PeptideGathering Mechanical Arm ” Tames Wild “ TrojanHorse TrojanHorse TrojanHorse ” Peptides for the Controlled Delivery of Cancer

Cell-penetrating peptides (CPPs), also called “Trojan-Horse” peptides, have been used for facilitating intracellular delivery of numerous diverse cargoes and even nanocarriers. However, the lack of targeting specificity (“wildness” or nonselectivity) of CPP-nanocarriers remains an intractable challenge for many in vivo applications. In this work, we used an intelligent “peptide-gathering mechanical arm” (Int PMA) to curb CPPs’ wildness and enhance the selectivity of R9-liposome-based cargo delivery for tumor targeting. The peptide NGR, serving as a cell-targeting peptide for anchoring, and peptide PLGLAG, serving as a substrate peptide for deanchoring, were embedded in the Int PMA motif. The Int PMA construct was designed to be sensitive to tumor microenvironmental stimuli, including aminopeptidase N (CD13) and matrix metalloproteinases (MMP-2/9). Moreover, Int PMA could be specifically recognized by tumor tissues via CD13-mediated anchoring and released for cell entry by MMP-2/9-mediated deanchoring. To test the Int PMA design, a series of experiments were conducted in vitro and in vivo. Functional conjugates Int PMA-R9-poly(ethylene glycol) (PEG)2000-distearoylphosphatidyl-ethanolamine (DSPE) and R9PEG2000-DSPE were synthesized by Michael addition reaction and were characterized by thin-layer chromatography and matrixassisted laser desorption ionization-time-of-flight mass spectrometry. The Int PMA-R9-modified doxorubicin-loaded liposomes (Int PMA-R9-Lip-DOX) exhibited a proper particle diameter (approximately 155 nm) with in vitro sustained release characteristics. Cleavage assay showed that Int PMA-R9 peptide molecules could be cleaved by MMP-2/9 for completion of deanchoring. Flow cytometry and confocal microscopy studies indicated that Int PMA-R9-Lip-DOX can respond to both endogenous and exogenous stimuli in the presence/absence of excess MMP-2/9 and MMP-2/9 inhibitor (GM6001) and effectively function under competitive receptor-binding conditions. Moreover, Int PMA-R9-Lip-DOX generated more significant subcellular dispersions that were especially evident within endoplasmic reticulum (ER) and Golgi apparatus. Notably, Int PMAR9-Lip-DOX could induce enhanced apoptosis, during which caspase 3/7 might be activated. In addition, Int PMA-R9-Lip-DOX displayed enhanced in vitro and in vivo antitumor efficacy versus “wild” R9-Lip-DOX. On the basis of investigations at the molecular level, cellular level, and animals’ level, the control of Int PMA was effective and promoted selective delivery of R9liposome cargo to the target site and reduced nonspecific uptake. This Int PMA-controlled strategy based on aminopeptidaseguided anchoring and protease-triggered deanchoring effectively curbed the wildness of CPPs and bolstered their effectiveness for in vivo delivery of nanotherapeutics. The specific nanocarrier delivery system used here could be adapted using a variety of intelligent designs based on combinations of multifunctional peptides that would specifically and preferentially bind to tumors versus nontumor tissues for tumor-localized accumulation in vivo. Thus, CPPs have a strong advantage for the development of intelligent nanomedicines for targeted tumor therapy.