GSH-activated Porphyrin Sonosensitizer Prodrug for Fluorescence Imaging-guided Cancer Sonodynamic Therapy

Abstract

Sonodynamic therapy (SDT), which uses ultrasound to trigger a sonosensitizer to generate reactive oxygen species (ROS), is a promising form of cancer therapy with outstanding tissue penetration depth. However, the sonosensitizer may inevitably spread to surrounding healthy tissue beyond the tumor, resulting in undesired side effects under an ultrasound stimulus. Herein, as glutathione (GSH) is overexpressed in the tumor microenvironment, a GSH-activatable sonosensitizer prodrug was designed by attaching a quencher to tetraphydroxy porphyrin for tumor therapy. The prodrug exhibited poor fluorescence and low ROS generation capacity under ultrasound irradiation but it can be activated by GSH to simultaneously switch on fluorescence emission and ROS generation in tumor site. Compared with the non-quenched sonosensitizer, the designed prodrug exhibited significantly higher tumor/healthy organ fluorescence ratios, due to the specific fluorescence and ROS activation by overexpressed GSH in the tumor. Finally, the prodrug exhibited efficient tumor growth inhibition under ultrasound irradiation, further demonstrating its promise as a GSH-activated sonosensitizer prodrug for highly effective cancer treatment.

Keywords: Glutathione; activatable; fluorescence image; sonodynamic therapy; sonosensitizer.

Figure 1.

(a) Schematic of GSH activation of prodrug NPs to simultaneously switch on fluorescence emission and ROS generation capacities. (b) HPLC profiles of the prodrug, the drug, and the prodrug + GSH. (c) Fluorescence emission spectra of the prodrug at 1 min and 1 h after incubation with GSH (6 mM), respectively. (d) Fluorescence emission spectra of the drug at 1 min and 1 h after incubation with GSH (6 mM), respectively. (e) Fluorescence emission spectra of the prodrug 1 h after incubation with GSH (6 mM); a series of inorganic salts (KCl (50 mM), NaCl (50 mM), MgCl₂ (50 mM)); organic compounds (glucose (10 mM); urea (10 mM); sucrose (10 mM)); amino acids (glycine (10 mM), glutamic acid (10 mM)); reduzate (NADH (1 mM), TrxR (1 μM)); and hydrogen peroxide (H₂O₂,10 mM), confirming that the prodrug is specifically activated by GSH. (f) Fluorescence image of 4T1 and HUVEC cells after incubation with prodrug (10 μM) for 24 h, suggesting the GSH activation of prodrug in cancer cells. (g) Quantization of fluorescence values in (f).

Figure 2.

(a) Absorption value of DBPF in different DMSO solutions (DMSO only, DMSO + prodrug, DMSO + drug) across several timepoints (days), with and without ultrasound irradiation (1.7 W/cm², 30 KHZ, 50% Cycle duty). Each data was repeated for 3 times. (b) Detection of ROS generation (indicated by a strong green fluorescence signal) using a DCFH-DA probe (10 μM) in 4T1 breast cancer cells after incubation with prodrug and treatment with US irradiation. (c) ROS generation was not particularly detected using a DCFH-DA probe (10 μM) in normal HUVEC after incubation with prodrug and treatment with US irradiation. (d) Viability of 4T1 breast cancer cells after treatment with the GSH preincubation prodrug at different concentrations without (blue) and with (red) US irradiation for 5 min (0.85 W/cm², 50% cycle duty, 30 KHZ). (e) Fluorescence imaging of live-dead cells stained by PI/AM after different treatments (no treatment, US irradiation only, prodrug only, prodrug + US irradiation).

Figure 3.

(a) Schematic of prodrug NPs preparation. (b) TEM characterization of prodrug NPs. (c) Size and PDI of prodrug NPs and drug NPs in water, as measured by DLS. (d) Absorption spectra of the prodrug in DMSO, prodrug NPs in water, and prodrug NPs in DMSO (3 μM). (e) Fluorescence emission spectra of the prodrug in DMSO, prodrug NPs in water, and prodrug NPs in DMSO (3 μM). (f) Fluorescence emission spectra of prodrug NPs after 1 h incubation with GSH. (g) Absorption value of DPBF (410 nm) after US irradiation in mixture solutions (DMSO/H₂O = 7/3).

Figure 4.

(a) Cytoviability of 4T1 breast cancer cells after incubation with different concentrations of prodrug NPs for 24 hours. (b) Fluorescence image of 4T1 cancer cells, after incubation with prodrug NPs only, and with prodrug NPs + BSO. (c) Fluorescence image demonstrating ROS generation in 4T1 cancer cells after different treatments (prodrug incubation and US irradiation, BSO and prodrug incubation and US irradiation, prodrug incubation only, US irradiation only). (d) Quantification of ROS fluorescence intensity in (c). (e) Cytoviability of 4T1 cancer cells after incubation with different concentrations of prodrug NPs and treatment with US irradiation (0.85 W/cm², 50% cycle duty, 30 KHZ).

Figure 5.

(a) Fluorescence imaging of tumor-bearing mice after tail vein injection of BSO + prodrug NPs, prodrug NPs, and drug NPs (5 mg/kg, 200 μL). (b) In vivo tumor quantification of fluorescence signal. (c) Biodistribution of drug NPs, prodrug NPs, and BSO + prodrug NPs at different time points. (d) Quantified biodistribution fluorescence signal 24 hours after injection with drug NPs, prodrug NPs, and BSO + prodrug NPs. (e) Fluorescence imaging of tumor sections at 24 h after tail vein injection. (f) Quantified fluorescence signal of tumor sections in (e). (g) Ratios of fluorescence values of tumor-to-normal muscle (T/N) at different time points in drug NPs, prodrug NPs, and BSO + prodrug NPs group.

Figure 6.

(a) The scheme of SDT in vivo. (b) Photographs of tumor-bearing mice 14 days after treatment. Groups 1, 2, 3, and 4 refer to control, prodrug NPs, US and prodrug NPs + US, respectively. (c) Collected tumors of the different groups. (d) Tumor growth curves of the different groups after treatment. Statistical significance was calculated by two-way ANOVA with GraphPad (p < 0.01). (e) Tumor weight of the different groups. Statistical significance was calculated by two-way ANOVA with GraphPad (p < 0.01). (f) Body weight changes in the different groups after treatment. (g) Immunofluorescence TUNEL staining and DAPI staining of tumor sections. The nucleus was imaged at DAPI channel and FITC-dUTP was imaged at GFP channel. Groups 1, 2, 3 and 4 refer to control, prodrug NPs, US and prodrug NPs + US group, respectively. (h) H&E staining of the heart, liver, spleen, lung, and kidney. Groups 1, 2, 3, and 4 refer to control, prodrug NPs, US and prodrug NPs +US, respectively. Scale bar = 50 μm.