Cascade-amplifying synergistic effects of chemo-photodynamic therapy using ROS-responsive polymeric nanocarriers

Abstract

The simple integration of chemotherapeutic drugs and photosensitizers (PSs) into the same nanocarriers only achieves a combination of chemo-photodynamic therapy but may not confer synergistic effects. The boosted intracellular release of chemotherapeutic drugs during the photodynamic therapy (PDT) process is necessary to achieve a cascade of amplified synergistic therapeutic effects of chemo-photodynamic therapy. Methods: In this study, we explored an innovative hyperbranched polyphosphate (RHPPE) containing a singlet oxygen (SO)-labile crosslinker to boost drug release during the PDT process. The photosensitizer chlorin e6 (Ce6) and doxorubicin (DOX) were simultaneously loaded into RHPPE nanoparticles (denoted as ^SOHNP_Ce6/DOX). The therapeutic efficacy of ^SOHNP_Ce6/DOX against drug-resistant cancer was evaluated in vitro and in vivo. Results: Under 660-nm light irradiation, ^SOHNP_Ce6/DOX can produce SO, which not only induces PDT against cancer but also cleaves the thioketal linkers to destroy the nanoparticles. Subsequently, boosted DOX release can be achieved, activating a chemotherapy cascade to synergistically destroy the remaining tumor cells after the initial round of PDT. Furthermore, ^SOHNP_Ce6/DOX also efficiently detected the tumor area by photoacoustic/magnetic resonance bimodal imaging. Under the guidance of bimodal imaging, the laser beam was precisely focused on the tumor areas, and subsequently, ^SOHNP_Ce6/DOX realized a cascade of amplified synergistic chemo-photodynamic therapeutic effects. High antitumor efficacy was achieved even in a drug-resistant tumor model. Conclusion: The designed ^SOHNP_Ce6/DOX with great biocompatibility is promising for use as a co-delivery carrier for combined chemo-photodynamic therapy, providing an alternative avenue to achieve a cascade of amplified synergistic effects of chemo-photodynamic therapy for cancer treatment.

Keywords: ROS responsive; chemo-photodynamic therapy; drug-resistant cancer; on-demand drug release; synergistic therapy.

Figure 1

(A) Schematic illustration of ^SOHNP_Ce6/DOX with PDT-activated cascade chemotherapy to synergistically treat cancer cells. The PS-generated SO would selectively cleave the thioketal linkers under 660-nm laser irradiation, leading to nanoparticle destruction and triggering DOX release into the cell nuclei. (B) Hydrodynamic diameters of HNP_Ce6/DOX or ^SOHNP_Ce6/DOX. (C) The UV-Vis absorption spectra of free Ce6, free DOX, HNP, ^SOHNP, HNP_Ce6/DOX and ^SOHNP_Ce6/DOX.

Figure 2

(A) Fluorescence recovery of DOX after light irradiation at pH 7.4 or 5.5. (B) The cumulative release of DOX from HNP_Ce6/DOX and ^SOHNP_Ce6/DOX with or without light irradiation. (C) The cumulative release of DOX from ^SOHNP_Ce6/DOX upon 660-nm laser irradiation at different power densities for 30 min. (D) Laser-stimulated pulsed release of DOX from HNP_Ce6/DOX and ^SOHNP_Ce6/DOX at pH 5.5. The samples were irradiated with 660-nm laser for 10 min at different time points indicated by the arrows.

Figure 3

(A) Fluorescence intensity changes of DCF at 525 nm in different groups (PBS, Ce6 in PBS after irradiation, ^SOHNP in PBS after irradiation, HNP_Ce6 in PBS after irradiation and ^SOHNP_Ce6 in PBS after irradiation). Vitamin C acts as an ROS scavenger. (B) The degradation rates of ^SOHNP, HNP_Ce6 and ^SOHNP_Ce6 after irradiation with different power densities (L: 0.2 W/cm², L': 0.1 W/cm²) detected by Ellman's test. (C) Changes in HNP_Ce6/DOX and ^SOHNP_Ce6/DOX diameter after light irradiation. (D) Transmission electron microscopy images of HNP_Ce6/DOX and ^SOHNP_Ce6/DOX with or without light irradiation. The scale bar is 200 nm.

Figure 4

(A) Total intracellular DOX in MCF-7/ADR cells after incubation with free DOX, HNP_Ce6/DOX, or ^SOHNP_Ce6/DOX for 1, 2, 4 or 8 h. The dose of DOX (free DOX or equivalent) was 4 μg/mL in the cell culture. *p < 0.05. (B) Retention of DOX in MCF-7/ADR cells after preincubation with DOX, ^SOHNP_Ce6/DOX or HNP_Ce6/DOX. The concentration of total DOX in the free DOX preincubation was 50 μg/mL, while it was 10 μg/mL in the ^SOHNP_Ce6/DOX or HNP_Ce6/DOX groups. *p < 0.05 compared with HNP_Ce6/DOX or ^SOHNP_Ce6/DOX. (C) Geometric mean fluorescence intensity (GMFI) of MCF-7/ADR cells after treatment with free DOX, HNP_Ce6/DOX, HNP_Ce6/DOX plus laser, ^SOHNP_Ce6/DOX or ^SOHNP_Ce6/DOX plus laser. The power density of the 660-nm laser was 0.1 W/cm². Data were collected from flow cytometric analyses (n = 3). *p < 0.05. (D) Assessment of the intracellular DOX release and biodistribution of HNPCe6/DOX or SOHNPCe6/DOX in MCF-7/ADR cells with continuous 660-nm laser irradiation (0.1 W/cm²) (scale bar: 50 μm). The concentration of DOX in the cell culture was 6 μg/mL. Acidic endosomes/lysosomes and cell nuclei were stained with LysoTracker^TM Green (green) and DAPI (blue), respectively.

Figure 5

Cytotoxicity of DOX, HNP_Ce6/DOX, or ^SOHNP_Ce6/DOX against MCF-7/ADR cells. The cells were incubated with nanoparticles for 12 h. After laser exposure for 30 min, the cells were further incubated with fresh medium for 12 h (A) or 60 h (B). The laser power density was 0.1 W/cm². *p < 0.05. (C) Flow cytometry analysis of MDA-MB-231 cell apoptosis induced by different formulations based on Annexin V-FITC/PI staining. Early apoptotic cells are shown in the lower right quadrant, and late apoptotic cells are shown in the upper right quadrant.

Figure 6

(A) Pharmacokinetic profiles of DOX after intravenous administration of different DOX formulations (mean±SD, n = 4). (B) In vivo fluorescence images of the MCF-7/ADR tumor-bearing mice at 0.5, 1, 2, 6, 12 and 24 h after i.v. injection of ^SOHNP_Ce6/DOX or HNP_Ce6/DOX. The tumor site was circled with a white line. Semiquantitative biodistribution of ^SOHNP_Ce6/DOX (C) and HNP_Ce6/DOX (D) in various organs at 24 h determined by the Ce6 fluorescence intensity. The data are shown as the mean±SD.

Figure 7

(A) PA imaging of tumor regions imaged before and 12 h post-injection of ^SOHNP_Ce6/DOX nanoparticles. The tumor site was circled with a white line. (B) T₁-weighted tumor contrast enhancement before and 12 h post-injection of ^SOHNP_Ce6/DOX nanoparticles. The tumor site was circled with a white line. Semiquantitative analysis of the PA (C) and MR signal (D) in the tumor site, as performed in (A) and (B). *p < 0.05.

Figure 8

(A) Tumor growth inhibition in MCF-7/ADR tumor xenograft-bearing nude mice after different treatments (n = 5). The injections were performed on days 0, 7 and 14 with an equivalent DOX dose of 2.5 mg/kg or a Ce6 dose of 2.0 mg/kg (mean±SD, n = 5). *p < 0.05. (B) The weight of the MCF-7/ADR xenograft tumor mass excised after the treatment. (C) Body weight monitoring of the mice that received treatment with various samples. (D) Enzyme-linked immunosorbent examination of mouse alanine aminotransferase (ALT, U/L), aspartate transaminase (AST, U/L) and blood urea nitrogen (BUN, 10 μmol/L) in the serum after receiving different treatments. *p < 0.05, vs. DOX.