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A disulfide-bridged cyclic RGD peptide, named iRGD (internalizing RGD, c(CRGDK/RGPD/EC)), is known to facilitate tumor targeting as well as tissue penetration. After the RGD motif-induced targeting on αv integrins expressed near tumor tissue, iRGD encounters proteolytic cleavage to expose the CendR motif that promotes penetration into cancer cells via the interaction with neuropilin-1. Based on these proteolytic cleavage and internalization mechanism, we designed an iRGD-based monolithic imaging probe that integrates multiple functions (cancer-specific targeting, internalization and fluorescence activation) within a small peptide framework. To provide the capability of activatable fluorescence signaling, we conjugated a fluorescent dye to the N-terminal of iRGD, which was linked to the internalizing sequence (CendR motif), and a quencher to the opposite C-terminal. It turned out that fluorescence activation of the dye/quencher-conjugated monolithic peptide probe requires dual (reductive and proteolytic) cleavages on both disulfide and amide bond of iRGD peptide. Furthermore, the cleavage of the iRGD peptide leading to fluorescence recovery was indeed operative depending on the tumor-related angiogenic receptors (αvβ3 integrin and neuropilin-1) in vitro as well as in vivo. Compared to an ‘always fluorescent’ iRGD control probe without quencher conjugation, the dye/quencher-conjugated activatable monolithic peptide probe visualized tumor regions more precisely with lower background noise after intravenous injection, owing to the multifunctional responses specific to tumor microenvironment. All these results, along with minimal in vitro and in vivo toxicity profiles, suggest potential of the iRGD-based activatable monolithic peptide probe as a promising imaging agent for precise tumor diagnosis.

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The general practice of photodynamic therapy (PDT) comprises repeated multiple sessions, where photosensitizers are repeatedly administered prior to each operation of light irradiation. To address potential problems arising from the total overdose of photosensitizer by such repeated injections, we here introduce an internalizing RGD peptide (iRGD) derivative (Ppa-iRGDC-BK01) that self-aggregates into an injectable single-component supramolecular depot. Ppa-iRGDC-BK01 is designed as an in situ self-implantable photosensitizer so that it forms a depot by itself upon injection, and its molecular functions (cancer cell internalization and photosensitization) are activated by sustained release, tumor targeting, and tumor-selective proteolytic/reductive cleavage of the iRGD segment. The experimental and theoretical studies revealed that when exposed to body temperature, Ppa-iRGDC-BK01 undergoes thermally accelerated self-assembly to form a supramolecular depot through the hydrophobic interaction of the Ppa pendants and the reorganization of the interpeptide hydrogen bonding. It turned out that the self-aggregation of Ppa-iRGDC-BK01 into a depot exerts a multiple-quenching effect on the photosensitivity to effectively prevent nonspecific phototoxicity and protect it from photobleaching outside the tumor, while enabling autonomous tumor rephotosensitization by long sustained release, tumor accumulation, and intratumoral activation over time. We demonstrate that depot formation through a single peritumoral injection and subsequent quintuple laser irradiations at intervals resulted in complete eradication of the tumor. During the repeated PDT, depot-implanted normal tissues around the tumor exhibited no phototoxic damage under laser exposure. Our approach of single-component photosensitizing supramolecular depot, combined with a strategy of tumor-targeted therapeutic activation, would be a safer and more precise operation of PDT through a nonconventional protocol composed of one-time photosensitizer injection and multiple laser irradiations.