Cell-specific, activatable, and theranostic prodrug for dual-targeted cancer imaging and therapy

Abstract

Herein we describe the design and synthesis of a folate-doxorubicin conjugate with activatable fluorescence and activatable cytotoxicity. In this study we discovered that the cytotoxicity and fluorescence of doxorubicin are quenched (OFF) when covalently linked with folic acid. Most importantly, when the conjugate is designed with a disulfide bond linking the targeting folate unit and the cytotoxic doxorubicin, a targeted activatable prodrug is obtained that becomes activated (ON) within the cell by glutathione-mediated dissociation and nuclear translocation, showing enhanced fluorescence and cellular toxicity. In our novel design, folic acid acted as both a targeting ligand for the folate receptor as well as a quencher for doxorubicin's fluorescence.

Figure 1

Activation of the fluorescent prodrug Doxo-S-S-Fol by GSH (5 mM) treatment. A) Enhanced fluorescence emission (λ_ex = 497 nm, λ_em = 594 nm) was observed after treatment of the Doxo-S-S-Fol (5 mM) in PBS (pH = 7.4) with glutathione (GSH) at 37 °C. B) The fluorescence emission of Doxo-C-C-Fol (5 mM) in PBS (pH = 7.4) does not change in the presence of GSH, leaving the prodrug in its initial quenched state.

Figure 2

A) Doxo-SH release from Doxo-S-S-Fol in 1.0 mL of PBS (pH = 7.4) in the presence (▼) or absence (●) of GSH (1:1 molar ratio) at 37 °C. B) Fluorescence emission (λ_ex = 497 nm, λ_em = 594 nm) of a Doxo-S-S-Fol solution in 1.0 mL of PBS (pH = 7.4) in the presence (▼) or absence (●) of GSH (1:1 molar ratio) at 37 °C.

Figure 3

A, B) Time-dependent and C, D) dose-dependent viabilities of A549 cells (A, C) and MCF 7 cells (B, D) treated with synthesized prodrugs and small molecule drugs (1.2 μM in PBS pH = 7.4).

Figure 4

A, B) Time-dependent and C, D) dose-dependent intra-cellular fluorescence emissions from FR(+) cells (A, C) and FR(−) cells (B, D) treated with synthesized prodrugs and small molecule drugs (1.2 μM in PBS pH = 7.4).

Figure 5

In vitro activation of a fluorescent prodrug. Left column: Fluorescence microscopy images showing internalization of the prodrug Doxo–S-S–Fol (1.2 μM in PBS pH = 7.4) by folate-receptor-expressing A549 cells (10,000 cells) leads to enhanced fluorescence emission within 12 h and induction of apoptosis. Middle columns: Internalization of Doxo and Doxo-SH (1.2 μM in PBS pH = 7.4) into A549 cells showing intercalation with nucleus within 24 h and cell death after 48 h. Right column: Minimal fluorescence emission and absence of apoptosis is observed in A549 cells treated with the control probe Doxo-C-C-Fol (1.2 μM in PBS pH = 7.4). Nucleus stained with DAPI (blue).

Figure 6

In vitro flow cytometry analysis of activation of fluorescent prodrug. Left column: Enhanced fluorescence emission due to activation and then decreased fluorescence emission due to induction of apoptosis is observed in A549 cells (10,000 cells) incubated with Activatable Doxo-S-S-Fol probe (1.2 μM in PBS pH = 7.4). Right column: Minimal fluorescence emission and absence of apoptosis is observed in A549 cells treated with the control probe Doxo-C-C-Fol (1.2 μM in PBS pH = 7.4).

Scheme 1

Intracellular reduced glutathione (GSH) activates a targetable theranostic prodrug, leading to target-specific cytotoxicity and fluorescence emission. A) Syntheses of activatable Doxo-S-S-Fol prodrug and non-activatable control Doxo-C-C-Fol probe. B) Once in the extra-cellular milieu, fluorescence emission and cytotoxicity of the prodrug (Doxo-S-S-Fol) are quenched (OFF). Upon target-specific internalization, enhanced fluorescence emission and cytotoxicity (ON) occurs due to cleavage of the disulfide bond. C) The non-cleavable control Doxo-C-C-Fol probe, although it undergoes internalization, remains biologically inactive (OFF) with quenched fluorescence, due to the absence of a cleaving mechanism.