Direct Fluorescence Monitoring of the Delivery and Cellular Uptake of a Cancer-Targeted RGD Peptide-Appended Naphthalimide Theragnostic Prodrug

Abstract

Presented here is a multicomponent synthetic strategy that allows for the direct, fluorescence-based monitoring of the targeted cellular uptake and release of a conjugated therapeutic agent. Specifically, we report here the design, synthesis, spectroscopic characterization, and preliminary in vitro biological evaluation of a RGD peptide-appended naphthalimide pro-CPT (compound 1). Compound 1 is a multifunctional molecule composed of a disulfide bond as a cleavable linker, a naphthalimide moiety as a fluorescent reporter, an RGD cyclic peptide as a cancer-targeting unit, and camptothecin (CPT) as a model active agent. Upon reaction with free thiols in aqueous media at pH 7.4, disulfide cleavage occurs. This leads to release of the free CPT active agent, as well as the production of a red-shifted fluorescence emission (λ(max) = 535 nm). Confocal microscopic experiments reveal that 1 is preferentially taken up by U87 cells over C6 cells. On the basis of competition experiments involving okadaic acid, an inhibitor of endocytosis, it is concluded that uptake takes place via RGD-dependent endocytosis mechanisms. In U87 cells, the active CPT payload is released within the endoplasmic reticulum, as inferred from fluorescence-based colocalization studies using a known endoplasmic reticulum-selective dye. The present drug delivery system (DDS) could represent a new approach to so-called theragnostic agent development, wherein both a therapeutic effect and drug uptake-related imaging information are produced and can be readily monitored at the subcellular level. In due course, the strategy embodied in conjugate 1 could allow for more precise monitoring of dosage levels, as well as an improved understanding of cellular uptake and release mechanisms.

Synthesis and Biological Evaluation of RGD Peptidomimetic–Paclitaxel Conjugates Bearing Lysosomally Cleavable Linkers

“…The synthetic procedures for the preparation of compounds 7 and 8 are included in the Supporting Information, along with the 1 H NMR and 13 C NMR spectra, the HPLC traces of the final RGD-PTX conjugates, and the procedures for the evaluation of the cellular expression of a v b 3 integrin.…”

“…[6] Several peptides and peptidomimetics bearing the RGD sequence have been reported to show high affinity for the receptor, [7] and the structural basis for this recognition was provided by a v b 3 -integrin cocrystallization with the cyclopentapeptide Cilengitide. [8] Due to their ability to selectively bind integrin receptors, RGD ligands have been conjugated to a number of cytotoxic agents (for example, doxorubicin, [9,10] doxsaliform, [11] monomethylauristatin E, [12] camptothecin, [13] cisplatin, [14] paclitaxel [15] ) and to proapoptotic compounds, [16,17] with the aim to target these drugs at integrin-rich tumor tissues.…”

Fluorescent chemical probes for accurate tumor diagnosis and targeting therapy

“…Kim et al reported a therapeutic agent 67 that allowed direct, fluorescence-based monitoring of targeted cellular uptake and release of the antitumor drug camptothecin. 206 The results of cellular experiments indicated that conjugate 67 bearing a cyclic RGD peptide targeting subunit was selectively internalized into U87 tumor cells via a v b 3 integrinmediated endocytosis. Disulfide bond cleavage occurred in the ER, permitting fluorescence changes of the naphthalimide moiety within 60 min.…”

Fluorescent Dyes Used in Polymer Carriers as Imaging Agents in Anticancer Therapy

“…In this case linear correlation between the concomitantly increasing fluorescence intensity at λ=540 nm and increasing concentration of Trx was retained. So, the naphthalimide unit provides a suitable fluorescent scaffold owing to its excellent spectroscopic characteristics and structural flexibility for introducing various modifications [124].…”

“…In order to monitor and validate the RME of drug release and drug binding to the target protein, three different probes with fluorescent or fluorogenic molecular chromophores were synthesized: biotin-fluorescein (FITC), biotinlinker-coumarin, and biotin-linker-taxoid-fluorescein. Kim et al [124] studied direct fluorescence monitoring of a delivery and cellular uptake of a cancer-targeted cyclic peptide RGD (with sequence Arg-Gly-Asp). The multifunctional molecule containing a disulfide bond as a cleavable linker was built from a naphthalimide moiety as a fluorescent reporter, RGD as a cancer targeting unit and camptothecin (CPT) as a model active anticancer reagent.…”