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


Materials and measurements

ART and ISMN were purchased from Energy Chemical (Shanghai, China), mPEG5000 was sourced from AVT Pharmaceutical Tech Co., Ltd (Shanghai, China), apoptosis detection kit, ROS assay kit, and NO fluorescence probe were obtained from Beyotime (Shanghai, China), and RNS fluorescence probe was provided by BestBio (Nanjing, China). The polyclonal antibody was bought from Bioss (Beijing, China). Cell culture vessels were purchased from Nest Biotechnology (Wuxi, China).

1H NMR spectra were recorded on a 400 MHz NMR spectrometer (Bruker, Switzerland). NP size and zeta potential were obtained via dynamic light scattering (DLS) analysis (Malvern, UK). The morphology and diameter were characterized using a transmission electron microscope (TEM). Drug loading rates for different NP formulations were measured using the ultraviolet (UV)-vis spectrum (Metash, UV-5500PC, China). The stability of PSSP@ART-ISMN under physiological conditions was investigated in 10% fetal bovine serum (Excell Bio, China) over the course of seven days using DLS.

Synthesis of ART-ISMN prodrug and PSSP

ART (384 mg), EDC (288 mg), and DMAP (12.2 mg) were dissolved in dry dichloromethane (4 mL), the mixture was stirred in an ice bath for 30 min, and the solution was stirred at room temperature overnight after addition of ISMN (286.5 mg). The ligation reaction was stopped by adding water (10 mL), and the crude products were filtered and dried in a rotary steamer and purified via silica gel flash column chromatography (EA/PE = 2:1) to yield the desired compound. PSSP was synthesized based on our previous work [21, 29, 30]. Detailed synthetic routes are described in the supporting information section (Additional file 1: Schemes S1, S2).


1H NMR (400 MHz, CDCl3) δ 5.77 (d, J = 9.8 Hz, 1H), 5.43 (s, 1H), 5.35 (d, J = 2.8 Hz, 1H), 5.23 (d, J = 2.3 Hz, 1H), 4.99 (s, 1H), 4.50 (d, J = 4.8 Hz, 1H), 4.14–3.95 (m, 3H), 3.90 (dd, J = 11.2, 5.5 Hz, 1H), 2.81–2.69 (m, 2H), 2.65 (dd, J = 12.7, 6.6 Hz, 2H), 2.59–2.48 (m, 1H), 2.35 (dd, J = 13.9, 3.8 Hz, 1H), 2.02 (d, J = 12.5 Hz, 1H), 1.89 (dd, J = 8.8, 4.9 Hz, 1H), 1.61 (d, J = 10.7 Hz, 4H), 1.52–1.45 (m, 1H), 1.43 (s, 3H), 1.43 (s, 2H), 1.32–1.21 (m, 3H), 0.96 (d, J = 5.8 Hz, 3H), 0.84 (d, J = 7.1 Hz, 3H).


1H NMR (400 MHz, DMSO) δ 12.24 (s, 112H), 4.30–4.09 (m, 281H), 3.52 (s, 825H), 2.91 (s, 131H), 2.80 (d, J = 6.3 Hz, 125H), 2.34 (s, 81H), 2.09–1.97 (m, 77H).

Preparation and characterization of PSSP@ART-ISMN

First, 10 mg of PSSP and 2 mg of ART-ISMN were dissolved in 0.8 mL of DMF. The mixture was stirred for 30 min and PSSP@ART-ISMN was obtained via drop-wise precipitation in deionized water (2.4 mL). The obtained PSSP@ART-ISMN was purified by dialysis (MW = 3500 Da) to remove the non-encapsulated drugs.

Drug release studies

The GSH responsiveness of nanoparticles was measured by adding 10 mM GSH for 1 h. The morphology and particle size of nanoparticles after response were measured by transmission electron microscopy and DLS, respectively. A total of 2 mL of PSSP@ART-ISMN (2 mg/mL) was placed in a dialysis bag, which was immersed in 18 mL of phosphate-buffered saline (PBS, pH = 6.5 and pH = 7.4) and 10 mM GSH. At the given times, the withdrawn external solution (0.2 mL) was replenished with the same volume of fresh medium. The cumulative amount of ART-ISMN released in vitro was determined using UV–vis spectra.

Cell lines and animals

SKOV3, HO8910, and IOSE-80 cells (Wuhan Sunncell Biotechnology Co., Ltd, Chian) were cultured in RPMI-1640 medium (Gibco) containing 10% FBS (HAKATA, China) and 1% penicillin/streptomycin. BALB/c nude mice (20 g) were obtained from Guangdong Medical Laboratory Animal Center.

Cellular uptake

SKOV3 cells were cultured overnight on 6-well plates (5 × 105/well). After incubated with PSSP@Rh B for 2, 4, and 6 h, the nucleus of SKOV3 were visualized by DAPI (Beijing Solarbio Science & Technology Co., Ltd, China). Then, the cells were observed using confocal laser scanning microscopy (CLSM). To perform flow cytometry, the cells were collected for the intracellular uptake analysis after being treated with PSSP@Rh B for 2, 4, and 6 h, respectively.

Cell viability studies

Two ovarian cancer cell types (SKOV3, HO8910) and a normal ovarian epithelial cell line (IOSE-80) were cultured on 96-well plates (5 × 103/well) overnight. Since PSSP was demonstrated to be safe in previous work [6, 31], it was not necessary to test their biocompatibility in the present study. The cells were treated with PSSP@ART-ISMN, PSSP@ART, ART-ISMN, ART, and PBS at ART concentrations ranging from 0.3 µM to 40.0 µM. After incubating for 72 h, the cellular viability was assessed via an methyl thiazolyl tetrazolium (MTT) colorimetric assay.

Apoptosis analysis

SKOV3 cells (8 × 103/well) were cultured on 12-well plates overnight. After incubation with PSSP@ART-ISMN, PSSP@ART, ART-ISMN, ART, and PBS for 48 h (ART: 5 µM, ART-ISMN: 5 µM), the cells were collected for double staining with FITC/PI (Yeason, China) according to the manufacturer’s instructions.

Intracellular ROS, NO, and RNS release

The intracellular NO generation was detected by DAF-FM DA (Beyotime, S0019). The intracellular levels of ROS and RNS were measured by DCFH-DA (Beyotime, S0033S) and O52D (BestBio, BB-460567). SKOV3 cells (1 × 105/well) were incubated on 12-well plates overnight, followed by incubation with PBS, ART, ART-ISMN, PSSP@ART, and PSSP@ART-ISMN for 48 h (ART: 5 µM, ART-ISMN: 5 µM). The cells were then washed with PBS three times, harvested, and incubated with a fluorescence probe (DAF-FM DA 1:1,000 dilutions, DCFH-DA 1:800 dilutions, and O52D fluorescent probe 1:100 dilutions) for 30 min. Finally, the cells were rinsed with PBS thrice before imaging, and quantitative analysis was performed using flow cytometry.

Cell cycle studies

SKOV3 cells were incubated on six-well plates (1.5 × 105/well) with serum-free media to synchronize the cell cycle at the G0/G1 stage. After treatment with different agents for 48 h (ART: 5 µM, ART-ISMN: 5 µM). Cell cycle analysis was performed using propidium iodide (PI) staining and analyzed via flow cytometry. Cell population percentages were calculated using ModFit LT 5 software (Verity Software House, Topsham, ME, USA).

Tissue distribution

SKOV3 cell transplantation model was established in female nude mice. Cy5.5-loaded PSSP was prepared to assess the bio-distribution of PSSP@ART-ISMN. An IVIS Spectrum live animal imaging system (IVIS Lumina, USA) was used to capture the fluorescence signal 0, 2, 4, 8, 12, 24, and 48 h post-injection (Eex = 640 nm, Eem = 670 nm). Mice were sacrificed at the end of the experiments, and tumors and major tissues were resected for in vitro fluorescence imaging and histological staining.

Western blotting

SKOV3 cells were cultured on 6-well plates overnight, after being treated with ART, ART-ISMN, PSSP@ART, and PSSP@ART-ISMN for 48 h (ART: 5 µM, ART-ISMN: 5 µM), the total proteins were collected from SKOV3 cells and quantified by a BCA protein assay kit. Then equal quantities of these proteins were loaded on 10% SDS-PAGE (GenScript) and subjected to polyvinylidene difluoride (PVDF) membrane. Then, the membranes were blocked with 5% non-fat milk for 2 h and followed by overnight incubation with primary antibodies (Cyclin D,γ-H2A.X, Caspase-3, Bak, Bcl2, P53, Cytochrome C, and β-actin). Then the secondary IgG antibody was applied and incubated at RT for 2 h. Finally, each band was visualized using an image analysis system (Protein Simple, USA).

Therapeutic effect and systemic toxicity in vivo

The SKOV3 tumor-bearing mice were used to evaluate the therapeutic effect and safety of NPs. The tumor volume (mm3) and body weight were measured daily. The mice were sacrificed at the end of the treatment, and tumors and major organs were harvested for H&E and immunofluorescence staining, which was performed by Servicebio Biological Technology.