Activatable theranostic prodrug scaffold with tunable drug release rate for sequential photodynamic and chemotherapy

Abstract

Glutathione (GSH)-activated prodrugs are promising for overcoming the limitations of conventional anti-tumor drugs. However, current GSH-responsive disulfide groups exhibit unregulated reactivity, making it impossible to precisely control the drug release rate. We herein report a series of GSH-responsive prodrugs with a "three-in-one" molecular design by integrating a fluorescence report unit, stimuli-responsive unit and chemodrug into one scaffold with tunable aromatic nucleophilic substitution (S_NAr) reactivity. The drug release rate of these prodrugs is tailored by modification of substituent groups with different electron-withdrawing or -donating abilities on the BODIPY core. Furthermore, the prodrugs self-assemble in water to form nanoparticles that serve as photosensitizers to produce reactive oxygen species upon irradiation for photodynamic therapy (PDT). The PDT process also increases the concentration of GSH in cells, further promoting the release of drugs for chemotherapy. This strategy provides a powerful platform for sequential photodynamic and chemotherapy with tunable drug release rates and synergistic therapeutic effects.

Keywords: combinational therapy; fluorescent probes; photodynamic therapy; prodrugs; theranostic agents.

SCHEME 1

(a) Reaction mechanism of the conventional disulfide‐based prodrugs: the drug release rate is uncontrollable through thiol‐triggered disulfide bond cleavage. (b) GSH‐responsive prodrugs with tunable aromatic nucleophilic substitution (S_NAr) reactivity. (c) Schematic illustration of sequential photodynamic‐chemo combination of theranostic prodrugs.

FIGURE 1

(a) Absorption spectra of 10 μM BP1 upon addition of 0–8 mM GSH in PBS (10 mM, containing 30 vol% CH₃CN) at 37°C; (b) Time‐dependent absorption spectra of 10 μM BP1 treated with 1 mM GSH; (c) Time‐dependent fluorescence spectra of 10 μM BP1 treated with 1 mM GSH; (d) Ratio of maximum fluorescence intensities of BP1‐5 before and after the reaction with GSH as a function of time. GSH, Glutathione; PBS, phosphate buffered saline.

FIGURE 2

(a) Size distribution of BP1 NPs using DLS (inset: SEM image of BP1 NPs); (b) Absorption spectra of ABDA in the presence of aqueous dispersion of BP1 NPs upon light irradiation for different times; (c) Absorption spectra of ABDA and the reaction mixture of aqueous dispersion of BP1 NPs with GSH (5 mM) for 24 h upon light irradiation. (d) Plots of A/A₀ of ABDA at 378 nm in the presence of BP1‐5 upon irradiation for different time intervals. ABDA, 9,10‐anthracenediyl‐bis(methylene)‐dimalonic acid; DLS, dynamic light scattering; GSH, Glutathione.

FIGURE 3

(a–d) Confocal fluorescence image of HeLa cell incubated with BP1 for different times; (a) BP1 channel; (b) BP1‐SG channel; (c) Merge; (d) Ratio of BP1‐SG channel to BP1 channel; (e–g) Average fluorescence intensity of (e) BP‐1, (f) BP‐2 and (g) BP‐3 in green (BP channel Em: 500–550 nm), red (BP‐SG channel Em: 570–620 nm) and ratio channels at different time points. Ex: 487 nm. Scale bar: 100 μm.

FIGURE 4

Cell viability of HeLa cells subjected to a range of BP1‐5 (a) in the dark and (b) upon light irradiation (White LED light, 40 mW/cm²).

FIGURE 5

(a, b) Confocal ratio fluorescence image of HeLa cells incubated with BP1 NPs for different times; (a) Ratio of BP1‐SG channel to BP1 channel under dark conditions; (b) Ratio of BP1‐SG channel to BP1 channel under illumination; (c) Average fluorescence intensity ratio of the imaging results in (a, b); (d) The viability of Hela cell after treatment with various doses of BP1. (Gray column: blank group; Blue column: +GSH; White LED light, 40 mW/cm²) Ex: 487 nm, BP1 channel, Em: 500–550 nm, BP1‐SG channel, Em: 570–620 nm. Scale bar: 50 μm.