In vivo monitoring of the biodistribution and activation of prodrugs is urgently required. Near infrared (NIR) fluorescence-active fluorophores with excellent photostability are preferable for tracking drug release in vivo. Herein, we describe a NIR prodrug DCM-S-CPT and its polyethylene glycol-polylactic acid (PEG-PLA) loaded nanoparticles as a potent cancer therapy. We have conjugated a dicyanomethylene-4H-pyran derivative as the NIR fluorophore with camptothecin (CPT) as the anticancer drug using a disulfide linker. In vitro experiments verify that the high intracellular glutathione (GSH) concentrations in tumor cells cause cleavage of the disulfide linker, resulting in concomitantly the active drug CPT release and significant NIR fluorescence turn-on with large Stokes shift (200 nm). The NIR fluorescence of DCM-S-CPT at 665 nm with fast response to GSH can act as a direct off-on signal reporter for the GSH-activatable prodrug. Particularly, DCM-S-CPT possesses much better photostability than ICG, which is highly desirable for in situ fluorescence-tracking of cancer chemotherapy. DCM-S-CPT has been successfully utilized for in vivo and in situ tracking of drug release and cancer therapeutic efficacy in living animals by NIR fluorescence. DCM-S-CPT exhibits excellent tumor-activatable performance when intravenously injected into tumor-bearing nude mice, as well as specific cancer therapy with few side effects. DCM-S-CPT loaded in PEG-PLA nanoparticles shows even higher antitumor activity than free CPT, and is also retained longer in the plasma. The tumor-targeting ability and the specific drug release in tumors make DCM-S-CPT as a promising prodrug, providing significant advances toward deeper understanding and exploration of theranostic drug-delivery systems.

A prodrug forms nanocapsules responsive to tumor GSH/ROS heterogeneity releasing the parent drug SN38 via thiolysis in the presence of GSH (glutathione) or via enhanced hydrolysis due to ROS (reactive oxygen species)-oxidation of the linker, giving rise to high in vitro cytotoxicity and in vivo anticancer therapeutic activity. The nanocapsules are a suitable size for tumor targeting by means of the EPR effect and have a fixed SN38 loading content of 35 wt%, ideal for translational nanomedicine.

A "cluster-bomb"-like lipid-dendrimer nanoassembly synergizes the functions of its components and thereby efficiently accomplishes the drug delivery cascade for high efficacy in treating cancer. The nanoassembly successfully circulates in the blood and accumulates in the tumor. Once in the tumor, it releases small dendrimers that act like "bomblets", enabling tumor penetration, cell internalization, and drug release.

Cell-penetrating peptides (CPPs) such as transactivator of transcription (TAT) peptide have long been explored for promoting in vitro cell penetration and nuclear targeting of various cargos, but their positive charges cause strong nonspecific interactions, making them inapplicable for many in vivo applications. In this work, we used TAT to demonstrate a molecular modification approach for inhibiting nonspecific interactions of CPPs in the bloodstream while reactivating their functions in the targeted tissues or cells. The TAT lysine residues' amines were amidized to succinyl amides ((a)TAT), completely inhibiting TAT's nonspecific interactions in the blood compartment; once in the acidic tumor interstitium or internalized into cell endo/lysosomes, the succinyl amides in the (a)TAT were quickly hydrolyzed, fully restoring TAT's functions. Thus, (a)TAT-functionalized poly(ethylene glycol)-block-poly(ε-caprolactone) micelles achieved long circulation in the blood compartment and efficiently accumulated and delivered doxorubicin to tumor tissues, giving rise to high antitumor activity and low cardiotoxicity. This amidization strategy effectively and easily enables in vivo applications of CPPs.