Hypoxia and Singlet Oxygen Dual-Responsive Micelles for Photodynamic and Chemotherapy Therapy Featured with Enhanced Cellular Uptake and Triggered Cargo Delivery

Abstract

Introduction: Combination therapy provides better outcomes than a single therapy and becomes an efficient strategy for cancer treatment. In this study, we designed a hypoxia- and singlet oxygen-responsive polymeric micelles which contain azo and nitroimidazole groups for enhanced cellular uptake, repaid cargo release, and codelivery of photosensitizer Ce6 and hypoxia-activated prodrug tirapazamine TPZ (DHM-Ce6@TPZ), which could be used for combining Ce6-mediated photodynamic therapy (PDT) and PDT-activated chemotherapy to enhance the therapy effect of cancer.

Methods: The hypoxia- and singlet oxygen-responsive polymeric micelles DHM-Ce6@TPZ were prepared by film hydration method. The morphology, physicochemical properties, stimuli responsiveness, in vitro singlet oxygen production, cellular uptake, and cell viability were evaluated. In addition, the in vivo therapeutic effects of the micelles were verified using a tumor xenograft mice model.

Results: The resulting dual-responsive micelles not only increased the concentration of intracellular photosensitizer and TPZ, but also facilitated photosensitizer and TPZ release for enhanced integration of photodynamic and chemotherapy therapy. As a photosensitizer, Ce6 induced PDT by generating toxic singlet reactive oxygen species (ROS), resulting in a hypoxic tumor environment to activate the prodrug TPZ to achieve efficient chemotherapy, thereby evoking a synergistic photodynamic and chemotherapy therapeutic effect. The cascade synergistic therapeutic effect of DHM-Ce6@TPZ was effectively evaluated both in vitro and in vivo to inhibit tumor growth in a breast cancer mice model.

Conclusion: The designed multifunctional micellar nano platform could be a convenient and powerful vehicle for the efficient co-delivery of photosensitizers and chemical drugs for enhanced synergistic photodynamic and chemotherapy therapeutic effect of cancer.

Keywords: combination therapy; hypoxia-responsive; photodynamic therapy; singlet oxygen-responsive.

Scheme 1

Hypoxia- and singlet oxygen-responsive polymeric micelles for in tegration of photodynamic and chemotherapy therapy in a tumor-bearing mice model.

Figure 1

Physicochemical properties of hypoxia and singlet oxygen-responsive micelles (n = 3). (A) The hydrodynamic size of DHM-Ce6 and DHM-Ce6@TPZ. Transmission electron microscope images of DHM-Ce6 (B) and DHM-Ce6@TPZ (C) (scale bar: 500 nm). (D) UV-vis absorption spectra of Ce6, TPZ, DHM, DHM-Ce6, and DHM-Ce6@TPZ. (E) Normalized absorbance of DPBF at 411 nm of different formulations with light irradiation. (F) TEM image of DHM-Ce6@TPZ micelles post laser treatment (660 nm, 200 mW/cm², 10 min). Scale: 500 nm. (G) The UV-vis spectra of DHM-Ce6@TPZ treated with sodium dithionite (10 mM). TPZ release from DHM-Ce6@TPZ micelles under mimicked hypoxia conditions. (H) (10 mM sodium dithionite), and under laser irradiation conditions (I) (660 nm, 200 mW/cm², 10 min).

Figure 2

Confocal images of cellular uptake by 4T1 cells under normoxia or hypoxia at 2, 4, and 6 h post DHM-Ce6@TPZ micelles incubation (scale bar: 20 μm, n = 3).

Figure 3

Viability of 4T1 cells in response to different formulations treatment under normoxia or hypoxia.(n = 4) (A) DHM-Ce6@TPZ without laser irradiation under normoxia or hypoxia. (B) DHM-Ce6 with laser irradiation under normoxia or hypoxia. (C) DHM-Ce6@TPZ without laser irradiation under normoxia or hypoxia. (D) Imaging of living and dead cells upon formulation treatment with the fixed TPZ dose at 1 μg/mL. The cells were stained with Calcein AM (green, live cells) and PI (red, dead cells) (scale bar: 20 μm).

Figure 4

Fluorescence imaging of total reactive oxygen species (ROS) in 4T1 cells upon formulation treatment under different conditions, ROS was performed using a DCFH-DA probe (scale bar: 50 μm) with the dose of TPZ fixed at 1 μg/mL (n = 3).

Figure 5

Kinetic biodistribution of micellar formulations in 4T1 tumor-bearing mice (n = 3). (A) In vivo kinetic fluorescence of Cy5 in 4T1 tumor-bearing mice up to 24 h after intravenous injection of free Cy5, DHM@Cy5 micelles. (B) Kinetic fluorescence quantification regarding Cy5 in tumors. (C) Semi-quantitative fluorescence summary of Cy5 level in healthy organs and tumors at the end of the biodistribution study. ***p < 0.001. (D) Ex vivo fluorescent analysis of Cy5 in tumors and other healthy organs 24 h post-dose administration.

Figure 6

In vivo antitumor efficacy of dual-responsive micelles (n = 5). (A) Tumor-growth inhibitory effects of seven formulations. (B) Quantitative analysis of tumor weight. (C) Images of tumors in the end of treatment. (D) Changes in body weight of mice during treatments. (E) TUNEL staining (scale bar: 50 μm) and H&E staining (scale bar: 100 μm) at the end of therapy. **p < 0.01; ***p < 0.001.

Figure 7

Histological analysis of major healthy organs (heart, liver, spleen, lung, and kidney) at the end of efficacy study (scale bar: 100 µm).