Cell membranes targeted unimolecular prodrug for programmatic photodynamic-chemo therapy

Abstract

Photodynamic therapy (PDT) has emerged as one of the most up-and-coming non-invasive therapeutic modalities for cancer therapy in rencent years. However, its therapeutic effect was still hampered by the short life span, limited diffusion distance and ineluctable depletion of singlet oxygen (¹O₂), as well as the hypoxic microenvironment in the tumor tissue. Such problems have limited the application of PDT and appropriate solutions are highly demand. Methods: Herein, a programmatic treatment strategy is proposed for the development of a smart molecular prodrug (D-bpy), which comprise a two-photon photosensitizer and a hypoxia-activated chemotherapeutic prodrug. A rhodamine dye was designed to connect them and track the drug release by the fluorescent signal generated through azo bond cleavage. Results: The prodrug (D-bpy) can stay on the cell membrane and enrich at the tumor site. Upon light irradiation, the therapeutic effect was enhanced by a stepwise treatment: (i) direct generation of ¹O₂ on the cell membrane induced membrane destruction and promoted the D-bpy uptake; (ii) deep tumor hypoxia caused by two-photon PDT process further triggered the activation of the chemotherapy prodrug. Both in vitro and in vivo experiments, D-bpy have exhabited excellent tumor treatment effect. Conclusion: The innovative programmatic treatment strategy provides new strategy for the design of follow-up anticancer drugs.

Keywords: cell membrane; combination therapy; glutathione; singlet oxygen; two-photon photodynamic therapy.

Figure 1

Schematic illustration of photoactivatable cancer treatment of D-bpy. (A) Illustration of the combined treatment of D-bpy upon light irradiation. (B) Structure and mechanism of action of D-bpy.

Figure 2

¹O₂ production and drug release test in vitro. (A) Fluorescent spectra of the mixture of D-bpy and SOSG upon irradiation. (B) Measurement of ¹O₂ production efficiency via changes in the fluorescence by SOSG at 540 nm versus irradiation time (irr = 450-470 nm) in the presence of adjusted concentrations of D-bpy and Bpy in MeOH. The absorption (C) and fluorescence intensity (D) of D-bpy (5 µM) reacted with sodium dithionite in PBS (1% DMSO, pH = 7.4) for 20 min at 37 °C. λ_ex = 488 nm.

Figure 3

Drug release under anoxic condition. (A) UV-vis and (B) fluorescent spectra of D-bpy before and after reduction by rat liver microsomes and NADPH.

Figure 4

Intracellular ¹O₂ production and drug release in living tissues. (A) Confocal fluorescent images of HeLa cells incubated with DCFH-DA and D-bpy before and after TP irradiation. Scale bars = 10 µm. (B) Fluorescence images and (C) Mean fluorescence intensity of tumor tissues from BALB/c mice with 4T1 tumor after intratumoral injection of D-bpy with/without light irradiation. Scale bars = 25 μm. ****p < 0.0001.

Figure 5

Cell localization and real-time imaging of D-bpy and Dio in cells under different conditions. (A) Colocalization images of D-bpy in HeLa cells. Cells were incubated with D-bpy (5 µM) for 1 h and then Dio (10 µM) was added and incubated for another 40 min. (Pearson's coefficient: R = 0.90) (B) Confocal images of living HeLa cells firstly incubated with D-bpy (5 µM) for 1 h and then Dio (10 µM) was added and co-incubated for another 40 min or (C) only incubated with Dio and then through continuous irradiation or without irradiation. Scale bars = 10 µm.

Figure 6

In vitro and in vivo therapeutic effect evaluation. (A) Cell viability of D-bpy, Bpy, R-drug with/without light irradiation in HeLa cells. (B) Fluorescence imaging of tumor tissue from 4T1 tumor-bearing BALB/c mice after intravenous injection of D-bpy and Bpy respectively. (C) Relative tumor volume changes of mice with different treatments (hv = 800 nm). (D) Relative body weight of different treatments. (E) Tumor weight of the mice with different treatment. (F) H&E staining of tumors with different treatments after PDT. Scale bars = 100 μm. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.