Synergetic delivery of artesunate and isosorbide 5-mononitrate with reduction-sensitive polymer nanoparticles for ovarian cancer chemotherapy

Abstract

Ovarian cancer is a highly fatal gynecologic malignancy worldwide. Chemotherapy remains the primary modality both for primary and maintenance treatments of ovarian cancer. However, the progress in developing chemotherapeutic agents for ovarian cancer has been slow in the past 20 years. Thus, new and effective chemotherapeutic drugs are urgently needed for ovarian cancer treatment. A reduction-responsive synergetic delivery strategy (PSSP@ART-ISMN) with co-delivery of artesunate and isosorbide 5-mononitrate was investigated in this research study. PSSP@ART-ISMN had various effects on tumor cells, such as (i) inducing the production of reactive oxygen species (ROS), which contributes to mitochondrial damage; (ii) providing nitric oxide and ROS for the tumor cells, which further react to generate highly toxic reactive nitrogen species (RNS) and cause DNA damage; and (iii) arresting cell cycle at the G0/G1 phase and inducing apoptosis. PSSP@ART-ISMN also demonstrated excellent antitumor activity with good biocompatibility in vivo. Taken together, the results of this work provide a potential delivery strategy for chemotherapy in ovarian cancer.

Keywords: Artesunate; Cell cycle arrest; DNA damage; Isosorbide 5-mononitrate; Mitochondrial damage; Ovarian cancer.

Fig. 1

Characterization of PSSP@ART-ISMN. A Schematic diagram of PSSP@ART-ISMN decomposition process. B UV–vis spectra of ART, ISMN, ART-ISMN, and PSSP@ART-ISMN. C, D DLS data and representative TEM images of PSSP@ART-ISMN, scale bar = 200 nm. Diameter (E) and PDI (F) of PSSP@ART-ISMN in PBS with or without 10% FBS. G HPLC spectra of ART-ISMN incubated in PBS containing esterase. H Cumulative ART release profiles for PBS (pH = 7.4), PBS (pH = 6.5), and in the presence of GSH (10 mM) with or without esterase (30 U/mL). DLS data (I) and representative TEM images J of PSSP@ART-ISMN with 10 mM GSH

Fig. 2

Cell internalization of PSSP@ART-ISMN and its cytotoxicity in vitro. A CLSM images of SKOV3 cells treated with PSSP@Rh B for 2, 4, and 6 h, scale bar = 25 µm. Flow cytometry (B) and semi-quantitative analysis (C) of SKOV3 cells after incubation with PSSP@Rh B for 2, 4, and 6 h. D Relative cell viability of SKOV3, HO8910, and IOSE-80 cells after treatment with ART, ART-ISMN, PSSP@ART, and PSSP@ART-ISMN for 72 h. Statistical analyses were carried out between free ART group and PSSP@ART-ISMN NPs group. E SKOV3 cell apoptosis images after different treatments for 24 h. *P < 0.05, **P < 0.01, and ***P < 0.001; ns indicates P > 0.05

Fig. 3

Anticancer mechanism of PSSP@ART-ISMN. A–C Intracellular ROS, NO, and RNS levels were detected by DCFH-DA, DAF-FM-DA, and O52D bio-probes, respectively, scale bar = 50 µm. D–F Flow cytometry analysis of intracellular ROS, NO, and RNS generation. G Western blot analysis of proteins related to mitochondria-dependent apoptosis, DNA damage, and cell cycle arrest. H Cell cycle study and quantitative analysis of SKOV3 cells after various treatments

Fig. 4

In vivo imaging, therapy, and biocompatibility of PSSP@ART-ISMN. A In vivo fluorescence biodistribution of Cy5.5-labeled PSSP@ART-ISMN in SKOV3 tumor-bearing mice. B Tumor growth curves of SKOV3 tumor-bearing mice receiving different therapies (n = 5). Representative photographs (C) and tumor weight (D) after treatment for 12 days. (E) Mouse body weight change curve over the duration of treatment. F H&E and TUNEL staining analysis of tumor sections, scale bar: 200 µm. G Immunofluorescence staining of tumor sections. *P < 0.05, **P < 0.01, and ***P < 0.001; ns indicates P > 0.05

Fig. 5

In vivo systemic toxicity study. A Evaluation of serum biochemical content in SKOV3 tumor-bearing mice after treatment with saline, ART, ART-ISMN, PSSP@ART, and PSSP@ART-ISMN (n = 3). B Histological analysis of main organs after treatment with different agents. Data are presented as mean ± SD, scale bar: 200 µm