Intelligent "Peptide-Gathering Mechanical Arm" Tames Wild "Trojan-Horse" Peptides for the Controlled Delivery of Cancer Nanotherapeutics.

Abstract

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 R9-PEG2000-DSPE were synthesized by Michael addition reaction and were characterized by thin-layer chromatography and matrix-assisted 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 PMA-R9-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 R9-liposome cargo to the target site and reduced nonspecific uptake. This Int PMA-controlled strategy based on aminopeptidase-guided 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.